CA1292946C - Method for inhibiting post-surgical adhesion formation by the topical administration of non- steroidal anti-inflammatory drug - Google Patents
Method for inhibiting post-surgical adhesion formation by the topical administration of non- steroidal anti-inflammatory drugInfo
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- CA1292946C CA1292946C CA000523793A CA523793A CA1292946C CA 1292946 C CA1292946 C CA 1292946C CA 000523793 A CA000523793 A CA 000523793A CA 523793 A CA523793 A CA 523793A CA 1292946 C CA1292946 C CA 1292946C
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- ibuprofen
- drug
- tolmetin
- composition
- vesicle
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Abstract
Abstract Postsurgical adhesion formation is inhibited by the topical administration to the site of surgical trauma of a non-steroidal anti-inflammatory drug, preferably ibuprofen, suprofen, or tolmetin. The drug may be contained in a controlled release vehicle such as an absorbable polymer microsphere or a phospholipid vesicle.
Description
lZ92946 METHOD FOR INHIBITING POST-SURGICAL ADHESION FORMATION aY
THE TOPICAL ADMINISTRATION OF NON-STEROIDAL
ANTI-INFLAMMATORY DRUG
The ~nvention relates to a me~hod for inhibiting post-surgical adhesion formation.
~ackaround of the Invention AdhQsion formation is a ma30r post-surgical complication wlth no ~ract~cal solution, The incidence o~ adhQsion rormation ~ollowing sur~ery a~proaches 100 per aent, ac¢ording ~o ~ome sources, with a clln~cally signif~cant ¢ompliaation rate oS about 5 to 10 per cent, de~end~ng on the type of surgery, Among such complications are bowel ob~truction, infsrtility, and pain. Oc¢as~onally, adhe6ions nQcQssitate a ~econd operative procedure to remove the adhesion, wh~ch may in turn further aggravate the problem.
Because o~ the serious~ess of the problem, much medical : 25 research has been performed in efforts to ~ind ways to combat adhesions. See, for instance, Stangel et al., ~Formation and Preventisn of Postoperative Abdominal Adhesions", the Journal of Reproductive M~dicine, Vol. 29, No. 3, March 19~4 (pages 143-156), and diZerega, "The Cause and Prevention of Postsurgical Adhesion~", published by Pregnancy ~e~earch Branch, National Institute of Child Health and Human ~evelopment, National In~titutes of Health, Building 18, Room 101, Bethesda, MD 20205.
Among the approaches that have been tried for preventing A~
~:z9Z~
post-surgical adhesion are the following:
Systemic administration of ibuprofen (e.g., see Singer, U.S. Patent No. 4,346,108);
Parenteral administration of antihistamines, corticosteroids, and antibiotics;
-Intraperitoneal administration of dextran solution and of polyvinylpyrrolidone solution; and Systemic administration of oxyphenbutazone, anon-steroidal anti-inflammatory drug that acts by inhibiting prostaglandin production.
Corticosteroids have been administered intraperitoneally as well as systemically in efforts to prevent adhesions.
(See the Stangel et al. article, cited above, on page 147, as well as the articles cited therein.) Some studies have questioned the efficacy of corticosteroids in adhesion prevention. In high doses, these materials may suppress the immune system and interfere with wound healing.
Therefore, the u~e of corticosteroids does not seem to be an acceptable solution to the post-operative adhesion problem.
on the basis of the results of animal studies and limited human clinical studies, the systemic administration of non-steroidal anti-inflammatory agents such as ibuprofen (usually in combination with other medicaments such as antibiotics) appears to be the most efficacious pharmacological means now known to reduce the incidence of post-surgical adhèsions. An objection to this means is that relatively large amounts of the drug must be administered over a period of several days, thereby ~Z9~946 subjecting the patient to the significant risk of experiencing adverse side effects. Also, this means has been shown to be effective only in a limited number of types of surgical procedures, e.g., gynecological surgery. As reported by Nishimura, Nakamura, and diZerega (Journal of Surgical Research 36, 115-124, February 1984), the minimum effective dose of systemically administered ibuprofen to inhibit postsurgical adhesion formation after abrasion or ischemia of the uterine horn of rabbits, is 70 mg/kg/day, administered once a day for at least 3 and preferably for 5 days post-operatively, with an additional dose 1 hour before surgery. In a similar series of experiments, Siegler et al. (Fertility and Sterility 4, No. 1, July 1980, pages 46-49) found an effective dose of systemically administered ibuprofen to be about 21 mg/kg/day, administered three times daily tin three 7 mg/kg doses) for two days post-operatively, with the initial in~ect~on being given 30 minutes before surgery.
The authors also reported that the best results were found in two rabbit~ that were each inadvertently given three extra 7 mg/kg doges. In order to deter adhesion formation in surgery to try to cure infertility, Corson et al. (The Journal of Reproductive Medicine, Vol. 29, No. 3, pages 143-156, March 1984) recommend a regimen including systemically administered ibuprofen, 400 mg per dose three to four doses per day, starting the night of surgery and continuing to the fifth postoperative day. A6suming that the average woman weighs about 48 kg 1110 pounds), this is a recommended do~age of 25 to 33 mg/kg/day. Singer, in U.S. Patent No. 4,346,108, recommends a dosage of from about 2.5 to 50 mg/kg/day in ~ingle or divided doses, for ibuprofen administered systemically to combat adhesions.
129Z9 ~6 Brief SummarY of the Invention The invention comprises a method for inhibitin~ the formation of post-surgical adhesions in mammals (including humans) which method comprises the topical administration of a non-steroidal anti-inflammatory drug ("NSAID") to the site of surgical trauma, over t~e critical wound healing period.
The Prior Art The Singer patent, and the journal articles by Nishimura et al., Siegler et al., and Corson et al., all cited above, disclose the 6ystemic administration of ibuprofen to combat adhesion formation, Lenk et al., in U. S, Patent No. 4,522,803, at column 17, lines 67 et seg,, disclose phospholipid vesicles containing anti-inflammatory agents. Only steroid anti-inflammatory agents are specifically disclosed. No anti-adhesion utility is disclosed.
Systemic administration of oxyphenbutazone to combat adhesions has been proposed. See, for example, Kapur et al., "Prevention of Reformation of Peritoneal Adhesions~, Arch. Surg., Vol. 105, Nov. 1972, Pages 761 - 764.
Detailed Description of the Invention The pharmacologically active compositions that are employed in this invention are the non-steroidal anti-inflammatory drugs such as ibuprofen, tolmetin, indomethacin, sulindac, suprofen, oxyphenbutazone, and ~329~6 s pharmaceutically acceptable salts or esters thereof.
NSAID's comprise a recognized class of compositions.
In accordance with the process of the invention, the active agent i8 applied topically to the site of surgical trauma in effective amounts for at least two and up to about seven days after the operation, to thereby inhibit the formation of post-surgical adhesions. By the term ~topically~, is meant that the NSAID is administered non-systemically to the surface of ehe tissue (internal or, in some cases, external) to be treated, for local effect. The term "site of surgical trauma~ is meant to include the site of tissue that has been injured in any way, and includes, for example, tissue sites that have undergone incision, excision, abrasion, contusion, laceration, anastomosis, manipulation, prosthetic surgery, curettaqe, orthopedic surgery, neurosurgery, cacdiovascular surgery, or plastic or recon~tructive surgery, "Site of surgical trauma~ also includes tissue that is ad3acent to the injured tissue.
The method of the invention is useful in any surgical procedure in which it is desired to inhibit the formation of post-surgical adhesions. It is thus broadly useful in all types of surgery in which adhesion formation can be a complication.
The NSAID may be administered to the site of surgical trauma by any convenient mode such as, for example, by lavage, by catheter, by coating directly on the site in a salve, ointment, gel, cream, aqueous surface active composition, emulsion, suspension, or foam, or by any other convenient mode. The site can be contacted directly, a~ by applying a salve, or i~ 60me ca6es the medicament can be introduced to a site near the site of trauma and natural migration of fluids will serve to carry the medicament to the desired site. Such natural migration of fluids can occur, for instance, intraperitoneally, in response to peristaltic contraction of the intestines.
The NSAID is ordinarily administered in a sterile - formulation in a pharmaceutically acceptable carrier or vehicle such as phosphate buffered saline ("PBS"), isotonic saline, purified water, an organic carrier such as a lipid, for example, a phospho}ipid micelle or vesicle, dextran, polymers (especially p-dioxanone, lactide, and/or glycolide based absorbable polymers), which may be in the form of microcapsules or which may be incorporated in a salve- or ointment-like formulation, or in an agueous solution of a surfactant such as a polyoxyethylene-polyoxypropylene block copolymer or a ~orbitan fatty acid ester-polyoxyethylene ether.
5terilization of the formulation may be accomplished in the usual ways, including aseptic preparation, filtration, exposure to gamma radiation, autoclaving, and the like.
In one preferred aspect of the invention, the NSAID is contained in a controlled release carrier that is capable of relea~ing the active drug for a period of at least one, preferably at least two, and up to about seYen days. The mode of delivery of the NSAID is preferably continually over the critical wound healing period, such as will be the case when the drug is applied to the site of surgical trauma in a single dose via a controlled releage carrier, or continually via a catheter.
A general procedure for preparing a polymeric microcapsule containing a drug, and which is applicable to incorporating NSAID' 8 in polymeric microcapsules, is the followi~.g:
129Z9'~6 1. The drug and the polymer are dissolved in a volatile organic solvent;
THE TOPICAL ADMINISTRATION OF NON-STEROIDAL
ANTI-INFLAMMATORY DRUG
The ~nvention relates to a me~hod for inhibiting post-surgical adhesion formation.
~ackaround of the Invention AdhQsion formation is a ma30r post-surgical complication wlth no ~ract~cal solution, The incidence o~ adhQsion rormation ~ollowing sur~ery a~proaches 100 per aent, ac¢ording ~o ~ome sources, with a clln~cally signif~cant ¢ompliaation rate oS about 5 to 10 per cent, de~end~ng on the type of surgery, Among such complications are bowel ob~truction, infsrtility, and pain. Oc¢as~onally, adhe6ions nQcQssitate a ~econd operative procedure to remove the adhesion, wh~ch may in turn further aggravate the problem.
Because o~ the serious~ess of the problem, much medical : 25 research has been performed in efforts to ~ind ways to combat adhesions. See, for instance, Stangel et al., ~Formation and Preventisn of Postoperative Abdominal Adhesions", the Journal of Reproductive M~dicine, Vol. 29, No. 3, March 19~4 (pages 143-156), and diZerega, "The Cause and Prevention of Postsurgical Adhesion~", published by Pregnancy ~e~earch Branch, National Institute of Child Health and Human ~evelopment, National In~titutes of Health, Building 18, Room 101, Bethesda, MD 20205.
Among the approaches that have been tried for preventing A~
~:z9Z~
post-surgical adhesion are the following:
Systemic administration of ibuprofen (e.g., see Singer, U.S. Patent No. 4,346,108);
Parenteral administration of antihistamines, corticosteroids, and antibiotics;
-Intraperitoneal administration of dextran solution and of polyvinylpyrrolidone solution; and Systemic administration of oxyphenbutazone, anon-steroidal anti-inflammatory drug that acts by inhibiting prostaglandin production.
Corticosteroids have been administered intraperitoneally as well as systemically in efforts to prevent adhesions.
