Patent Abstract:
the present invention comprises novel preparations of polyoxypropylene / polyoxyethylene copolymers which retain the therapeutic activity of the commercial preparations , but are substantially free from the undesirable effects which are inherent in the prior art preparations . because the preparations of polyoxypropylene / polyoxyethylene copolymers which comprise the present invention are a less polydisperse population of molecules than the prior an polyoxypropylene / polyoxyethylene copolymers , the biological activity of the copolymers is better defined and more predictable .

Detailed Description:
although the prior art preparations of polyoxypropylene / polyoxyethylene block copolymers may have been suitable for industrial uses , it has been determined that the newly discovered uses for the copolymers as therapeutic agents require less polydisperse populations of molecules in the preparations . the present invention comprises polyoxypropylene / polyoxyethylene copolymers that have a polydisperse value of less than 1 . 05 . the novel copolymers can be prepared by removing disparate molecules from the prior art preparation or by preparing the copolymer according to the method that is contemplated as part of the present invention . the method of preparation of the copolymers of the present invention is the purification of the polyoxypropylene block of the polyoxypropylene / polyoxyethylene copolymer before the polyoxyethylene blocks are added to the molecule . in this way , the partially polymerized polyoxypropylene polymers are removed before the addition of polyoxyethylene polymers to the molecule . this results in a block copolymer that is within the physical parameters which are contemplated as the present invention . the present invention also comprises a polyoxypropylene / polyoxyethylene block copolymer which has the following formula : wherein the molecular weight represented by the polyoxypropylene portion of the copolymer is between approximately 900 and 1500 daltons with a more preferred molecular weight of between 1 , 200 and 6500 daltons and the molecular weight represented by the polyoxyethylene portion of the copolymer constitutes between approximately 5 % and 95 % of the copolymer with a more preferred range of between approximately 10 % and 90 % of the copolymer and the polydispersity value is less than approximately 1 . 07 . the present invention also comprises a polyoxypropylene / polyoxyethylene block copolymer which has the following formula : wherein the molecular weight of the hydrophobe ( c 3 h 6 o ) is approximately 1750 daltons and the average molecular weight of the compound is approximately 8300 to 9400 daltons . the compound has a molecular weight distribution ranging from approximately 5 , 000 to 15 , 000 daltons with a preferred molecular weight range of between approximately 7 , 000 to 12 , 000 daltons . in addition , the copolymer has substantially no unsaturation as measured by nuclear magnetic resonance . the nomenclature of the poloxamer compounds is based on a poloxamer grid ( fig1 ). the poloxamer grid is the relationship between nomenclature and composition of the various polymer members . the hydrophobe ( polyoxypropylene ) molecular weights are given as approximate midpoints of ranges . the first two digits of a poloxamer number on the grid , multiplied by 100 , gives the approximate molecular weight of the hydrophobe . the last digit , times 10 , gives the approximate weight percent of the hydrophile ( polyoxyethylene ) content of the surfactant . 20 for example , poloxamer 407 , shown in the upper right hand quadrant of the grid ( fig1 ), is derived from a 4000 molecular weight hydrophobe with the hydrophile comprising 70 % of the total molecular weight of the copolymer . another example is poloxamer 760 . 5 which has a hydrophobe with a molecular weight of 7600 daltons and has a hydrophile which comprises 5 % of the total molecular weight of the copolymer . the representative poloxamers that are described in this patent application along with their pluronic ® numbers are shown in table i . although molecular weight averages are important and useful when characterizing polymers in general , it is important to know the molecular weight distribution of a polymer . some processing and end - use characteristics ( melt flow , flex life , tensile strength , etc .) are often predicted or understood by observing the values and / or changes occurring in specific molecular weight averages . these values can also be assigned to biological properties of the polyoxypropylene / polyoxyethylene copolymers . a list of the processing characteristics follows . for example , the breadth of the distribution is known as the polydispersity ( d ) and is usually defined as mw / mn . a monodisperse sample is defined as one in which all molecules are identical . in such a case , the polydispersity ( mw / mn ) is 1 . 