Abstract:
Provided is a pharmaceutical composition for permeabilizing fetal membranes including an active ingredient having a log K in the range of 2 to 4, where K is the octanol/water partition coefficient. The active ingredient may be, for example, bupivacaine, sodium lauryl sulfate or oleic acid. Further provided is a system for transfetal membrane transport. The system includes a probe unit adapted for insertion into a female reproductive tract and releasing a substance onto fetal membranes that permeabilizes the membranes. The system is also configured to apply ultrasound radiation to the fetal membranes to further increase the membrane permeability.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention relates to pharmaceutical compositions and to medical devices, and more particularly to gynecological compositions and medical devices. 
       BACKGROUND OF THE INVENTION 
       [0002]    The following prior art publications are considered relevant for an understanding of the invention.
   1. Karande P., Jain A. &amp; Mitragotry S., Discovery of transdermal penetration enhancers by high-throughput screening, J Nature Biotechnology, 22, 2 (2004).   2. Suhonen M., Bouwstra J., &amp; Urtti A., Chemical enhancers of percutaneous absorption in relation to stratum corneum structural alteration. J Control Release 59, 146-161 (1999).   3. Mitragotri S., Effect of bilayer disruption on transdermal transport of low molecular weight hydrophobic solutes. Pharm. Res. 18, 1022-1028 (2001).   4. Williams A C, Barry B W. Chemical permeation enhancement, in: Elka Touitou, Brian W. Barry (Ed.), Enhancement in Drug Delivery, CRC Press, 233-254 (2006).   5. U.S. Pat. No. 6,773,418 to Sharrow et al.   6. U.S. Pat. No. 6,264,638 to Contente.   7. U.S. Pat. No. 6,139,538 to Houghton et al.   8. U.S. Pat. No. 5,988,169 to Anderson et al.   9. U.S. Pat. No. 7,425,340 to Grenier et al.   
 
         [0012]    Prenatal testing involves testing a fetus for the presence of various hereditary or spontaneous genetic disorders, such as Down syndrome. One of the most common procedures for detecting abnormalities before birth is amniocentesis in which a sample of the fluid surrounding the fetus (amniotic fluid) is obtained. In amniocentesis, after anesthetizing an area of abdominal skin, a needle is inserted through the abdominal wall into the amniotic cavity. During the procedure, ultrasonography is performed so that the position of the fetus can be monitored and the needle guided into place without touching the fetus. Amniotic fluid is aspirated through the needle into a syringe, and the needle is then removed. Another method of fetal examination is chorionic villus sampling (CVS). Both amniocentesis and CVS are invasive, and as such carry a small but definite risk to the mother and fetus. After amniocentesis, the chance of miscarriage due to the procedure is about 1-2 in 100. Also these invasive tests for evaluating fetus health can be preformed only at relatively late stages of the pregnancy (from week 13) and two more weeks are required to obtain the results. Moreover, many women fear of these invasive tests because of the pain and risk of miscarriage. 
         [0013]    In recent years, numerous efforts have been made to find alternatives to invasive procedures. One of these methods involves applying a chemical penetrating enhancer (CPE) onto a biological membrane in order to enhance the permeability of the membrane. CPEs alter the partition coefficient of substances to be transported across the membrane, either by modification of the substances to be transported or altering the membrane structure. In transdermal applications, CPEs usually enhance skin permeability by altering the stratum corneum structure [1-2]. Since the stratum corneum consists of dead keratinized cells, disruption of its structure does not induce irritation [3]. 
         [0014]    CPEs can be divided into major classes based on their chemical structure. The major classes of CPEs are: water, sulfoxides, azone, pyrrolidones, fatty acids, alcohols and glycols, surfactants, urea, essential oils, terpenes and terpenoids, phospholipids, and ceramide analogs [4]. 
         [0015]    Ultrasound has been used in a number of medical applications. Examples of clinical applications of ultrasound include imaging, stimulation of the healing of soft tissue, during topical application of a medication, and for enhancement of transdermal drug delivery into the circulatory system. In addition, ultrasound has also been used for selectively altering the permeability of cell membranes. This alteration is reversible and the effect can be controlled as to its extent and rate. 