(See the Stangel et al. article, cited above, on page 147, as well as the articles cited therein.) Some studies have questioned the efficacy of corticosteroids in adhesion prevention. In high doses, these materials may suppress the immune system and interfere with wound healing.
Therefore, the u~e of corticosteroids does not seem to be an acceptable solution to the post-operative adhesion problem.
on the basis of the results of animal studies and limited human clinical studies, the systemic administration of non-steroidal anti-inflammatory agents such as ibuprofen (usually in combination with other medicaments such as antibiotics) appears to be the most efficacious pharmacological means now known to reduce the incidence of post-surgical adhèsions. An objection to this means is that relatively large amounts of the drug must be administered over a period of several days, thereby ~Z9~946 subjecting the patient to the significant risk of experiencing adverse side effects. Also, this means has been shown to be effective only in a limited number of types of surgical procedures, e.g., gynecological surgery. As reported by Nishimura, Nakamura, and diZerega (Journal of Surgical Research 36, 115-124, February 1984), the minimum effective dose of systemically administered ibuprofen to inhibit postsurgical adhesion formation after abrasion or ischemia of the uterine horn of rabbits, is 70 mg/kg/day, administered once a day for at least 3 and preferably for 5 days post-operatively, with an additional dose 1 hour before surgery. In a similar series of experiments, Siegler et al. (Fertility and Sterility 4, No. 1, July 1980, pages 46-49) found an effective dose of systemically administered ibuprofen to be about 21 mg/kg/day, administered three times daily tin three 7 mg/kg doses) for two days post-operatively, with the initial in~ect~on being given 30 minutes before surgery.
The authors also reported that the best results were found in two rabbit~ that were each inadvertently given three extra 7 mg/kg doges. In order to deter adhesion formation in surgery to try to cure infertility, Corson et al. (The Journal of Reproductive Medicine, Vol. 29, No. 3, pages 143-156, March 1984) recommend a regimen including systemically administered ibuprofen, 400 mg per dose three to four doses per day, starting the night of surgery and continuing to the fifth postoperative day. A6suming that the average woman weighs about 48 kg 1110 pounds), this is a recommended do~age of 25 to 33 mg/kg/day. Singer, in U.S. Patent No. 4,346,108, recommends a dosage of from about 2.5 to 50 mg/kg/day in ~ingle or divided doses, for ibuprofen administered systemically to combat adhesions.
129Z9 ~6 Brief SummarY of the Invention The invention comprises a method for inhibitin~ the formation of post-surgical adhesions in mammals (including humans) which method comprises the topical administration of a non-steroidal anti-inflammatory drug ("NSAID") to the site of surgical trauma, over t~e critical wound healing period.
The Prior Art The Singer patent, and the journal articles by Nishimura et al., Siegler et al., and Corson et al., all cited above, disclose the 6ystemic administration of ibuprofen to combat adhesion formation, Lenk et al., in U. S, Patent No. 4,522,803, at column 17, lines 67 et seg,, disclose phospholipid vesicles containing anti-inflammatory agents. Only steroid anti-inflammatory agents are specifically disclosed. No anti-adhesion utility is disclosed.
Systemic administration of oxyphenbutazone to combat adhesions has been proposed. See, for example, Kapur et al., "Prevention of Reformation of Peritoneal Adhesions~, Arch. Surg., Vol. 105, Nov. 1972, Pages 761 - 764.
Detailed Description of the Invention The pharmacologically active compositions that are employed in this invention are the non-steroidal anti-inflammatory drugs such as ibuprofen, tolmetin, indomethacin, sulindac, suprofen, oxyphenbutazone, and ~329~6 s pharmaceutically acceptable salts or esters thereof.
NSAID's comprise a recognized class of compositions.
In accordance with the process of the invention, the active agent i8 applied topically to the site of surgical trauma in effective amounts for at least two and up to about seven days after the operation, to thereby inhibit the formation of post-surgical adhesions. By the term ~topically~, is meant that the NSAID is administered non-systemically to the surface of ehe tissue (internal or, in some cases, external) to be treated, for local effect. The term "site of surgical trauma~ is meant to include the site of tissue that has been injured in any way, and includes, for example, tissue sites that have undergone incision, excision, abrasion, contusion, laceration, anastomosis, manipulation, prosthetic surgery, curettaqe, orthopedic surgery, neurosurgery, cacdiovascular surgery, or plastic or recon~tructive surgery, "Site of surgical trauma~ also includes tissue that is ad3acent to the injured tissue.
The method of the invention is useful in any surgical procedure in which it is desired to inhibit the formation of post-surgical adhesions. It is thus broadly useful in all types of surgery in which adhesion formation can be a complication.
The NSAID may be administered to the site of surgical trauma by any convenient mode such as, for example, by lavage, by catheter, by coating directly on the site in a salve, ointment, gel, cream, aqueous surface active composition, emulsion, suspension, or foam, or by any other convenient mode. The site can be contacted directly, a~ by applying a salve, or i~ 60me ca6es the medicament can be introduced to a site near the site of trauma and natural migration of fluids will serve to carry the medicament to the desired site. Such natural migration of fluids can occur, for instance, intraperitoneally, in response to peristaltic contraction of the intestines.
The NSAID is ordinarily administered in a sterile - formulation in a pharmaceutically acceptable carrier or vehicle such as phosphate buffered saline ("PBS"), isotonic saline, purified water, an organic carrier such as a lipid, for example, a phospho}ipid micelle or vesicle, dextran, polymers (especially p-dioxanone, lactide, and/or glycolide based absorbable polymers), which may be in the form of microcapsules or which may be incorporated in a salve- or ointment-like formulation, or in an agueous solution of a surfactant such as a polyoxyethylene-polyoxypropylene block copolymer or a ~orbitan fatty acid ester-polyoxyethylene ether.
5terilization of the formulation may be accomplished in the usual ways, including aseptic preparation, filtration, exposure to gamma radiation, autoclaving, and the like.
In one preferred aspect of the invention, the NSAID is contained in a controlled release carrier that is capable of relea~ing the active drug for a period of at least one, preferably at least two, and up to about seYen days. The mode of delivery of the NSAID is preferably continually over the critical wound healing period, such as will be the case when the drug is applied to the site of surgical trauma in a single dose via a controlled releage carrier, or continually via a catheter.
A general procedure for preparing a polymeric microcapsule containing a drug, and which is applicable to incorporating NSAID' 8 in polymeric microcapsules, is the followi~.g:
129Z9'~6 1. The drug and the polymer are dissolved in a volatile organic solvent;
2. The solvent containing the drug and polymer is dispersed in water with a dispersing agent:
3. The organic solvent is evaporated from the dispersion product of step 2, either by mild heating or by vacuum evaporation, or by a combination of the two; and 4. The resulting microcapsules are recovered from the aqueous dispersion by customary procedures such as filtration, centrifugation, or the like, usually coupled with one or more washing steps.
This procedure is illustrated for this invention by Examples 1, 2, 9, and 10, below:
Exam~le 1 (a) To a small vial was added 9.39 grams of poly~lactide-co-glycolide-65:35) (a 65/35, by weight, copolymer of laceide and glycolide having an inherent viscosity in chloroform of 0.5 dlJg), 0.75 gram refined sesame seed oil, 1.140 grams ibuprofen, and 40 milliliters of dichloromethane. The solution obtained wa~ added to 400 milliliters of aqueous 5% wt/vol poly(vinyl alcohol) (Air Products Vinol Grade 523) in a one liter resin kettle equipped with a mechanical stirrer and vacuum take off, which was being cooled in an ice/water bath and stirred at 500 rpm. After allowing 10 minutes for emulsification, vacuum was slowly applied by means of an aspirator to an absolute pressure of 500 mm of mercury over 1.5 hours.
The vacuum was then maintained for an additional 19.5 hours (to remove the dichloromethane solvent), at which time vacuum and stireing wa~ stopped and the contents of lZ~29~6 the flask was poured into a one liter beaker and diluted to 800 milliters total volume with water. (An alternative method for removing the dichloromethane solvent is to heat at 30C. for two hours at atmospheric pressure.) The contents of the beaker were added to centrifuge tubes and they were centrifuged at about 1000 rpm for two minutes.
The liquid was deranted, fresh water was added, and centrifugation was repeated. This procedure was repeated one more time, and after the third wash, the last trace~
of water were removed by freeze drying. The dried sample was a free flowing white powder which weighed 2.03 grams.
The powder was examined at lOOX and was found to contain microcapsules which ranged in size from about l to 10 microns. No free drug crystals were noted. Subsequent NMR
analysis indicated that 8.6 wt. % ibuprofen was present.
(b) To make control microcapsules, the foregoing procedure i8 repeated without adding the ibuprofen.
~c) Microcapsules produced as described above, both with and without ibuprofen, are disperged in an aqueous dispersion of lecithin in proportions of 1.5 grams of microcapsules per 50 milliliters of 0.05 weight per cent aqueous lecithin. After freeze drying, 1.4 grams of microcapsules are obtained which contain about 1.4 weight per cent of lecithin.
- ExamPle 2 To a small vial was added 1.50 grams of poly(lactide-co-glycolide - 65:35), 0.50 gram ibuprofen, and 5 milliliters of dichloromethane. The resulting solution was added to 50 milliliters of 3~ aqueous poly(vinyl alcohol) solution which was being cooled in an ice/water bath and stirred at 500 rpm. Vacuum was applied as before and after washing and freeze-drying, l.lO0 grams 129Z94~
of microcapsules was obtained. The microcapsules ranged in size from 10 to 120 microns and were found to contain 17.1% by weight ibuprofen by NMR.
ExamPle 3 Ipuprofen/poly(lactide-co-glycolide-65:35) microcapsules (Example 2) which were found to contain 17.1~ by weight ibuprofen (NMR) were investigated. In five separate 4 ounce amber jars was placed 100 milligrams of microcapsules and 100 milliliters of pH 7.27 phosphate buffer. The caps were tightly closed and the jars incubated at 37 C., with no agitation. After 15 minutes, 1 day, 2 days, 7 days, and 14 days, a jar was removed, the microcapsules collected by filtration, washed well with water, dried under vacuum, and analyzed for ibuprofen content by NMR~ The results ~how~ in the attached table ~ndicate that approximately 50% of the drug wa~ relea6ed in the fir~t 7 days and the remainder was Z0 completely released by day 14. (Negligible polymer weight lo~s occurred over the 14 day period.) Table I
In-Vitro ~elease of Ibuprofen From Poly(lactide-co-glycolide) Microcapsules Sample Time WT % IBF REMAINING
(~ays) ~ of TOT CAPSULE WT) __________________________________________________________ 1 0 17.0 2 1 14.3 3 2 13.6 4 7 10.0 14 0.0 i2~2946 Methods for incorporating drugs in lipid caLriers are known in the art. For instance, one procedure for encapsulating a drug in a phospholipid vesicle is the following:
a lipid or mixture of lipids such as lecithin or other phospholipid, which may be mixed with cholesterol or other lipoid substance, is dissolved in an organic solvent such as diethyl ether; and an aqueous phase containing the material to be encapsulated (in this case, an NSAID) is added to the lipid solution, and the mixture is agitated as by exposing it to ultrasonic sound waves (sonicated). Preferably, the organic solvent is removed during sonication, as by u6e of heat or vacuum or both, although in some caseg the solvent can be removed after the sonication.