0 . narrow molecular weight standards have a value of d near 1 and a typical polymer has a range of 2 to 5 . some polymers have a polydispersity in excess of 20 . the equations for expressing polydispersity are as follows : m _ n = σarea i σarea / m i m _ w = σ  [ ( area i )  ( m i ) 2 ] σ  ( area i ) m _ k = σ  [ ( area i )  ( m i ) 2 ] σ  [ ( area i )  ( m i ) ] m _ k + l = σ  [ ( area i )  ( m i ) 3 ] σ  [ ( area i )  ( m i ) 2 ] polydispersity   ( d ) = m _ w m _ n thus , by calculating the parameters listed above , one can specify a certain polydispersity that is acceptable for a pharmaceutical preparation . a high polydispersity value indicates a wide variation in size for the population of molecules in a given preparation while a lower polydispersity value indicates less variation . because molecular size is an important determinant of biological activity , it is important to restrict the dispersity of the molecules in the preparation in order to achieve a more homogeneous biological effect . thus , the polydispersity measurement can be used to measure the dispersity of molecules in a preparation and correlates to that compound &# 39 ; s potential for variation in biological activity . it is to be understood that the polydispersity values that are described herein were determined from chromatograms which were obtained using a model 600e powerline chromatographic system equipped with a column heater module , a model 410 refractive index detector , maxima 820 software package ( all from waters , div . of millipore , milford , mass . ), two lichrogel ps - 40 columns and a lichrogel ps - 20 column in series ( em science , gibbstown , n . j . ), and polyethylene glycol molecular weight standards ( polymer laboratories , inc ., amherst , mass .). polydispersity values obtained using this system are relative to the chromatographic conditions , the molecular weight standards and the size exclusion characteristics of the gel permeation columns . polydispersity measurements using different separation principles may give absolute polydispersibyt values which are different from those described herein . however , one of ordinary skill in the art can easily convert any polydispersity value that is obtained using a different separation method to the values described herein simply by running a single sample on both systems and then comparing the polydispersity values from each chromatogram . in accordance with the present invention , a composition is provided that is a polyoxypropylene / polyoxyethylene block copolymer that has a polydispersity value of less than 1 . 07 . preferably , the polydispersity value is less than approximately 1 . 05 , with a most preferable polydispersity value of 1 . 03 . it is to be understood that the present invention includes , but is not limited to , poloxamer compounds and poloxamine compounds . also in accordance with the present invention , a composition is provided that is a surface - active copolymer comprising a polyoxypropylene / polyoxyethylene block copolymer with the following general formula : wherein the total molecular weight of the copolymer is between approximately 5 , 000 and 15 , 000 daltons , preferably a molecular weight of between approximately 7 , 000 and 12 , 000 daltons and the molecular weight represented by the polyoxyethylene portion of the copolymer constitutes approximately 80 % of the copolymer . one embodiment of the present invention comprises a polyoxypropylene / polyoxyethylene copolymer which has the following formula : wherein the molecular weight of the hydrophobe ( c 3 h 6 o ) is approximately 1750 daltons and the average molecular weight of the compound is approximately 8300 to 9400 daltons . the polydispersity value is less than approximately 1 . 05 . a block copolymer corresponding to at least these physical parameters has the beneficial biological effects of the prior art poloxamer 188 but does not exhibit the unwanted side effects which have been reported for the prior art compound . by reducing the polydispersity value of the surface - active copolymer , it has been found that the toxicity associated with the prior art poloxamer 188 is significantly reduced . however , the beneficial therapeutic activity of the modified poloxamer 188 is retained . the surface - active copolymers of the present invention can be prepared in a number of ways . the polydispersity value can be reduced by subjecting the prior art compounds to gel permeation chromatography . in addition , the compounds can be subjected to molecular sieving techniques that are known to those of ordinary skill in the art . the surface - active copolymer of the present invention can be prepared in several ways . in the first method , commercially available poloxamer 188 is subjected to gel permeation chromatography . the chromatogram that is obtained from this procedure is shown in fig1 . as can be seen in fig1 commercial poloxamer 188 is composed of a broad distribution of molecules with a peak molecular weight of approximately 7900 to 9500 daltons . this corresponds generally to