         [0016]    U.S. Pat. No. 6,773,418 to Sharrow et al discloses a device for delivering an agent to the uterine cervix. The device includes a chamber that is engaged with the cervix. An agent delivery port in fluid communication with the chamber is provided for delivery of the agent to the uterine cervix. A vacuum port in fluid communication with the chamber allows application and retention of vacuum pressure to the chamber to seal the chamber to the cervix and prevent leakage of the agent away from the cervix. 
         [0017]    U.S. Pat. No. 6,264,638 to Contente discloses a device for introducing agents, including drugs and other substances, into the vaginal canal. The device may also be used to collect discharges from the canal. The device has an elastic rim surrounding a flexible film and is lodged in the vaginal canal. 
         [0018]    U.S. Pat. No. 6,139,538 to Houghton et al discloses an apparatus for iontophoretically delivering an agent to a uterus or cervix. The apparatus comprises a probe sized to fit within the cervical canal. The device includes a reservoir for containing the agent to be delivered and a pair of electrodes for iontophoretically delivering the agent into the uterus or cervix. 
         [0019]    U.S. Pat. No. 5,988,169 to Anderson et al discloses a vaginal insert for delivering an agent into a female urogenital tract. The insert has first and second portions projecting outward from a main portion where at least one of the projecting ends has means for containing the agent. The projecting ends of the first and second portions are configured to engage the anterior vaginal wall while the main portion engages the posterior vaginal wall, thereby positioning the projecting end of the first portion proximal to one side of the urogenital tract and positioning the projecting end of the second portion proximal to an opposite side of the urogenital tract. 
         [0020]    U.S. Pat. No. 7,425,340 to Grenier et al discloses a composition for transdermal transmucosal administration comprising a therapeutically effective amount of an anticholinergic or antispasmodic agent, and a urea-containing compound in an amount sufficient for enhancing permeation of the anticholinergic agent, and a carrier system suitable for topical or transdermal drug delivery. The composition may be used for treating urinary incontinence. The composition may be administered via buccal and sublingual tablets, suppositories, vaginal dosage forms, or other passive or active transdermal devices for absorption through the skin or mucosal surface. 
       SUMMARY OF THE INVENTION 
       [0021]    In its first aspect, the present invention provides a pharmaceutical composition for permeabilizing fetal membranes. The pharmaceutical composition of the invention comprises an active ingredient and a physiologically acceptable carrier wherein the active ingredient comprises any one or more ingredients having a log K in the range of 2-4, where K is the octanol/water partition coefficient of the active ingredient. The inventors have found that substances having a log K in the range of 2 to 4 are capable of permeabilizing fetal membranes. 
         [0022]    For example, the composition may contain bupivacaine in a concentration from 0.1% to 1% (wt/vol), or sodium lauryl sulfate (SLS) in a concentration from 0.1% to 10%, or oleic acid in a concentration from 0.1% up to 2%. Alternatively, the pharmaceutical composition may comprise a mixture of bupivacaine, SLS, and limonene. In this case, the bupivacaine may have a concentration from 0.1% to 10%, the limonene may have a concentration from 1% to 10%, and the SLS may have a concentration from about 0.1% to 10%. 
         [0023]    The pharmaceutical composition of the invention may be in the form of a liquid or a paste in order to allow it to be applied to the amniotic membrane. 
         [0024]    In its second aspect, the invention provides a system for permeabilizing a fetal membrane. The system of the invention comprises a probe unit adapted for insertion through a vagina into a female reproductive tract. The probe unit has a reservoir and a slender shaft. A delivery system is configured to release a substance stored in the reservoir from the distal end of the shaft. The system further comprises an ultrasound source located at the distal end of the shaft, and a control unit configured to activate the ultrasound source. The system is used to release a pharmaceutical composition of the invention from the reservoir and apply the composition to the fetal membrane to be permeabilized. Activation of the ultrasound enhances the permeabilization caused by the active ingredient in a synergistic manner. 
         [0025]    The shaft may be curved or bent to form a vaginal portion and a cervical portion in order to facilitate insertion into the female reproductive tract and delivery of the distal end of the shaft to the cervix. Alternatively, the shaft may be flexible. 