This procedure typically produces a unilamellar vesicle.
Another procedure for producing a phospholipid vesicle (in this case a multilamellar vesicle "MLV") containing a medicament is to form a film of dry lipid, as by evaporating the solvent from an organic solvent 601ution containing a lipid to form a film on the walls of the vessel containing the solution, and then stirring in the agueous phase containing the NSAID to be encapsulated.
(The evaporation can be done by spray drying or by vacuum evaporation, or by any other convenient method.) Free unencapsulated NSAID can be separated from MLV~s by centrifugation at, e. g., 12,000 rpm.
Preferably, the vesicle containing the NSAID is dehydrated, as by freeze drying, after preparation, in order to insure long term storage stability. The agueous vesicle suspension can be reconstituted just prior to use by adding sterile phosphate buffered saline, sterile water, or the like.
1~2929~6 The use of multilamellar vesicles of comparatively large size (that is, greater than 1 micron, e. g., from about 1 to about 10 microns) appears to be preferable in order to increase the dwell time of the vesicle containing the NSAID in the peritoneal cavity (or other body cavity). It i8 also preferred to~use a pure or synthetic phosphatidylcholine in which the fatty acid moieties in - the phosphatidylcholine molecule are derived from a single fatty acid, in preparing the vesicle instead of natural lecithin, which is ordinarily a mixture of compounds, Example 13, below, illustrates the preparation of a multilamellar vesicle containing an NSAID.
The following United States patents describe the preparation, by various procedur~s, of phospholipid vesicles containing various medicaments:
Lenk et al. No. 4,522,803 ~alde~chwieler et at. No. 4,310,505 Mezei et al. No. 4,485,054 Gersonde et al. No. 4,452,747 Kelly No. 4,356,167 Papahadjopoulo~ et al. No. 4,241,046 Suzuki et al. No. 4,016,100 Sache et al. No. 4,239,754 MacDonald No. 4,532,089 Rahman et al. No. 3,993,754 See also Callahan et al., European Patent Application No.
0126580, published November Z8, 1984, and Gregoriadis, "The Carrier Potential of Lipo~omes In Biology and Medicine", New England Journal of Medicine, V~l. 295, pp. 704-710 and pp.
765~770 (Sept. 23 and 30, lg76).
.
The foregoing are incorporated herein by reference a~ general proeedure~ which can be utilized for the incorporation of ~AID's ln lipo~omes.
S Oth-~ procodu~s ~or aontaining drug~ in ~ho~p~olipid0 ~micelles or liposomes) ~ and which are applicable to NSAID~s, are described in Sears, U.S. Patent Nos. 4,426,330 and 4;1~5,410, and ~ears et al., U.S. P~tent No. 4,298,594 .
It is not essential that the NSAID medicament u6ed in the invention be encapsulated in an inside aompartment or compartments of the carrier as will normally be the case when the carrier i~ a phospholipid vesicle. In some ca6es it i8 a¢co~table for tho NSAID to be d~ssolYed or otherwise di~tributed more or le~s evenly ~hroughout the carrler.
Thé non-steroidal anti-inflammatory drug i8 delivered to the ~ite of ~urgiaal trauma in effe¢tive quantitie~ over the ¢ritical wound healing period ~whi¢h period varies from patlent to patient and with the type of ~urgical trauma encountered, but i8 u~ually from about two to five days, and in some case~
up to seven days or more, post-operatively.) The example6 below illustrate the order of magnitude of effective quantities : 25 of the drug and the period of time over which the drug is administered for effective results.
The following ~tudies u~e rabbit models to illustratQ the adhesion inhibition effectiveness of the topical admini~tration of a non-steroidal anti-inflammatory drug to the site of ~urgical trauma:
, 12~Z946 ExamPle 9 New Zealand white female rabbits (1.8-2.0 kg) underwent midline laparotomy using acelepromazine and ketamine anaesthesia. A
3x5 centimeter abrasion was produced over the right-lateral peritoneal side-wall by ~craping the surface peritoneum with a scalpel until punctate bleeding developed over the entire 3x5 centimeter area. A second abrasion covering the same total area (15 cm2) using the same technique was developed 1.5-2.0 centimeters inferior to the initial site along the right-lateral peritoneal side-wall. This second site was used as an untreated control. The serosal surface of the large bowel adjacent to the peritoneal abrasion sites was also similarly abraded.
Ibuprofen contained in poly~lactide-co-glycolide-65:35) microcapsule~ produced as described in Example 1, were ~uspended in 15 we~ght per cent aqueous poly(vinyl pyrrolidone) "PVP" (GAF Povidone C-15). The proportions were 15 grams of microcapsules per 100 grams of the PVP solution.
The lecithin-containing microcapsules were used in half of the experiments. The suspension was dripped on the wound in an amount such that about 40 milligrams of ibuprofen (about 470 milligrams of microcapsules) was applied to the wound site. One control was the PVP solution containing ibuprofen-free microcapsules, and the other was the PVP
solution containing ibuprofen-free microcapsules with lecithin.
Seven days after the day of abrasion, the rabbitg were sacrificed by pentobarbital overdose. The extent of adhesions was evaluated as follows:
1~32~46 1. No adhesions 2. Filmy adhesions (separable) 3. Mild adhesions (not separable - covering up to about 35%
of the test area) 4. Moderate adhegions (not separable - covering about 35 to 60 % of the test area) 5. Severe adhesions (not separable - covering greater than about 60% of the test area) The evaluation ratings set forth above are useful in the context of comparing the efficacy of various means for inhibiting the formation of adhesions. However, it should be mentioned that ultimately only a rating of ~'1" can be considered to be completely acceptable. Clinical complications can result from even mild adhesions, although such complications are considered to be more likely to occur with severe adhe~ions than with mild or moderate adhegions.
The following Table II presentg the results:
Table II
Evaluation Ibuprofen Untreated Rabbit No. IbuProfen Lecithin Treatment Controls 1 yes no 4 4 2 yes no 4 5 3 yes no 1 1 *
4 yes yes 3 5 yes yes 4 5 6 yes yes 3 5 7 no no 5 5 8 no no 5 5 9 no no 5 5 no yes 5 5 11 no yes 5 5 12 no yes 5 s ____________________________________________________________ * ~he surgical procedure followed in this experiment induces adhesion formation in about 87 per cent of untreated rabbits.
Evidently this rabbit was one of the approximately 13 per cent that do not develop adhesions.
________________________________ The results indicate some anti-adhesion activity exhibited by the microcapsules containing ibuprofen.
It is relevant to note that when the vehicle control site is in the same rabbit as the test site, migration of fluid in the peritoneal cavity can carry some of the medicament from the NSAID treatment site to the vehicle control site. Therefore, it i8 possible that the untreated (by "untreated" is meant no actiYe medicament) control ~ites of rabbits Nos. 1-6 in Table II and analogous untreated control sites reported below could 2~ have received small amounts of NSAID owing to migration or circulation of fluid within the peritoneal cavity. However, if any of the untreated control sites did receive some o the active medicament by such fluid migration, it would have been significantly less than that received at the treatment site in the same rabbit. Therefore, differences in results between the treatment sites and the control sites in the same rabbit can - confidently be interpreted as being caused by the adhesion inhibition effect of the NSAID.
ExamDle 5 In order to carry out dose response studies and time response studies, miniature osmotic pump8 (Alzet mini pumps, model 2MlL
or model 2002 - these pumps are described in Higuchi et al., l~Z9~6 .
U.S. Patent No. 3,995,631) were used to de,liver the antl-adhe~lon agent ~n a continuou~ stream at a very low, controlled Slow rate, to the s~te o~ the surgical trauma in the te~t rabbits over a period of time, up to ~even days. The mini-pumps therefore deliver the medicament in a manner analogou~ to a catheter delivery modQ.
New Zealand white ~emale rabbits (1.8 to 2.0 kg) underwent midline laparotomy u6ing acelepromazine and ketamine anaesthesia. A 3 x 5 cm flap of parietal peeitoneum (about 1 mm thic~) was sharply dissected from the right lateral peritoneal side-wall, The serosal surf'ace of the ad~acent large bowe? was abraided with a scalpel until punctate bleeding developed. This area between the excised parietal peritoneum and ad~acent large b~wel serosa was then used for evaluating th~ e~fica¢y o~ the te~t medicament ~or adhe~ion inhib~tion. A
~econd oxais~on o~ parie~al peritoneum co~er~ng the ~ame total area ~about 15 ¢m ) wa~ ~qr~ormed 1.5 to 2.0 cm inferior to, the initial to~t site along the right lateral peritoneal s~de-wall. Abrasion of the ad3acent larqe bowel serosa was p-r~orm-~ a~ de~aribe~ above'for the tr--tm-nt 8ite . Thls ~-cond area wa~ u~ed to dotermine the e~fectiveness ot the 6uryical procedure in producing adhe6ions and the response to ~ vehicle controls.
: 25 Alzet mini pumps containing ibuprofen dissolved in pho&phate bufered saline ("PBS") were sewn into the right dorsal subcutaneous space of the test rabbi,t with VICRYL (Polyglactin 910) sutures placed 3 to 5 millimeters from each end of the, pump. The polyethylene catheter tip leadihg from the pump into the pe~itoneal cavity of ,each rabbit was placed 2 to 3 ,millimeter8 over the in3ury test 8ite. The catheter was securëd in place by two 3/0 VICRYL 6uture8 which did not , , involve the site of the injury. A similar ~ump and catheter , , *Trademark, ET~-694 , , ' A
, lZ9Z9~6 6ystem containing PBS vehicle only was implanted in the middle portion of the inferior (vehicle control) abrasion site.
Two different mini pumps were used in these experiments. The first (a 2 ml pump~ had a pumping rate of 10 microliters per hour and the second (a 0.2 ml pump) had a pumping rate of 0.5 microliter per hour. Each pump contained phosphate buffered saline (pH 7.2, unless otherwise indicated), 2 milliliters in the larger pump and 0.2 milliliter in the smaller pump. The control pumps contained phosphate buffered saline alone, and the treatment pumps contained either 20 milligrams of ibuprofen (2 ml pump) or 2 milligrams of ibuprofen (0.2 ml pump).
The results seven days after the operation were as shown below in Table III. The evaluation procedure was the same as the one de~cribed in Example 4.
Table III
Evaluation Ibuprofen Vehicle Rabbit No. PumP Size Treatment Control 1 0.2 ml 5 5 2 0.2 ml 4 5 3 0.2 ml 4 5 4 2 ml 4 5 2 ml 2 5 6 2 ml 1 5 The smaller of the two pumps gave a slight positive response in two of three rabbits, whereas the larger pump gave significant positive responses in two out of three rabbits and a 61ight positive re6ponse in the other.