the published molecular weight for poloxamer 188 of 8400 daltons . the published molecular weight for poloxamer 188 is determined by the hydroxyl method . the end groups of polyether chains are hydroxyl groups . the number averaged molecular weight can be calculated from the analytically determined “ oh number ” expressed in mg koh / g sample . it should be understood that the molecular weight of a polydisperse compound can be different depending upon the methodology used to determine the molecular weight . fig1 also shows small secondary peaks or shoulders lying to the left and fight of the primary peak . these areas of the poloxamer 188 chromatogram represent the high and low molecular weight molecules respectively . the high molecular weight species range in size from approximately 24 , 000 to 15 , 000 daltons . it is believed that these larger molecules have a greater capacity to activate complement compared to the lower molecular weight species . the shoulder on the right or lower molecular weight side of the chromatogram is composed of molecules between approximately 2 , 300 daltons and 5 , 000 daltons . this species represents compounds which have more detergent - like properties and are cytotoxic to cells . using the gel permeation chromatography procedure , it has been determined that a fraction of poloxamer 188 with molecules ranging from approximately 5 , 000 daltons to 15 , 000 daltons , preferably between approximately 6 , 000 daltons and 13 , 000 daltons , with a peak at approximately 8 , 700 daltons , represents a population of surface - active copolymers which are essentially devoid of toxic activities while still retaining the beneficial therapeutic activity of the commercially available poloxamer 188 . this new composition is a much more homogeneous preparation than those currently available and unexpectedly has fewer side effects than the prior art preparation . it should be understood that the molecular weight range that is described as the optimum range for the copolymer is to be considered the outside range and that any population of molecules that fall within that range are considered as embodiments of the present invention . the present invention also includes a novel method of preparing a surface - active copolymer composition with the specifications described herein . the novel method involves the preparation of a uniform hydrophobic polyoxypropylene polymer and then proceed with the addition of the hydrophilic polyoxyethylene as is normally done . it is believed that the toxic copolymers that are the result of the standard commercial method of preparing poloxamer 188 are due to truncated polymer chains and to unsaturation in the polymer . in practicing the present invention , the hydrophobic polyoxypropylene polymer is purified to obtain a substantially uniform population of polyoxypropylene polymers . the purification can be performed using gel permeation chromatography . however , any method known to one of ordinary skill in the art which gives the desired range of polyoxypropylene polymers can be used . in preparing the improved rheologic reagent , the polyoxypropylene polymer should have an average molecular weight of approximately 1750 daltons with an approximate molecular weight range between 1 , 000 and 2 , 600 daltons . the preferred molecular weight range is between 1 , 200 and 2 , 400 daltons . after the desired polyoxypropylene copolymer has been obtained , the ethylene portion of the copolymer is added to both ends of the molecule by standard methods well known to those of ordinary skill in the art . the final polymer population should have a polyoxyethylene composition of approximately 20 % of the total molecular weight of the molecule . this invention is further illustrated by the following examples , which are not to be construed in any way as imposing limitations upon the scope thereof . on the contrary , it is to be clearly understood that resort may be had to various other embodiments , modifications , and equivalents thereof which , after reading the description herein , may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims . poloxamer 188 ( basf corporation , parsippany n . j .) is dissolved in tetrahydrofuran at a concentration of 20 mg / ml . a model 600e powerline chromatographic system equipped with a column header module , a model 410 refractive index detector and maxima 820 software package ( all from waters , div . of millipore , milford , mass .) is used to fractionate the commercially prepared poloxamer 188 copolymer . the chromatographic system is equipped with two lichrogel ps - 40 columns and a lichrogel ps - 20 column in series ( em science , gibbstown , n . j .). the lichrogel ps - 40 columns are 10 μm particle size and the lichrogel ps - 20 columns is 5 μm particle size . all columns are 7 mm by 25 cm in size . 