         [0026]    The system of the invention may further comprise a collecting system configured to collect substances around the distal end of the shaft. This may be used to collect substances released from the amniotic sac after permeabilization in accordance with the invention. The collection system may comprise a vacuum system or a solution of high osmotic pressure that collects fluids by osmosis or vacuum 
         [0027]    The invention also provides a method for permeabilizing a fetal membrane comprising applying to the fetal membrane a pharmaceutical composition of the invention. The pharmaceutical composition may be applied to the fetal membrane by releasing the pharmaceutical composition from the distal end of the shaft of a system of the invention. The method may further comprise applying ultrasound radiation to the fetal membrane. The ultrasound radiation may have a frequency in the range of 20 kHz to 100 kHz. 
         [0028]    Further provided by the invention is a method for delivering one or more substances into an amniotic sac comprising permeabilizing fetal membranes of the amniotic sac by the method of the invention and applying the one or more substances to the permeabilized membrane. The one or more substances may be applied (with or without ultrasound) to the membrane by releasing the one or more substances from the distal end of the shaft of a system of the invention. 
         [0029]    The invention further provides a method for collecting a fluid from an amniotic sac comprising permeabilizing fetal membranes of the amniotic sac by the method of the invention and collecting fluid released from the amniotic sac. The fluid released from the amniotic sac may be collected into the reservoir of a system of the invention or detected in situ by a sensor at the distal end of the shaft. 
         [0030]    Thus, in its first aspect, the present invention provides a pharmaceutical composition for permeabilizing fetal membranes comprising one or more active ingredients and a physiologically acceptable carrier wherein the one or more active ingredients have a log K in the range of 2 to 4, where K is the octanol/water partition coefficient. 
         [0031]    In its second aspect, the invention provides a system for transfetal membrane transport comprising:
       (a) a probe unit adapted for insertion through a vagina to a into a female reproductive tract comprising a shaft having a proximal end and a distal end;   (b) an ultrasound source located at the distal end of the shaft;   (c) a reservoir;   (d) a delivery system configured to release a substance in the reservoir from the distal end of the shaft; and   (e) a control unit configured to activate the ultrasound source.       
 
         [0037]    In another of its aspects, the invention provides a method for permeabilizing a fetal membrane comprising applying to the fetal membrane a pharmaceutical composition of the invention. 
         [0038]    In still another of its aspects, the invention provides a method for delivering one or more substances into an amniotic sac comprising permeabilizing fetal membranes of the amniotic sac by the method of the invention and applying the one or more substances to the permeabilized membrane. 
         [0039]    In yet another aspect, the invention provides a method for collecting a fluid from an amniotic sac comprising permeabilizing fetal membranes of the amniotic sac by the method of the invention and collecting fluid released from the amniotic sac. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0040]    In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: 
           [0041]      FIG. 1  shows a system for trans-fetal membranes transport according to one embodiment of the invention; 
           [0042]      FIG. 2  shows a probe unit for use in the embodiment of  FIG. 1 ; 
           [0043]      FIGS. 3   a  and  3   b  show the probe of  FIG. 2  inserted in a vagina and a cervix; 
           [0044]      FIG. 4  shows a system for trans-fetal membranes transport according to another embodiment of the invention; 
           [0045]      FIG. 5  shows a probe unit for use in the embodiment of  FIG. 1 ; 
           [0046]      FIGS. 6   a  and  6   b  show the probe of  FIG. 2  inserted in a vagina and a cervix; 
           [0047]      FIG. 7  shows a system for in vitro trans-fetal membranes transport; 
           [0048]      FIG. 8  shows the effect of different CPEs on postpartum human fetal membranes permeability; and 
           [0049]      FIG. 9  shows a synergistic effect on the permeability of fetal membranes of a substance having a log K in the range of 2 to 4 and ultrasound radiation. 