ETH-69~
lZ~Z946 ExamPle 6 In order to try to better define the threshold dosage rate for ibuprofen ("IBF") in this experimental model, similar experiments were carried out with the two sizes of pumps, using different concentrations of IBF per pump. Table IV, below, sets forth the concentrations of IBF per pump and responses in this serie~ of experiments:
Table IV
Evaluation Concentration of Vehicle Rabbit No. pumP Size IBF, mo~ml Treatment Control 1 0.2 ml 10 1 4 2 0,Z ml 10 4 5 3 0.2 ml 10 5 5 4 2 ml 10 3 5 2 ml 10 4 4 6 2 ml 10 3 5 7 0.2 ml 3 4 5 8 0.2 ml 3 5 5 9 0.2 ml 3 4 5 2 ml 3 3 5 11 2 ml 3 5 5 12 2 ml 3 4 5 13 0.2 ml 1 5 5 14 0.2 ml 1 5 5 0.2 ml 1 1 4 16 2 ml 1 5 5 17 2 ml 1 5 5 18 2 ml 1 4 5 19 0.2 ml 0.3 5 5 0.2 ml 0.3 5 5 21 0.2 ml 0.3 5 5 lZ~Z9~16 22 2 ml 0.3 5 5 23 2 ml 0.3 5 5 24 2 ml 0.3 4 5 This series of experiments indicates that, in this model, the threshold dosage at which significant anti-adhesion effects began to be noted occurred when pumps containing concentrations between 1 and 3 milligrams of IBF per milliliter were used. No significant difference was noted here between the two different sized pumps.
An effective dose of a topically applied drug is normally expressed in terms of concentration of the drug in the carrier.
coupled with the number of times per day the drug is applied.
In the present invention, the effective dose will be dependent upon factors such as nature of specific NSAID used, nature of vehicle, nature of tissue to be treated, type of trauma, and mode of delivery (i,e~, continuous delivery by catheter or a one-time application in a vehicle such a6 a controlled release vehicle). Therefore, no hard and fast rule can be formulated that will apply in all cases, and experiments analogous to those reported in this Example 6 will have to be performed in order to precisely define the threshold dosage for each different NSAID, for specific vehicle system6, and for specific modes of delivery. It is well within the ability of the,person skiIled in the art to carry out the necessary experiments to determine threshold dosages, after having read this disclosure.
Exam~le 7 In order to determine the time period over which the anti-adhesion agent must be administered in order to have a significant anti-adhesion effect, the following series :: :
12~Z946 of experiments were carried out (using the experimental procedure described above in Example 5):
The 2 ml pump was used containing 10 mg/ml of IBF, and the catheter delivering the treatment solution to the site of the surgical trauma was disconnected 1, 2, 3, 4, and 5 days post-operatively. The rabbits were sacrificed 7 days post-operatively, and evaluated a~ above. The results are displayed in Table V, below.
Table V
Post-Op Day Evaluation RabbitCatheter Ibuprofen Vehicle No.Disconnected Treatment Control With ibuprofen, in this model, it appears that administration of the NSAID via the 2 ml mini pump in PBS
for at least three days i~ required in order for the drug to have significant anti-adhe6ion effects, With other 12~Z~346 -2~-NSAID~s and/or other delivery modes, and with other types of surgical trauma, it is reasonable to expect that the minimum period of time will differ. In any given case, the minimum period of time over which the NSAID must be administered can be determined by experiments analogous to that described in this Example 7. As a general rule, in most cases, a minimum of one day is recommended, and in some cases, longer periods (e. g., up to 5 to 7 days or longer) may be required, The NSAID active ingredient is administered to the site of surgical trauma topically. Such topical administration can be by spraying, lavage, dripping on the site, by catheter administration, or the like The exact method of administration chosen is not critical, as long as an ef~ective do~e is administered over the critical wound healing period, which can be determ~ned by a series of experiments analogous to that deficribed above in Example 7. The NSAID should be administered immediately post-operatively, that i8, before wound healing has begun ExamDle 8 By a procedure analogous to that described above in Example 5, the 2 ml Alzet mini pump was used to deliver suprofen to t~e test site in the rabbits, The concentration of suprofen was 2.5 mg/ml of phosphate buffered saline (pH = 7.4). The rabbits were sacrificed 7 days post-operatively, and evaluated as above. The results are set forth in Table VI, below:
lZ~Z946 Table VI
2.5 m~/ml Su~rofen Evaluation Suprofen Vehicle Rabbit No. Treatment Control ExamPle 9 To a small vial was added 2,78 grams of poly(lactide-co-glycolide - 65:35), 0,30 gram suprofen, and 8 milliliter~ of dichloromethane. The resulting 601ution was added to 120 milliliters of 3~ aqueous poly(vinyl alcohol) solution which was heated to 30 C.
while being mechanically stirred at 500 rpm. After two hours, the microcapsules were isolated as described in Example 1, and after washing and freeze-dryinq, 1.61 grams of microcapsules were obtained. The microcapsules ranged in size from >10 to 250 microns and were found to contain 7.0% by weight suprofen by NMK.
ExamPle 10 To a small vial was addéd 1.25 grams of poly~D,L-Lactic acid), 0,12 grams refined ~esàme seed oil, 0,152 gram ibuprofen, and 3 milliliters of dichloromethane, The resulting solution was added to 30 milliliters of 3%
~Z~Z946 a~ueous poly(vinyl alcohol) solution whic~ was being cooled in an ice/water bath and stirred at 5~0 rpm.
Vacuum was applied as described in Example 1, and after washing and freeze-drying, 1.12 grams of microcapsules were obtained. The microcapsules ranged in size from 10 to 75 microns and were found to contain 8.7~ by weight ibuprofen by NMR.
ExamPle 11 By a procedure similar to that described above in Example 5, the 2 ml mini pump was used to deliver varying quantities of suprofen sodium (i. e., the sodium salt of suprofen) to the rabbit model. The quantities of medicament contained in the phosphate buffered saline (pH
= 6.9) varied from 3,0 mg/ml down to 0.03 mg/ml, The rabbit~ were sacrificed seven days post-operatively and evaluated as above, The results are set forth below in Table VII:
Table VII
Suprofen, Dosage ResDonse Studies ConcentrationEvaluation of Suprofen,Vehicle Rabbit No. ma~mlTreatment Control 12~Z946 ~ 1 1 5 *
11 0.3 1 5 12 0.3 3 4 13 0.3 3 5 14 0.3 3 3 0.3 3 4 16 0.1 3 3 17 0.1 3 9 18 0.1 3 5 19 0.1 1 5 0.1 5 5 21 0.03 5 Z2 0.03 23 0.03 4 4 24 0.03 1 4 0.03 4 5 __________ _____________________________ ________________ * This rabbit exhibited slight bleeding. (In many cases, in this model, when bleeding occurs no adhesions develop.) __________________ _________ ____________________________ This series of experiments indicates that the threshold dosage at which significant anti-adhesion effects begin to be noted in this model with suprofen sodium as the NSATD
occurs when pumps containing a concentration of about 0.1 mg~ml of drug are used. Thus, on a weight basis, suprofen appears to be slightly more active than ibuprofen.
: 35 129Z9 ~6 ExamPle 12 By a procedure analogous to that described above in Example 5, the 2 milliliter mini pump was used to deliver varying amounts of tolmetin. (The sodium dihydrate salt of tolmetin was used.) The concentrations of drug contained in the phosphate buffered saline (pH = 7.4) varied from 3.0 mg/ml down to 0.01 mg/ml. The treatment pumps contained the concentrations of tolmetin displayed below in Table VIII, and the control pumps contained vehicle only. The rabbits were sacrificed 7 days post-operatively, with results being di6played below in Table VIII:
Table VIII
Tolmetin, ~o~age ResDonse Studie~
Concentration Evaluation of Tolmetin, Vehicle Rabbit No. ma/ml_ Treatment Control 3 3 l 5 6 l 1 4 9 1 l 3 1 Thi8 rabbit died **
lZ9Z946 11 0.3 3 3 12 0.3 1 3 *
13 0.3 1 3 14 0.3 3 5 0.3 3 4 16 0.1 3 5 17 0.1 1 5 18 0.1 1 5 19 0.1 1 5 200.1 This rabbit died (snuffles)***
21 0.03 22 0.03 1 5 23 0.03 3 s 24 0.03 1 5 0.03 1 4 26 0.01 5 5 27 0.01 1 5 28 0.01 1 5 29 0.01 1 4 0.01 25--________________________________ * Slight bleeding occurred.
** The cause of death was unknown, but there was no suggestion that it was drug-related.
*** "Snuffles" is a viru~-cau~ed upper re~piratory disease that affects rabbits. There i~ no sugge~tion that it was drug-related.
~: _____________________________ As can be seen from the data presented in Table VIII, tolmetin appears to be more effective. on a weight basis.
lZ~2`~3~6 than either ibuprofen or suprofen in this rabbit model experiment. The thre6hold dosage, a~ indlcated by the re~ult~ trom thia experiment, wa~ appar~ntly 1Q~8 than the dosage admln~stered to Rabblt Nos. 26 - 30, wherein the S concentration of drug wa~ 0.01 mg/ml.
ExamPle 13 ~he following is a typical preparstion of a lipo~ome (MLV) containing an NSAID:
(In this preparation, all materiais and aquipment used are sterile and pyrogen-free) a) P~e~arati~n o~ l~P~d ~lm L-alpha-di6tearoyl phosphatidylcholine ("DSPC"), 1.21 gm., and 0.29 gm. cholesterol ~molar ratio of DSPC to cholestero,l is 2:1), are dis~olved in 45 ml. of chloroform. ~he re~ulting solution i8 div~ded into nine 5 ml portions, and each ~uch portion is placed in a lOO ml ~la~k. The 601vent is evaporated from each fla~k using a rotary vacuum evaporator. Sterility i8 maintained by attaching a 0.22 micron Millex filter to the air intake of : 25 the evaporator prior to flask removal. Starile septa are placed on the flasks after solvent evaporation. The surface of each septum is ~iped with 70% alcohol, and a 19 gauge sterile needle affixed to a 0.,22 micron filter is pa~sed through each septum. All flasks are then placed in a large vacuum desiccator and kept there ove~night. ~ach flask contains about 167 mg. of lipid.
*Trademark .:
.
ATy-694 ', .
12~Z~46 b) Pre~aration of NSAID solution The sodium salt of ibuprofen (Na-IBF), 0.202 gm., is dissolved in 40 ml of sterile, pyrogen-free water. The solution is then passed through a 0.22 micron Millex filter.
c) Pre~aration of MLV containina Na-IBF
3.9 Ml of the Na-IBP solution is injected into each flask containing lipid film. The flasks are vortex-stirred for 40 to 60 minutes in a 65 C. water bath under nitrogen.
(The nitrogen purge is first passed through a 0.22 micron filter.) To the contents of each flask are added sterile, pyrogen-free phosphate buffered saline, and the flask6 are centrifuged for 6 to 10 minutes at 15,000 rpm. This wa~hing procedure i~ repeated for a total of five times to remove unencapsulated ~SAID. The contents of the flasks are then combined and PBS (5mM P04 in 0.15 NaCl) is added to a total of 3Z ml. The liposome suspension thus produced comprises MLV's of about 1 micron in size ~uspended in the PBS. It is storage stable for a period of several months, but for long term storage is preferably dehydrated and remixed with sterile, pyrogen-free water just before use.
To prepare drug-free controls, the procedure is repeated substituting pure water for the water/Na-IB~ ~olution.
The procedure is repeated using ta) the free-acid form of IBF, and (b) the sodium salt of tolmetin. Similar result~
are obtained and liposome MLV's containing IB~ or sodium tolmetin are produced.
MLV's containing IBP or tolmetin, and produced as lZ9Z9 ~6 described herein, were found to have particle sizes of about 1-1/2 to 2 microns (by optical microscope) and about 5 to 8 microns (by Coulter counter).