200 μl ( 4 mg ) of the poloxamer 188 in tetrahydrofuran is added to the column and the sample is run with the columns and the detector at 40 ° c . the resulting chromatogram is shown in fig2 . the sample that was collected in example i was fractionated into five fractions and each fraction was run on the column as described in example i . the chromatograms from the various chromatographic runs are shown in fig3 through 8 . the fraction that demonstrates the least toxicity while retaining the therapeutic activity of the poloxamer 188 is shown in fig5 . as can be clearly seen , the shoulders on either side of the peak in fig5 are absent . the average molecular weight for each fraction is shown in table ii . the chromatogram for each fraction is indicated in fig3 through 8 . the polydispersity value for the unfractionated poloxamer 188 is 1 . 0896 . the fraction that most closely corresponds to poloxamer 188 is fraction 3 which has a polydispersity value of approximately 1 . 0280 . in a one - liter 3 neck round bottom flask equipped with a mechanical stirrer , reflux condenser , thermometer and propylene oxide feed inlet , there is placed 57 grams ( 0 . 75 mol ) of propylene glycol and 7 . 5 grams of anhydrous sodium hydroxide . the flask is purged with nitrogen to remove air and heated to 120 ° c . with stirring until the sodium hydroxide is dissolved . sufficient propylene oxide is introduced into the mixture as fast as it reacts until the product possesses a calculated molecular weight of approximately 1750 daltons . the product is cooled under nitrogen and the naoh catalyst is neutralized with sulfuric acid and the product is then filtered . the final product is a water - insoluble polyoxypropylene glycol . the polyoxypropylene glycol from example iii is dissolved in tetrahydrofuran at a concentration of 20 mg / ml . a model 600e powerline chromatographic system equipped with a column heater module , a model 410 refractive index detector and maxima 820 software package ( all from waters , div . of millipore , milford , mass .) is used to fractionate the commercially prepared poloxamer 188 copolymer . the chromatographic system is equipped with two lichrogel ps - 40 columns and a lichrogel ps - 20 column in series ( em science , gibbstown , n . j .). the lichrongel ps - 40 columns are 10 μm particle size and the lichrogel ps - 20 column is 5 μm particle size . all columns are 7 mm by 25 cm in size . 200 μl ( 4 mg ) of the polyoxypropylene glycol in tetrahydrofuran is added to the column and the sample is run with the columns and the detector at 40 ° c . the fraction which corresponded to an average molecular weight of 1750 daltons with a molecular weight distribution between 1 , 000 and 2 , 600 daltons was collected . other fractions were discarded . the purified polyoxypropylene glycol from example iv was placed in the same apparatus as described in example iii with an appropriate amount of anhydrous sodium hydroxide . an appropriate amount of ethylene oxide was added at an average temperature of 120 ° c . using the same technique described in example iii . the amount of added ethylene oxide corresponded to 20 % of the total weight of the polyoxypropylene glycol base plus the weight of added ethylene oxide . this procedure results in a polyoxypropylene / polyoxyethylene block copolymer composed of molecules which are far more homogeneous relative to molecular size and configuration compared to commercial preparations . fractions of poloxamer 760 . 5 prepared by gel permeation chromatography and were analyzed for weight percent of oxyethylene and for unsaturation by nmr analysis as follows : poloxamer 760 . 5 ( basf corporation , parsippany n . j .) is dissolved in tetrahydofuran at a concentration of 20 mg / ml . a model 600e powerline chromatographic system equipped with a column header module , a model 410 refractive index detector and maxima 820 software package ( all from waters , div . of millipore , milford , mass .) is used to fractionate the commercially prepared poloxamer 760 . 5 copolymer . the chromatographic system is equipped with ultrastyragel 10 3 a and 500 a in series ( waters , div . of millipore , milford , mass .). column size 7 . 89 mm internal diameter by 30 cm . precolumn filters # a - 315 with removable 2 μm fits ( upchurch scientific , oak harbor , wash .) were used for protection of the columns . 200 μl ( 4 mg ) of the poloxamer 760 . 5 in tetrahydrofuran is added to the column and the sample is run with the columns at 40 ° c . and the detector at 45 ° c . sample one is an unfractionated sample of the polaxamer 760 . 5 is obtained from basf corporation ( parsippany , n . j .) and is shown in fig9 a . fraction one is an early fraction from the chromatographic system and is shown in fig9 b . fraction two is a late fraction and is shown in fig9 c . all proton nmr analyses were performed in accordance with the nf procedure “ weight percent oxyethylene ” on a bruker 300 mhz instrument . the proton nuclear magnetic resonance spectra from fig9 b and 9c showed slight ban broadening in the spectra when compared to the unfractionated sample . the late eluting fraction ( fraction 2 ) contains the largest amount of unsaturation as noted by a doublet signal at about 4 . 