       
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Permeabilization of Amniotic Membranes 
       [0050]    The permeability of postpartum human fetal membranes (obtained from Hillel Yaffe Medical Center, Israel) upon exposure to different substances was determined. The experimental set-up used is shown schematically in  FIG. 7 . As shown in  FIG. 7 , a fetal membrane  171  (a piece of a gestational sac) was mounted on a vertical glass diffusion cell  172 . The membrane  171  was first incubated with a substance  176  added to the donor compartment  173 . After 30 minutes of incubation, the substance was removed from the donor compartment  173 . The donor compartment was then washed with PBS, and the donor and receiver compartments were filled with 0.01M phosphate buffered Saline (PBS)  175 . 5 mL of a 0.5 mg/mL Dextran solution in PBS (average molecular weight of the Dextran 77 KDa) conjugated to the fluorescent label FITC (fluorescein isothiocyanate-Dextran))  179  was added to the donor compartment  173 . The diffusion cell  172  was protected from light in order to prevent fluorescent bleaching of the FITC. 1 mL samples were withdrawn from the receiver compartment  174  at various times with constant stirring of the receiver compartment, and the concentration of the FITC in the samples was determined spectofluorimetrically. 
         [0051]    From the rate of increase in the concentration of the fluorescence in the receiver compartment  174 , the extent of permeability enhancement of the membrane, in comparison to a control experiment was calculated.  FIG. 8  shows the enhancement in permeability of the membrane treated by several substances having a log K in the range of 2 to 4 compared to a control in which the incubation step was omitted (column a in  FIG. 8 ). The results shown in  FIG. 8  were obtained with the maternal side facing the donor compartment. Of the substances tested, a maximal effect was observed with a mixture of 1% sodium lauryl sulfate (SLS), 2% limonene, and 0.5% bupivacaine (b). Enhanced permeability was also observed with 10% iso-stearic acid (c), 1% lidocaine (d), 0.5% bupivacaine (e), 1% SLS (f), 1.5% Oleic acid (g), 10% ethylene glycol (h), 4% Cetyl trimethylammonium bromide (CTAB) (i), a mixture of 1% lidocaine and 1% SLS (j), and N-methy-2-pyrrolidone (NMP) (k). 
         [0052]    Results similar to those presented in  FIG. 8  were obtained with the maternal side facing the receiving compartment (not shown). 
         [0053]      FIG. 9  shows enhancement of permeability of fetal membranes after incubation with the above mentioned mixture comprising a combination of 1% Sodium Lauryl Sulfate (SLS), 2% limonene, and 0.5% bupivacaine. a substance having a log K in the range of 2 to 4 (b), after exposure to ultrasound radiation (c), and after exposure to both the mixture and ultrasound (c), in comparison with control membranes which were not exposed to the mixture or to ultrasound radiation (a). Exposure to the substance and the ultrasound radiation had a synergistic effect on the permeability. 
         [0054]      FIG. 1  shows a system generally indicated by  2  for transfetal membrane transport in accordance with one embodiment of the invention. As explained below, the system  2  may be used to generate transport through the fetal membranes from the interior of the gestational sac to the exterior, for example, to obtain a sample of amniotic fluid or coelomic fluid. Alternatively, the system  2  may be used to generate transport through the fetal membranes into the gestational sac, for example, to deliver a drug into the sac. 
         [0055]    The system  2  comprises a control unit  4  and a probe unit  6  which is attached to the control unit  4  via a harness  8 . The probe unit  6  has a handle  10 , a shaft  12 , a proximal end  13  and a distal end  15 . The shaft  12  may be rigid and permanently bent or curved to form a vaginal portion  16  and a cervical portion  18 . Alternatively, the shaft may be flexible so as to be bendable into an angled shape having a vaginal portion and a cervical portion to match the anatomy of the recipient of the treatment. 
         [0056]      FIG. 2  shows the probe unit  6  in greater detail. The cervical portion  18  comprises an ultrasound source  17  that emits ultrasound waves from the distal end  15  of the probe unit  6 . The ultrasound source  17  is contained in an outer sleeve  24  that extends along the length of the shaft  12 . The outer sleeve  24  is made from a biocompatible material such as Teflon or silicone. The outer sleeve  24  is attached to the handle  10  at a collar  25 . The outer sleeve is preferably detachable from the handle  10 , and is most preferably disposable. The probe unit also comprises a reservoir  122 . The reservoir  122  may be located in the distal end of the probe unit  6 , as shown in the embodiment of  FIG. 2 , or at the proximal end of the probe unit  6 . The probe unit  6  is also provided with a delivery system which releases a substance  124  stored in the reservoir  122  from the distal end of the probe  6 . The substance may be a pharmaceutical composition of the invention or an acoustic coupling medium for acoustic coupling of the ultrasound sonication to the body tissues, as explained below. Depressing a spring-biased push button  28  drives a piston  29  to create an elevated pressure in the reservoir  122  via a conduit  26  that urges the substance  124  to flow from the reservoir  122  out of the distal end  15  of the shaft  12 . 