Analogous procedures would be employed to produce vesicles from phosphatidylcholines in which the fatty acid moieeies were derived from other fatty acids, e.g., C12 to C24 fatty acids. C14 to ~20 saturated fatty acids are preferred.
ExamDle 14 This experiment evaluated the eficacy of a liposome/ibuprofen combination to combat post-surgical adhesions. The procedure used was similar to that described above in Example 4, except that the untreated control sites in those rabbits that were given the liposome/ibuprofen treatment were on the left-lateral peritoneal side-wall instead of being sited 1.5 to 2.0 centimeters inferior to the treatment sites in the right-lateral peritoneal side wall, and the wounding procedure described in Example 5 was used.
The liposomes used in this example were DSPC/cholesterol MLV~s (L-alpha-distearoyl phosphatidylcholine/cholesterol multilamellar vesicles), prepared in a manner analogous to that described above in Example 13. The treatment mixture consisted of 31 milliliters containing 1200 milligrams of MLV and 35 milligrams of the free acid form o ibuprofen, suspended in 5mM phosphate buffered saline. The vehicle control had the same composition, except that the ibuprofen was omitted. Each rabbit received 10 ml of suspension, which amounted to 3.5 mg o ibuprofen per rabbit. The treatment and vehicle control suspensiong were dripped on the traumatized ~ites, as described above lZS'29 ~6 in Example 4. Three rabbits received the~
liposome/ibuprofen treatment on the right side-wall site with the other site being untreated, and three rabbits received a vehicle control treatment (i. e., liposome without ibuprofen) on the right side-wall site with no treatment on the other site. The rabbits were sacrificed 7 days post-operatively, and evaluated as described above. The results are displayed below in Table IX:
Table IX
LiDosomeJIbuProfen Studies Rabbit Site No.IbuProfen Treated Untreated 1 yes 2 yes 3 yes 1 4 4 no 2 4 no 5 5 6 no 1 5 The experiment was repeated in exactly the same fashion, ;~ with the results as displayed below in Table X:
125~Z946 Table X
Rabbit Site No.IbuProfen Treated Untreated 1 yes 2 yes 3 yes 1 4 4 no 5 4 no 3 4 6 no 1 1*
_ ___________________________ ~ This rabbit exhibited ~light bleéding.
________________________________________________________ In the experiments reported above in Tables IX and X, the rabbits receiving treatment by the ibuprofen/liposome combination were virtually free of adhesions, even on the sides that received no direct application of medicament.
This is considered to be indicative of the fact that the medicament can migrate in the peritoneal cavity as a result of circulation of peritoneal fluid. Also, by gross observation, no tissue reaction or granulomas were found at the treated sites.
Exam~le l5 In this series of experiments, the procedure of ~xample 14 was repeated, with the following modifications:
The liposomes that were used were DSPC~cholesterol MLV~s prepared as described above in Example 13, and which lZ9Z9~6 contained Na-IBF. The control MLV~s (two batches -"MLV-l" and "MLV-2") contained no drug. The drug-containing lipo60mes were mixed with the control liposomes in varying proportions to obtain MLV's that contained varying amounts of NSAID. In Table XI, below, there is di6played the compositions of the drug-containing and tbe control liposomes:
-Table XI
LiDosome ComPosition Vol, Lipid, Total Lipid, Drug, Drug/Lipid, ml mq/ml mq ma/ml (bY weiqht~
Control MLV-l 50 33 1675 N/A N/A
MLV 50 33 1675 0.98 0.029 (49 gm) In Table XII, below, there is displayed the proportions of the drug-containing and control liposomes that were mixed together and then administered to the test rabbits in the manner described above in Example 14, along witb the results of the evaluations of the rabbits seven days post operatively:
lZ92~46 Table XII
Na-lBF/MLV Evaluations Rabbit IBF Control Evaluation No. MLV, MLV, Treatment Control ml ml Site Site 1~ 0.3 9.7 1 3 13 0.3 9.7 1 3 lZ9Z946 Exam~le 16 By a pro¢edure analogous to that described above in Example 14, aqueous ¢ompositions including a surface at1vo ~g-nt and t~e f roo aoid ~o~m o~ tolmetln we~e applied to the treatment sitQs in the rabbit model. The a~ueous compositions comprised various concenteations of "Tween 80", an ethoxylated sorbitan mono-oleate, in triple distilled water, and tolmetin at a concentration of either 10 1 or 2 mg~ml. The 601utions were sterilized by pa~sing them through a 0.22 micron filter. The tables below display the concentration of tolmetin and Tween 80, and the evaluation~ of the adhesions seven days post-operatively. In each case, 10 ml of the solution was tr1~pod on the treatmen~ s~to.
Table XIII
5 Wt, ~ Tween 80 *
~ ma/ml Tol~,e~
a~gi_~Q~ Evaluation Treatment Contr~l Site _Site 3 1 1 ~Bleeding) 4 1 , 5 (Control*) 3 3 9 ' : 10 (Control*) ' 4 3 ;*Trademark ETH-694 , Al , l~Z9'~6 * The control rabbits received no treatment.
___________________________________ Table XIV
20 Wt. % Tween 80 2 ma/ml Tolmetin Rabbit No. Evaluation Treatment Control Site Site 5 (Control*) 4 4 10 (Control*) 5 5 ____________________________________________________________ : ~ No Treatment ______________ lZ9Z9~6 Table XV
20 Wt. ~ Tween 80 2 mqJml Tolmetin Rabbit No.Evaluation Treatment Control Site Site 2 1 1 (Bleeding) Table XVI
1 ma/ml Tolmetin Sodium (1 No Tween Rabbit No~valuation Treatment Control Site Site 9 (~Control(2)) 4 3 lZ9Z946 ____________________________________________________________ (1) The sodium salt was used because the free acid form is guite insoluble in pure water.
(2) Water only; no Tolmetin ____________________________________________________________ The free acid form of tolmetin, which i6 guite insoluble in water, was maintained in agueou~ solution or colloidal lo suspension by the surface active agent.
Referring again to the question of the effective dose of NSAID in accordance with this invention, while no hard and fast numbers can be presented that will be applicable to all cases, the examples presented above can be referred to as a guide to determine the order of magnitude of drug to employ in certain cases. In pho~hate buffered saline admini~tered continually via the Alzet osmotic mini pump, the following dosages were found to be effective:
ibuprofen, 3 mg~ml, at a rate of 0.5 microliter/hr. ~from EX.6):
suprofen, 0.1 mg/ml, at a rate of 10 microliters~hr. (from Ex. 11); and tolmetin, 0.01 mg/ml, at a rate of 10 microliters~hr. (from Ex. 12~.
Expres6ed in terms o mg~kq/day, and in term~ of mg~day/cm2 of traumatized tissue, these numbers are the ollowing:
1~9Z9~6 ibuprofen - 18 x 10 3 mg/~g/day 2.4 x 1- 3 mg/day/cm2 suprofen - 12 x 10 3 mg~kg/day 1.6 x 10 3 mg/day/cm2 tolmetin - 1.2 x 10 3 mg/kg/day 0.16 x 10-3 mg/day/cm2 These calculation~ are carried out as follow6 (using the ibuprofen numbers as illustrative): 0.5 microliter/hr. of golution containing 3 mg/ml equals 0.5 x 10 6 liter/hr.
x (3 mg/10 3 liter). This reduces to 0.5 x 3 x 10 6/10 3 3 1.5 x 10 3 mg/hr.
~his equals 36 x 10 3 mg/day or 18 x 10 3 mg/kg/day (each rabbit weighs about 2 kg).
The area of traumatized tissue i8 about 15 cm2, 80 the ~0 dose expressed in terms of ~g/day/cm2 = 36/15 x 10 3 mg/day/cm2, or 2.4 x 10 3 mg/day/cm2.
Compared with the minimum recommended dose of 2.5 mgikg/day (from the Binger patent cited above) for Z5 6ystemically ad~inistered ibuprofen, the effective dose for topically administered ibuprofen may be two to three orders of magnitude lower. Obviously, this greatly reduced do6age significantly reduces the chances for ; undesired side effects.
The minimu~ effective dose using an MLV or an a~ueous composition containing a surface active agent as a carrier has apparently not been approached. Howevèr, it is noted from Example 15, Rabbit Nos. 12-13, that a total dose of about 0.3 mg of ibuprofen in an MLV carrier ~as lZ9Z946 effective. This number was calculated as follows:
0.98 mg/ml x 0.3 ml/0.7 ml, eguals 0.0278 mg/ml. 10 ml of this preparation was admini~tered, 80 the total dosage was 0.3 mg or 0.15 mg/~g or 0.02 mg/cm2. It would appear that an even lower concentration would be effective in this ~odel.
In the Tween 80 solutions, the smallest amount of tolmetin applied was 10 mg (from the data presented in Table XIII). It would appear that an even smaller dosage administered in this manner would be effective.
A reasonable extrapolation of the data presented herein ifi that a minimum effective concentration for an NSAID
preparation applied in a ~ingle dose would be between about 0.025 mg and 5 mg of N5AID per ml of total vehicle.
(By "total vehicle" i6 meant an orqanic vehicle such as MLV or Tween 80 plus diluent ~uch as water or PB5.) 5imilarly, for an N5AID preparation that is administered continuou~ly, as by catheter, minimum effective concentrations of N5AID in total vehicle will usually be found within the range of from about 0.01 to about 10 mg~ml.
ExamDle 17 In this series, the uterine horn of New Zealand wbite female rabbits wa~ used as the model for adhesion development. It is believed that the trau~a induced in this type of surgical procedure i~ more apt to produce severe adhesion~ than any trau~a ordinarily associated with surgery, and therefore thi~ i~ a very severe te~t for evaluating the efficacy of a medica~ent in inhibiting the formation of po~t-~urgical adhe~ions.
12~Z946 The rabbits were anaesthesized using acelepromazine and ketamine, and then underwent a lower median laparotomy incision. Then, the 6erosal surface of both uterine horns were abraded by grasping them with a gauze surgical sponge and pulling them away from the uterus until punctate bleeding developed.
Immediately after the uterine horns were traumatized as described above, varying quantities of a MLV liposome Iprepared by a procedure analogous to that described above in ~xample 13) containing the sodium salt of tolmetin were dripped on the traumatized site, and the rabbits were then closed. Seven days post-operatively, the rabbits were sacrificed and the development of adhesions was evaluated, with the results discussed below.
The lipo~omes were ~u~pended in PBS, at a concentration of 40 mg. of liposome per ml of suspension and 1.46 mg. of tolmetin per ml of ~uspension.
The results of the evaluation were as follows, with the quantities indicated being the volume of liposome suspen~ion dripped on the site of surgical trauma:
10 ml - Scant adhesion 3 ml - Scant adhesion, but a few more than with the 10 ~1 dose. The adhesions were filmy 1 ml - Mild adhesions Control- Ino medicament) - Severe adhesions:
essentially unable to open the rabbit without tearing adhesions which developed between the uterus and the bowel and , 1~92~ ~6 between the uterus and the anterior peritoneal wall.
The above-described study was repeated several times with the liposome/tolmetin combination, with es6entially the same results.
When the free acid form of tolmetin contained in 15 milliliters of aqueous Tween 80 (5 wsight per cent Tween 80, 1 mg/ml of tolmetin) was substituted for the tolmetin/liposome suspension in the double uterine horn model study, the formation of post-surgical adhe6ions was substantially prevented.