0 ppm . the proton spectra for the early eluting peak ( fraction 1 ) showed no impurities except water . the weight percent oxyethylene was calculated for the samples . as can be seen from table iii , the early eluting fraction , which is the purest fraction , has the lowest percentage of oxyethylene . this fraction also showed no unsaturation as measured by nuclear magnetic resonance . using the poloxamer nomenclature system described above , the various fractions have the following characteristics and poloxamer number . poloxamer 188 ( pluronic ® f68 ) was fractionated on a gel permeation chromatography system according to example i . three fractions were collected . fig1 a shows fraction 1 , an early , high molecular weight fraction . fig1 b shows fraction ii , which is the major peak . fig1 c shows fraction iii , a late eluting , lower molecular weight population of molecules . the percent oxyethylene of each fraction was determined by proton nmr using a 200 mhz nmr spectrophotometer . approximately 10 mg of each sample was tested . samples were prepared by adding approximately 0 . 7 ml of cdcl 3 to each vial . the solution was filtered and transferred to a 5 - mm nmr tube . one drop of d 2 o was added , and the tube was shaken prior to treatment . as shown in table iv , the early eluting , the large molecular weight fraction had a high percentage of oxyethylene and corresponded to a poloxamer 258 . the middle fraction had the smallest percentage of oxyethylene while the late eluting , small molecular weight fraction had the highest percentage of oxyethylene . the middle fraction had a calculated poloxamer number of 178 which corresponds closely to the desired number of 188 . the late fraction had a calculated poloxamer number of 039 . thus , the commercially available poloxamer preparation has a significant population of polymers which may be harmful in a biological system . poloxamer 331 ( pluronic ® l101 ) was fractionated according to the protocol in example vi . the chromatographs for unfractionated poloxamer 331 , an early eluting fraction and a late eluting fraction are shown in fig1 a through 12c respectively . the nmr spectra for each sample was then determined as in example vi . the results of these spectra and chromatograms are summarized in table v . when the poloxamer number for each fraction is calculated based on the empirical data collected , it is seen that the late fraction polymer is a very different poloxamer than the unfractionated preparation . in addition , the unsaturated population of polymers has been removed by the fractionation procedure . it should be understood that the foregoing relates only to a preferred embodiment of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims . 1 . adamson , a . w ., physical chemistry of surfaces . 4th ed ., john wiley & amp ; sons , new york ( 1982 ). 2 . see generally , hemostasis and thrombosis , basic principles and clinical practice , ed . by colman , et al ., j . b . lippincott company ( 1987 ) 3 . atkinson , t . p ., et al ., ““, am . j . physiol . 254 : c20 ( 1988 ). 4 . brooks , d . e ., and evans , e . a ., rheology of blood cells in clinical hemorheology , applications in cardiovascular and hematological disease , diabetes , surgery and gynecology . s . chien , j . dormandy , e . ernst , and a . matrai , eds , martinus nijhoff publishers , dordrecth ( 1987 ). 5 . thompson , a . r ., and harker , l . a ., manual of hemostatis and thrombosis , edition 3 , f . a . davis company , philadelphia ( 1983 ). 6 . lee , l . h ., “ effect of surface energetics on polymer friction and wear ”, in advances in polymer friction and wear , polymer science and technology , vol . 5a . l . h . lee , editor , plenum press , new york ( 1974 ). 10 . grover , f . l ., et al ., “ a nonionic surfactant and blood viscosity ”, arch surg , 106 : 307 ( 1973 ). 11 . papadea , c . and hunter , r ., “ effect of rheothrx ® copolymer on blood viscosity related to fibrin ( ogen ) concentration ”, faseb j 2 : a384 ( 1988 ). 12 . wiman b . and rånby , m ., “ determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay ”, thromb . haemost ., 55 : 189 ( 1986 ). 13 . connaghan , d . g ., francis , c . w ., lane , d . a ., and marder , v . j ., “ specific identification of fibria polymers , fibrinogen degradation products , and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique ”, blood , 65 : 589 ( 1985 ). 14 . wiman , b . and rånby , m ., “ determination of soluble fibrin in plasma by a rapid and quantitative spectrophotometric assay ”, throm . haemost . 55 : 189 ( 1986 ). 15 . vercellotte , g . m ., et al ., “ activation of plasma complement by perfluorocarbon artificial blood : probable mechanism of adverse pulmonary reactions in treated patients and rationale for corticosteroid prophylaxis ”, blood , vol . 59 , pp . 1299 - 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