         [0057]    The system  2  is also provided with a vacuum system that draws into the probe  6  substances surrounding the distal end of the shaft  12 . A vacuum pump  28  may be located in the control unit  4 , as shown in  FIG. 1 , or may be external to the control unit  4 . The vacuum pump  28  creates a negative pressure in a receptacle  38  in the sleeve  24  via a vacuum hose  32  in the harness  8 , and a connecting channel  34  in the handle  10 . A normally closed valve  36  in the connecting channel  34  is opened by depressing a spring biased push-button switch  37  when it is desired to create a negative pressure in the receptacle  38 , as explained below. 
         [0058]    In an alternative embodiment (not shown) drawing of substances released from the gestational sac into the probe unit  6  utilizes a solution of high osmotic pressure that is applied to the external surface of the gestational sac. The high osmotic pressure solution draws amniotic and/or ceolomic fluid and dissolved or suspended substances across the fetal membranes by osmosis from the interior to the exterior of the gestational sac where the substances are collected in a receptacle. 
         [0059]    The control unit contains a power supply  40  that is connected to the ultrasound transducer  17  via wires  44  in the harness  8  that connect with wires  45  in the probe unit  6 . Closing a switch  41  on the handle  10  activates the ultrasound source  17  to the power supply  40 . The control unit also contains a user input device, such as a key pad  42  that allows a user to input values of various parameters relating to the ultrasound sonication, such as intensity, pulse duration, pulse repetition rate or wavelength, as well as details relating to the individual being examined or the treatment. 
         [0060]      FIGS. 3   a  and  3   b  show use of the system  2  to collect a body substance such as an amniotic fluid sample or a coelomic fluid sample from an individual  50 . The shaft  12  of the probe unit  6  is introduced into the vagina  51  and positioned with the vaginal portion  16  in the vagina  51  and the cervical portion  18  in the cervix  52 . Positioning of the probe unit  6  in the body may be monitored by external ultrasonography to ensure proper placement of the probe unit  6  in the body. A small amount of coupling medium  24  is then expelled from the distal end  15  of the probe unit  6  by depressing the push-button  28 . The distal end  15  of the probe is then apposed to a portion of the fetal membranes  56  adjacent to the cervix  52  in order to ensure acoustic coupling of ultrasound sonication to the portion  56  of the fetal membranes. Ultrasound sonication  54  emitted from the ultrasound source  17  is directed to the portion of the fetal membranes  56  adjacent to the cervix  52 . The ultrasound activation button  41  is then depressed to activate the ultrasound transducer  17 . Substances withdrawn from the gestational sac may be collected at any time by depressing the push-button  29  to open the vacuum valve  36 . As demonstrated above, exposure of the fetal membranes  56  to the pharmaceutical composition of the invention increases the permeability of the fetal membranes. The permeability of the fetal membranes  56  may be monitored at any time by measuring the conductivity of the membranes (not shown). Substances passing out of the gestational sac as a result of the increased permeability, such as amniotic or coelomic fluid, are drawn into the distal end  15  of the probe unit  6  under the influence of the vacuum system and/or osmotic pressure when present, and are collected in the receptacle  38 . After collection of substances passing through the fetal membranes, the vacuum is turned off, and the probe unit  6  is removed from the body. Substances collected in the receptacle  38  are then removed from the receptacle  16  and are analyzed. 
         [0061]      FIG. 4  shows a system generally indicated by  102  for transfetal membranes transport, in accordance with another embodiment of the invention. The system  102  may be used to transport substances such as drugs, from the exterior of the fetal membranes into the gestational sac. 
         [0062]    The system  102  comprises a control unit  104  and a probe unit  106  which is attached to the control unit  104  via a harness  108 . The probe unit  106  has a handle  110 , a shaft  112 , a proximal end  113  and a distal end  115 . The shaft  112  may be rigid and permanently bent, or may be bendable to form a vaginal portion  116  and a cervical portion  118 . 