This procedure is illustrated for this invention by Examples 1, 2, 9, and 10, below:
Exam~le 1 (a) To a small vial was added 9.39 grams of poly~lactide-co-glycolide-65:35) (a 65/35, by weight, copolymer of laceide and glycolide having an inherent viscosity in chloroform of 0.5 dlJg), 0.75 gram refined sesame seed oil, 1.140 grams ibuprofen, and 40 milliliters of dichloromethane. The solution obtained wa~ added to 400 milliliters of aqueous 5% wt/vol poly(vinyl alcohol) (Air Products Vinol Grade 523) in a one liter resin kettle equipped with a mechanical stirrer and vacuum take off, which was being cooled in an ice/water bath and stirred at 500 rpm. After allowing 10 minutes for emulsification, vacuum was slowly applied by means of an aspirator to an absolute pressure of 500 mm of mercury over 1.5 hours.
The vacuum was then maintained for an additional 19.5 hours (to remove the dichloromethane solvent), at which time vacuum and stireing wa~ stopped and the contents of lZ~29~6 the flask was poured into a one liter beaker and diluted to 800 milliters total volume with water. (An alternative method for removing the dichloromethane solvent is to heat at 30C. for two hours at atmospheric pressure.) The contents of the beaker were added to centrifuge tubes and they were centrifuged at about 1000 rpm for two minutes.
The liquid was deranted, fresh water was added, and centrifugation was repeated. This procedure was repeated one more time, and after the third wash, the last trace~
of water were removed by freeze drying. The dried sample was a free flowing white powder which weighed 2.03 grams.
The powder was examined at lOOX and was found to contain microcapsules which ranged in size from about l to 10 microns. No free drug crystals were noted. Subsequent NMR
analysis indicated that 8.6 wt. % ibuprofen was present.
(b) To make control microcapsules, the foregoing procedure i8 repeated without adding the ibuprofen.
~c) Microcapsules produced as described above, both with and without ibuprofen, are disperged in an aqueous dispersion of lecithin in proportions of 1.5 grams of microcapsules per 50 milliliters of 0.05 weight per cent aqueous lecithin. After freeze drying, 1.4 grams of microcapsules are obtained which contain about 1.4 weight per cent of lecithin.
- ExamPle 2 To a small vial was added 1.50 grams of poly(lactide-co-glycolide - 65:35), 0.50 gram ibuprofen, and 5 milliliters of dichloromethane. The resulting solution was added to 50 milliliters of 3~ aqueous poly(vinyl alcohol) solution which was being cooled in an ice/water bath and stirred at 500 rpm. Vacuum was applied as before and after washing and freeze-drying, l.lO0 grams 129Z94~
of microcapsules was obtained. The microcapsules ranged in size from 10 to 120 microns and were found to contain 17.1% by weight ibuprofen by NMR.
ExamPle 3 Ipuprofen/poly(lactide-co-glycolide-65:35) microcapsules (Example 2) which were found to contain 17.1~ by weight ibuprofen (NMR) were investigated. In five separate 4 ounce amber jars was placed 100 milligrams of microcapsules and 100 milliliters of pH 7.27 phosphate buffer. The caps were tightly closed and the jars incubated at 37 C., with no agitation. After 15 minutes, 1 day, 2 days, 7 days, and 14 days, a jar was removed, the microcapsules collected by filtration, washed well with water, dried under vacuum, and analyzed for ibuprofen content by NMR~ The results ~how~ in the attached table ~ndicate that approximately 50% of the drug wa~ relea6ed in the fir~t 7 days and the remainder was Z0 completely released by day 14. (Negligible polymer weight lo~s occurred over the 14 day period.) Table I
In-Vitro ~elease of Ibuprofen From Poly(lactide-co-glycolide) Microcapsules Sample Time WT % IBF REMAINING
(~ays) ~ of TOT CAPSULE WT) __________________________________________________________ 1 0 17.0 2 1 14.3 3 2 13.6 4 7 10.0 14 0.0 i2~2946 Methods for incorporating drugs in lipid caLriers are known in the art. For instance, one procedure for encapsulating a drug in a phospholipid vesicle is the following:
a lipid or mixture of lipids such as lecithin or other phospholipid, which may be mixed with cholesterol or other lipoid substance, is dissolved in an organic solvent such as diethyl ether; and an aqueous phase containing the material to be encapsulated (in this case, an NSAID) is added to the lipid solution, and the mixture is agitated as by exposing it to ultrasonic sound waves (sonicated). Preferably, the organic solvent is removed during sonication, as by u6e of heat or vacuum or both, although in some caseg the solvent can be removed after the sonication.
This procedure typically produces a unilamellar vesicle.
Another procedure for producing a phospholipid vesicle (in this case a multilamellar vesicle "MLV") containing a medicament is to form a film of dry lipid, as by evaporating the solvent from an organic solvent 601ution containing a lipid to form a film on the walls of the vessel containing the solution, and then stirring in the agueous phase containing the NSAID to be encapsulated.
(The evaporation can be done by spray drying or by vacuum evaporation, or by any other convenient method.) Free unencapsulated NSAID can be separated from MLV~s by centrifugation at, e. g., 12,000 rpm.
Preferably, the vesicle containing the NSAID is dehydrated, as by freeze drying, after preparation, in order to insure long term storage stability. The agueous vesicle suspension can be reconstituted just prior to use by adding sterile phosphate buffered saline, sterile water, or the like.
1~2929~6 The use of multilamellar vesicles of comparatively large size (that is, greater than 1 micron, e. g., from about 1 to about 10 microns) appears to be preferable in order to increase the dwell time of the vesicle containing the NSAID in the peritoneal cavity (or other body cavity). It i8 also preferred to~use a pure or synthetic phosphatidylcholine in which the fatty acid moieties in - the phosphatidylcholine molecule are derived from a single fatty acid, in preparing the vesicle instead of natural lecithin, which is ordinarily a mixture of compounds, Example 13, below, illustrates the preparation of a multilamellar vesicle containing an NSAID.
The following United States patents describe the preparation, by various procedur~s, of phospholipid vesicles containing various medicaments:
Lenk et al. No. 4,522,803 ~alde~chwieler et at. No. 4,310,505 Mezei et al. No. 4,485,054 Gersonde et al. No. 4,452,747 Kelly No. 4,356,167 Papahadjopoulo~ et al. No. 4,241,046 Suzuki et al. No. 4,016,100 Sache et al. No. 4,239,754 MacDonald No. 4,532,089 Rahman et al. No. 3,993,754 See also Callahan et al., European Patent Application No.
0126580, published November Z8, 1984, and Gregoriadis, "The Carrier Potential of Lipo~omes In Biology and Medicine", New England Journal of Medicine, V~l. 295, pp. 704-710 and pp.
765~770 (Sept. 23 and 30, lg76).
.
The foregoing are incorporated herein by reference a~ general proeedure~ which can be utilized for the incorporation of ~AID's ln lipo~omes.
S Oth-~ procodu~s ~or aontaining drug~ in ~ho~p~olipid0 ~micelles or liposomes) ~ and which are applicable to NSAID~s, are described in Sears, U.S. Patent Nos. 4,426,330 and 4;1~5,410, and ~ears et al., U.S. P~tent No. 4,298,594 .
It is not essential that the NSAID medicament u6ed in the invention be encapsulated in an inside aompartment or compartments of the carrier as will normally be the case when the carrier i~ a phospholipid vesicle. In some ca6es it i8 a¢co~table for tho NSAID to be d~ssolYed or otherwise di~tributed more or le~s evenly ~hroughout the carrler.
Thé non-steroidal anti-inflammatory drug i8 delivered to the ~ite of ~urgiaal trauma in effe¢tive quantitie~ over the ¢ritical wound healing period ~whi¢h period varies from patlent to patient and with the type of ~urgical trauma encountered, but i8 u~ually from about two to five days, and in some case~
up to seven days or more, post-operatively.) The example6 below illustrate the order of magnitude of effective quantities : 25 of the drug and the period of time over which the drug is administered for effective results.
The following ~tudies u~e rabbit models to illustratQ the adhesion inhibition effectiveness of the topical admini~tration of a non-steroidal anti-inflammatory drug to the site of ~urgical trauma:
, 12~Z946 ExamPle 9 New Zealand white female rabbits (1.8-2.0 kg) underwent midline laparotomy using acelepromazine and ketamine anaesthesia. A
3x5 centimeter abrasion was produced over the right-lateral peritoneal side-wall by ~craping the surface peritoneum with a scalpel until punctate bleeding developed over the entire 3x5 centimeter area. A second abrasion covering the same total area (15 cm2) using the same technique was developed 1.5-2.0 centimeters inferior to the initial site along the right-lateral peritoneal side-wall. This second site was used as an untreated control. The serosal surface of the large bowel adjacent to the peritoneal abrasion sites was also similarly abraded.
Ibuprofen contained in poly~lactide-co-glycolide-65:35) microcapsule~ produced as described in Example 1, were ~uspended in 15 we~ght per cent aqueous poly(vinyl pyrrolidone) "PVP" (GAF Povidone C-15). The proportions were 15 grams of microcapsules per 100 grams of the PVP solution.
The lecithin-containing microcapsules were used in half of the experiments. The suspension was dripped on the wound in an amount such that about 40 milligrams of ibuprofen (about 470 milligrams of microcapsules) was applied to the wound site. One control was the PVP solution containing ibuprofen-free microcapsules, and the other was the PVP
solution containing ibuprofen-free microcapsules with lecithin.
Seven days after the day of abrasion, the rabbitg were sacrificed by pentobarbital overdose. The extent of adhesions was evaluated as follows:
1~32~46 1. No adhesions 2. Filmy adhesions (separable) 3. Mild adhesions (not separable - covering up to about 35%
of the test area) 4. Moderate adhegions (not separable - covering about 35 to 60 % of the test area) 5. Severe adhesions (not separable - covering greater than about 60% of the test area) The evaluation ratings set forth above are useful in the context of comparing the efficacy of various means for inhibiting the formation of adhesions. However, it should be mentioned that ultimately only a rating of ~'1" can be considered to be completely acceptable. Clinical complications can result from even mild adhesions, although such complications are considered to be more likely to occur with severe adhe~ions than with mild or moderate adhegions.
The following Table II presentg the results:
Table II
Evaluation Ibuprofen Untreated Rabbit No. IbuProfen Lecithin Treatment Controls 1 yes no 4 4 2 yes no 4 5 3 yes no 1 1 *
4 yes yes 3 5 yes yes 4 5 6 yes yes 3 5 7 no no 5 5 8 no no 5 5 9 no no 5 5 no yes 5 5 11 no yes 5 5 12 no yes 5 s ____________________________________________________________ * ~he surgical procedure followed in this experiment induces adhesion formation in about 87 per cent of untreated rabbits.
Evidently this rabbit was one of the approximately 13 per cent that do not develop adhesions.
________________________________ The results indicate some anti-adhesion activity exhibited by the microcapsules containing ibuprofen.
It is relevant to note that when the vehicle control site is in the same rabbit as the test site, migration of fluid in the peritoneal cavity can carry some of the medicament from the NSAID treatment site to the vehicle control site. Therefore, it i8 possible that the untreated (by "untreated" is meant no actiYe medicament) control ~ites of rabbits Nos. 1-6 in Table II and analogous untreated control sites reported below could 2~ have received small amounts of NSAID owing to migration or circulation of fluid within the peritoneal cavity. However, if any of the untreated control sites did receive some o the active medicament by such fluid migration, it would have been significantly less than that received at the treatment site in the same rabbit. Therefore, differences in results between the treatment sites and the control sites in the same rabbit can - confidently be interpreted as being caused by the adhesion inhibition effect of the NSAID.
ExamDle 5 In order to carry out dose response studies and time response studies, miniature osmotic pump8 (Alzet mini pumps, model 2MlL
or model 2002 - these pumps are described in Higuchi et al., l~Z9~6 .
U.S. Patent No. 3,995,631) were used to de,liver the antl-adhe~lon agent ~n a continuou~ stream at a very low, controlled Slow rate, to the s~te o~ the surgical trauma in the te~t rabbits over a period of time, up to ~even days. The mini-pumps therefore deliver the medicament in a manner analogou~ to a catheter delivery modQ.
New Zealand white ~emale rabbits (1.8 to 2.0 kg) underwent midline laparotomy u6ing acelepromazine and ketamine anaesthesia. A 3 x 5 cm flap of parietal peeitoneum (about 1 mm thic~) was sharply dissected from the right lateral peritoneal side-wall, The serosal surf'ace of the ad~acent large bowe? was abraided with a scalpel until punctate bleeding developed. This area between the excised parietal peritoneum and ad~acent large b~wel serosa was then used for evaluating th~ e~fica¢y o~ the te~t medicament ~or adhe~ion inhib~tion. A
~econd oxais~on o~ parie~al peritoneum co~er~ng the ~ame total area ~about 15 ¢m ) wa~ ~qr~ormed 1.5 to 2.0 cm inferior to, the initial to~t site along the right lateral peritoneal s~de-wall. Abrasion of the ad3acent larqe bowel serosa was p-r~orm-~ a~ de~aribe~ above'for the tr--tm-nt 8ite . Thls ~-cond area wa~ u~ed to dotermine the e~fectiveness ot the 6uryical procedure in producing adhe6ions and the response to ~ vehicle controls.
: 25 Alzet mini pumps containing ibuprofen dissolved in pho&phate bufered saline ("PBS") were sewn into the right dorsal subcutaneous space of the test rabbi,t with VICRYL (Polyglactin 910) sutures placed 3 to 5 millimeters from each end of the, pump. The polyethylene catheter tip leadihg from the pump into the pe~itoneal cavity of ,each rabbit was placed 2 to 3 ,millimeter8 over the in3ury test 8ite. The catheter was securëd in place by two 3/0 VICRYL 6uture8 which did not , , involve the site of the injury. A similar ~ump and catheter , , *Trademark, ET~-694 , , ' A
, lZ9Z9~6 6ystem containing PBS vehicle only was implanted in the middle portion of the inferior (vehicle control) abrasion site.
Two different mini pumps were used in these experiments. The first (a 2 ml pump~ had a pumping rate of 10 microliters per hour and the second (a 0.2 ml pump) had a pumping rate of 0.5 microliter per hour. Each pump contained phosphate buffered saline (pH 7.2, unless otherwise indicated), 2 milliliters in the larger pump and 0.2 milliliter in the smaller pump. The control pumps contained phosphate buffered saline alone, and the treatment pumps contained either 20 milligrams of ibuprofen (2 ml pump) or 2 milligrams of ibuprofen (0.2 ml pump).
The results seven days after the operation were as shown below in Table III. The evaluation procedure was the same as the one de~cribed in Example 4.
Table III
Evaluation Ibuprofen Vehicle Rabbit No. PumP Size Treatment Control 1 0.2 ml 5 5 2 0.2 ml 4 5 3 0.2 ml 4 5 4 2 ml 4 5 2 ml 2 5 6 2 ml 1 5 The smaller of the two pumps gave a slight positive response in two of three rabbits, whereas the larger pump gave significant positive responses in two out of three rabbits and a 61ight positive re6ponse in the other.
ETH-69~
lZ~Z946 ExamPle 6 In order to try to better define the threshold dosage rate for ibuprofen ("IBF") in this experimental model, similar experiments were carried out with the two sizes of pumps, using different concentrations of IBF per pump. Table IV, below, sets forth the concentrations of IBF per pump and responses in this serie~ of experiments:
Table IV
Evaluation Concentration of Vehicle Rabbit No. pumP Size IBF, mo~ml Treatment Control 1 0.2 ml 10 1 4 2 0,Z ml 10 4 5 3 0.2 ml 10 5 5 4 2 ml 10 3 5 2 ml 10 4 4 6 2 ml 10 3 5 7 0.2 ml 3 4 5 8 0.2 ml 3 5 5 9 0.2 ml 3 4 5 2 ml 3 3 5 11 2 ml 3 5 5 12 2 ml 3 4 5 13 0.2 ml 1 5 5 14 0.2 ml 1 5 5 0.2 ml 1 1 4 16 2 ml 1 5 5 17 2 ml 1 5 5 18 2 ml 1 4 5 19 0.2 ml 0.3 5 5 0.2 ml 0.3 5 5 21 0.2 ml 0.3 5 5 lZ~Z9~16 22 2 ml 0.3 5 5 23 2 ml 0.3 5 5 24 2 ml 0.3 4 5 This series of experiments indicates that, in this model, the threshold dosage at which significant anti-adhesion effects began to be noted occurred when pumps containing concentrations between 1 and 3 milligrams of IBF per milliliter were used. No significant difference was noted here between the two different sized pumps.
An effective dose of a topically applied drug is normally expressed in terms of concentration of the drug in the carrier.
coupled with the number of times per day the drug is applied.
In the present invention, the effective dose will be dependent upon factors such as nature of specific NSAID used, nature of vehicle, nature of tissue to be treated, type of trauma, and mode of delivery (i,e~, continuous delivery by catheter or a one-time application in a vehicle such a6 a controlled release vehicle). Therefore, no hard and fast rule can be formulated that will apply in all cases, and experiments analogous to those reported in this Example 6 will have to be performed in order to precisely define the threshold dosage for each different NSAID, for specific vehicle system6, and for specific modes of delivery. It is well within the ability of the,person skiIled in the art to carry out the necessary experiments to determine threshold dosages, after having read this disclosure.
Exam~le 7 In order to determine the time period over which the anti-adhesion agent must be administered in order to have a significant anti-adhesion effect, the following series :: :
12~Z946 of experiments were carried out (using the experimental procedure described above in Example 5):
The 2 ml pump was used containing 10 mg/ml of IBF, and the catheter delivering the treatment solution to the site of the surgical trauma was disconnected 1, 2, 3, 4, and 5 days post-operatively. The rabbits were sacrificed 7 days post-operatively, and evaluated a~ above. The results are displayed in Table V, below.
Table V
Post-Op Day Evaluation RabbitCatheter Ibuprofen Vehicle No.Disconnected Treatment Control With ibuprofen, in this model, it appears that administration of the NSAID via the 2 ml mini pump in PBS
for at least three days i~ required in order for the drug to have significant anti-adhe6ion effects, With other 12~Z~346 -2~-NSAID~s and/or other delivery modes, and with other types of surgical trauma, it is reasonable to expect that the minimum period of time will differ. In any given case, the minimum period of time over which the NSAID must be administered can be determined by experiments analogous to that described in this Example 7. As a general rule, in most cases, a minimum of one day is recommended, and in some cases, longer periods (e. g., up to 5 to 7 days or longer) may be required, The NSAID active ingredient is administered to the site of surgical trauma topically. Such topical administration can be by spraying, lavage, dripping on the site, by catheter administration, or the like The exact method of administration chosen is not critical, as long as an ef~ective do~e is administered over the critical wound healing period, which can be determ~ned by a series of experiments analogous to that deficribed above in Example 7. The NSAID should be administered immediately post-operatively, that i8, before wound healing has begun ExamDle 8 By a procedure analogous to that described above in Example 5, the 2 ml Alzet mini pump was used to deliver suprofen to t~e test site in the rabbits, The concentration of suprofen was 2.5 mg/ml of phosphate buffered saline (pH = 7.4). The rabbits were sacrificed 7 days post-operatively, and evaluated as above. The results are set forth in Table VI, below:
lZ~Z946 Table VI
2.5 m~/ml Su~rofen Evaluation Suprofen Vehicle Rabbit No. Treatment Control ExamPle 9 To a small vial was added 2,78 grams of poly(lactide-co-glycolide - 65:35), 0,30 gram suprofen, and 8 milliliter~ of dichloromethane. The resulting 601ution was added to 120 milliliters of 3~ aqueous poly(vinyl alcohol) solution which was heated to 30 C.
while being mechanically stirred at 500 rpm. After two hours, the microcapsules were isolated as described in Example 1, and after washing and freeze-dryinq, 1.61 grams of microcapsules were obtained. The microcapsules ranged in size from >10 to 250 microns and were found to contain 7.0% by weight suprofen by NMK.
ExamPle 10 To a small vial was addéd 1.25 grams of poly~D,L-Lactic acid), 0,12 grams refined ~esàme seed oil, 0,152 gram ibuprofen, and 3 milliliters of dichloromethane, The resulting solution was added to 30 milliliters of 3%
~Z~Z946 a~ueous poly(vinyl alcohol) solution whic~ was being cooled in an ice/water bath and stirred at 5~0 rpm.
Vacuum was applied as described in Example 1, and after washing and freeze-drying, 1.12 grams of microcapsules were obtained. The microcapsules ranged in size from 10 to 75 microns and were found to contain 8.7~ by weight ibuprofen by NMR.
ExamPle 11 By a procedure similar to that described above in Example 5, the 2 ml mini pump was used to deliver varying quantities of suprofen sodium (i. e., the sodium salt of suprofen) to the rabbit model. The quantities of medicament contained in the phosphate buffered saline (pH
= 6.9) varied from 3,0 mg/ml down to 0.03 mg/ml, The rabbit~ were sacrificed seven days post-operatively and evaluated as above, The results are set forth below in Table VII:
Table VII
Suprofen, Dosage ResDonse Studies ConcentrationEvaluation of Suprofen,Vehicle Rabbit No. ma~mlTreatment Control 12~Z946 ~ 1 1 5 *
11 0.3 1 5 12 0.3 3 4 13 0.3 3 5 14 0.3 3 3 0.3 3 4 16 0.1 3 3 17 0.1 3 9 18 0.1 3 5 19 0.1 1 5 0.1 5 5 21 0.03 5 Z2 0.03 23 0.03 4 4 24 0.03 1 4 0.03 4 5 __________ _____________________________ ________________ * This rabbit exhibited slight bleeding. (In many cases, in this model, when bleeding occurs no adhesions develop.) __________________ _________ ____________________________ This series of experiments indicates that the threshold dosage at which significant anti-adhesion effects begin to be noted in this model with suprofen sodium as the NSATD
occurs when pumps containing a concentration of about 0.1 mg~ml of drug are used. Thus, on a weight basis, suprofen appears to be slightly more active than ibuprofen.