         [0063]      FIG. 5  shows the probe unit  106  in greater detail. The cervical portion  118  comprises an ultrasound source  117  that emits ultrasound waves from a distal end  115  of the probe unit  106 . The ultrasound source  117  is contained in an outer sleeve  124  that extends along the length of the shaft  112 . The outer sleeve  124  is attached to the handle  110  at a collar  125 . The outer sleeve is preferably detachable from the handle  110 , and is most preferably disposable. The probe unit  106  is also provided with a coupling medium delivery system which delivers an acoustic coupling medium to the distal end of the probe unit  106  for acoustic coupling of the ultrasound sonication to the body tissues, as explained below. A reservoir  122  is used to store an amount of an ultrasound coupling medium and/or a pharmaceutical composition of the invention  124 . Depressing a spring-biased push button  128  drives a piston  129  to create an elevated pressure in the reservoir  122  via a conduit  126  that urges the coupling medium and/or pharmaceutical composition of the invention  124  to flow out from the reservoir  122  though the delivery tube  126  to the distal end  115  of the probe unit  112 . 
         [0064]    The system  102  is provided with a delivery system for delivering one or more substances, such as drugs and a pharmaceutical composition of the invention  124 , to the external surface of the gestational sac. The drugs  160  are stored in a reservoir  165 . Depressing a spring-biased push button  168  drives a piston  169  to create an elevated pressure in the reservoir  165  via a conduit  166  that urges the drugs to flow from the reservoir  165  out of the distal end  115  of the probe unit  112 . 
         [0065]    The control unit contains a power supply  140  that is connected to the ultrasound transducer  117  via wires  144  in the harness  108 . Closing a switch  141  on the handle  110  activates the ultrasound source  117 . The control unit also contains a user input device, such as a key pad  142  that allows a user to input values of various parameters relating to the ultrasound sonication, such as intensity, pulse duration, pulse repetition rate or wavelength, as well as details of the individual being examined. 
         [0066]      FIGS. 6   a  and  6   b  show use of the system  102  to deliver the one or more substances  160 , such as a drug, into a gestational sac of an individual  150 . The shaft  112  of the probe unit  106  is introduced into the vagina  152  and is positioned with the vaginal portion  116  in the vagina  151  and the cervical portion  118  in the cervix  152 . A small amount of a pharmaceutical composition of the invention  124  is then delivered to the distal end  115  of the probe unit  106  by depressing the push-button  128 . The distal end  115  of the probe is then apposed to a portion of the fetal membranes  156  adjacent to the cervix  152  in order to ensure acoustic coupling of ultrasound sonication to the portion  156  of the fetal membranes. The ultrasound activation button  141  is then depressed to activate the ultrasound source  117 . The one or more substances  136  are delivered to the distal end  115  of the shaft  112  by depressing the push-button  168 , during or after the ultrasound sonication. Ultrasound sonication  154  emitted from the ultrasound source  117  is directed to the portion of the fetal membranes  156  adjacent to the cervix  52 . As demonstrated below, exposure of the fetal membranes  156  to the composition  124  the composition  124  with ultrasound sonication  154  increases the permeability of the fetal membranes. The permeability of the membranes  156  may be monitored during and after administration of the composition and sonication by measuring the electrical conductivity of the membranes (not shown). The substances  160  delivered to the distal end  115  of the shaft  112  are available to diffuse across the fetal membranes as a result of the increased permeability. After delivery of the substances  160 , the probe unit  106  is removed from the body. 
         [0067]    In one embodiment, the ultrasound sonication has a frequency of between about 20 kHz to about 3 MHz. In a preferred embodiment the ultrasound sonication has a frequency between about 20 kHz and about 500 kHz, and more preferably between about 20 kHz and 100 kHz. This range is referred to at times by the term “low frequency ultrasound sonication” (LFUS). In one embodiment, continuous ultrasound sonication for about 5 sec to about 30 min, more preferably, from about 30 sec to about 10 min, is used. 
         [0068]    In another embodiment, the pharmaceutical composition of the invention may be applied prior to ultrasound sonication or after ultrasound sonication. The pharmaceutical composition may be applied for about 1 min. to about 30 min.