: 35 129Z9 ~6 ExamPle 12 By a procedure analogous to that described above in Example 5, the 2 milliliter mini pump was used to deliver varying amounts of tolmetin. (The sodium dihydrate salt of tolmetin was used.) The concentrations of drug contained in the phosphate buffered saline (pH = 7.4) varied from 3.0 mg/ml down to 0.01 mg/ml. The treatment pumps contained the concentrations of tolmetin displayed below in Table VIII, and the control pumps contained vehicle only. The rabbits were sacrificed 7 days post-operatively, with results being di6played below in Table VIII:
Table VIII
Tolmetin, ~o~age ResDonse Studie~
Concentration Evaluation of Tolmetin, Vehicle Rabbit No. ma/ml_ Treatment Control 3 3 l 5 6 l 1 4 9 1 l 3 1 Thi8 rabbit died **
lZ9Z946 11 0.3 3 3 12 0.3 1 3 *
13 0.3 1 3 14 0.3 3 5 0.3 3 4 16 0.1 3 5 17 0.1 1 5 18 0.1 1 5 19 0.1 1 5 200.1 This rabbit died (snuffles)***
21 0.03 22 0.03 1 5 23 0.03 3 s 24 0.03 1 5 0.03 1 4 26 0.01 5 5 27 0.01 1 5 28 0.01 1 5 29 0.01 1 4 0.01 25--________________________________ * Slight bleeding occurred.
** The cause of death was unknown, but there was no suggestion that it was drug-related.
*** "Snuffles" is a viru~-cau~ed upper re~piratory disease that affects rabbits. There i~ no sugge~tion that it was drug-related.
~: _____________________________ As can be seen from the data presented in Table VIII, tolmetin appears to be more effective. on a weight basis.
lZ~2`~3~6 than either ibuprofen or suprofen in this rabbit model experiment. The thre6hold dosage, a~ indlcated by the re~ult~ trom thia experiment, wa~ appar~ntly 1Q~8 than the dosage admln~stered to Rabblt Nos. 26 - 30, wherein the S concentration of drug wa~ 0.01 mg/ml.
ExamPle 13 ~he following is a typical preparstion of a lipo~ome (MLV) containing an NSAID:
(In this preparation, all materiais and aquipment used are sterile and pyrogen-free) a) P~e~arati~n o~ l~P~d ~lm L-alpha-di6tearoyl phosphatidylcholine ("DSPC"), 1.21 gm., and 0.29 gm. cholesterol ~molar ratio of DSPC to cholestero,l is 2:1), are dis~olved in 45 ml. of chloroform. ~he re~ulting solution i8 div~ded into nine 5 ml portions, and each ~uch portion is placed in a lOO ml ~la~k. The 601vent is evaporated from each fla~k using a rotary vacuum evaporator. Sterility i8 maintained by attaching a 0.22 micron Millex filter to the air intake of : 25 the evaporator prior to flask removal. Starile septa are placed on the flasks after solvent evaporation. The surface of each septum is ~iped with 70% alcohol, and a 19 gauge sterile needle affixed to a 0.,22 micron filter is pa~sed through each septum. All flasks are then placed in a large vacuum desiccator and kept there ove~night. ~ach flask contains about 167 mg. of lipid.
*Trademark .:
.
ATy-694 ', .
12~Z~46 b) Pre~aration of NSAID solution The sodium salt of ibuprofen (Na-IBF), 0.202 gm., is dissolved in 40 ml of sterile, pyrogen-free water. The solution is then passed through a 0.22 micron Millex filter.
c) Pre~aration of MLV containina Na-IBF
3.9 Ml of the Na-IBP solution is injected into each flask containing lipid film. The flasks are vortex-stirred for 40 to 60 minutes in a 65 C. water bath under nitrogen.
(The nitrogen purge is first passed through a 0.22 micron filter.) To the contents of each flask are added sterile, pyrogen-free phosphate buffered saline, and the flask6 are centrifuged for 6 to 10 minutes at 15,000 rpm. This wa~hing procedure i~ repeated for a total of five times to remove unencapsulated ~SAID. The contents of the flasks are then combined and PBS (5mM P04 in 0.15 NaCl) is added to a total of 3Z ml. The liposome suspension thus produced comprises MLV's of about 1 micron in size ~uspended in the PBS. It is storage stable for a period of several months, but for long term storage is preferably dehydrated and remixed with sterile, pyrogen-free water just before use.
To prepare drug-free controls, the procedure is repeated substituting pure water for the water/Na-IB~ ~olution.
The procedure is repeated using ta) the free-acid form of IBF, and (b) the sodium salt of tolmetin. Similar result~
are obtained and liposome MLV's containing IB~ or sodium tolmetin are produced.
MLV's containing IBP or tolmetin, and produced as lZ9Z9 ~6 described herein, were found to have particle sizes of about 1-1/2 to 2 microns (by optical microscope) and about 5 to 8 microns (by Coulter counter).
Analogous procedures would be employed to produce vesicles from phosphatidylcholines in which the fatty acid moieeies were derived from other fatty acids, e.g., C12 to C24 fatty acids. C14 to ~20 saturated fatty acids are preferred.
ExamDle 14 This experiment evaluated the eficacy of a liposome/ibuprofen combination to combat post-surgical adhesions. The procedure used was similar to that described above in Example 4, except that the untreated control sites in those rabbits that were given the liposome/ibuprofen treatment were on the left-lateral peritoneal side-wall instead of being sited 1.5 to 2.0 centimeters inferior to the treatment sites in the right-lateral peritoneal side wall, and the wounding procedure described in Example 5 was used.
The liposomes used in this example were DSPC/cholesterol MLV~s (L-alpha-distearoyl phosphatidylcholine/cholesterol multilamellar vesicles), prepared in a manner analogous to that described above in Example 13. The treatment mixture consisted of 31 milliliters containing 1200 milligrams of MLV and 35 milligrams of the free acid form o ibuprofen, suspended in 5mM phosphate buffered saline. The vehicle control had the same composition, except that the ibuprofen was omitted. Each rabbit received 10 ml of suspension, which amounted to 3.5 mg o ibuprofen per rabbit. The treatment and vehicle control suspensiong were dripped on the traumatized ~ites, as described above lZS'29 ~6 in Example 4. Three rabbits received the~
liposome/ibuprofen treatment on the right side-wall site with the other site being untreated, and three rabbits received a vehicle control treatment (i. e., liposome without ibuprofen) on the right side-wall site with no treatment on the other site. The rabbits were sacrificed 7 days post-operatively, and evaluated as described above. The results are displayed below in Table IX:
Table IX
LiDosomeJIbuProfen Studies Rabbit Site No.IbuProfen Treated Untreated 1 yes 2 yes 3 yes 1 4 4 no 2 4 no 5 5 6 no 1 5 The experiment was repeated in exactly the same fashion, ;~ with the results as displayed below in Table X:
125~Z946 Table X
Rabbit Site No.IbuProfen Treated Untreated 1 yes 2 yes 3 yes 1 4 4 no 5 4 no 3 4 6 no 1 1*
_ ___________________________ ~ This rabbit exhibited ~light bleéding.
________________________________________________________ In the experiments reported above in Tables IX and X, the rabbits receiving treatment by the ibuprofen/liposome combination were virtually free of adhesions, even on the sides that received no direct application of medicament.
This is considered to be indicative of the fact that the medicament can migrate in the peritoneal cavity as a result of circulation of peritoneal fluid. Also, by gross observation, no tissue reaction or granulomas were found at the treated sites.
Exam~le l5 In this series of experiments, the procedure of ~xample 14 was repeated, with the following modifications:
The liposomes that were used were DSPC~cholesterol MLV~s prepared as described above in Example 13, and which lZ9Z9~6 contained Na-IBF. The control MLV~s (two batches -"MLV-l" and "MLV-2") contained no drug. The drug-containing lipo60mes were mixed with the control liposomes in varying proportions to obtain MLV's that contained varying amounts of NSAID. In Table XI, below, there is di6played the compositions of the drug-containing and tbe control liposomes:
-Table XI
LiDosome ComPosition Vol, Lipid, Total Lipid, Drug, Drug/Lipid, ml mq/ml mq ma/ml (bY weiqht~
Control MLV-l 50 33 1675 N/A N/A
MLV 50 33 1675 0.98 0.029 (49 gm) In Table XII, below, there is displayed the proportions of the drug-containing and control liposomes that were mixed together and then administered to the test rabbits in the manner described above in Example 14, along witb the results of the evaluations of the rabbits seven days post operatively:
lZ92~46 Table XII
Na-lBF/MLV Evaluations Rabbit IBF Control Evaluation No. MLV, MLV, Treatment Control ml ml Site Site 1~ 0.3 9.7 1 3 13 0.3 9.7 1 3 lZ9Z946 Exam~le 16 By a pro¢edure analogous to that described above in Example 14, aqueous ¢ompositions including a surface at1vo ~g-nt and t~e f roo aoid ~o~m o~ tolmetln we~e applied to the treatment sitQs in the rabbit model. The a~ueous compositions comprised various concenteations of "Tween 80", an ethoxylated sorbitan mono-oleate, in triple distilled water, and tolmetin at a concentration of either 10 1 or 2 mg~ml. The 601utions were sterilized by pa~sing them through a 0.22 micron filter. The tables below display the concentration of tolmetin and Tween 80, and the evaluation~ of the adhesions seven days post-operatively. In each case, 10 ml of the solution was tr1~pod on the treatmen~ s~to.
Table XIII
5 Wt, ~ Tween 80 *
~ ma/ml Tol~,e~
a~gi_~Q~ Evaluation Treatment Contr~l Site _Site 3 1 1 ~Bleeding) 4 1 , 5 (Control*) 3 3 9 ' : 10 (Control*) ' 4 3 ;*Trademark ETH-694 , Al , l~Z9'~6 * The control rabbits received no treatment.
___________________________________ Table XIV
20 Wt. % Tween 80 2 ma/ml Tolmetin Rabbit No. Evaluation Treatment Control Site Site 5 (Control*) 4 4 10 (Control*) 5 5 ____________________________________________________________ : ~ No Treatment ______________ lZ9Z9~6 Table XV
20 Wt. ~ Tween 80 2 mqJml Tolmetin Rabbit No.Evaluation Treatment Control Site Site 2 1 1 (Bleeding) Table XVI
1 ma/ml Tolmetin Sodium (1 No Tween Rabbit No~valuation Treatment Control Site Site 9 (~Control(2)) 4 3 lZ9Z946 ____________________________________________________________ (1) The sodium salt was used because the free acid form is guite insoluble in pure water.
(2) Water only; no Tolmetin ____________________________________________________________ The free acid form of tolmetin, which i6 guite insoluble in water, was maintained in agueou~ solution or colloidal lo suspension by the surface active agent.
Referring again to the question of the effective dose of NSAID in accordance with this invention, while no hard and fast numbers can be presented that will be applicable to all cases, the examples presented above can be referred to as a guide to determine the order of magnitude of drug to employ in certain cases. In pho~hate buffered saline admini~tered continually via the Alzet osmotic mini pump, the following dosages were found to be effective:
ibuprofen, 3 mg~ml, at a rate of 0.5 microliter/hr. ~from EX.6):
suprofen, 0.1 mg/ml, at a rate of 10 microliters~hr. (from Ex. 11); and tolmetin, 0.01 mg/ml, at a rate of 10 microliters~hr. (from Ex. 12~.
Expres6ed in terms o mg~kq/day, and in term~ of mg~day/cm2 of traumatized tissue, these numbers are the ollowing:
1~9Z9~6 ibuprofen - 18 x 10 3 mg/~g/day 2.4 x 1- 3 mg/day/cm2 suprofen - 12 x 10 3 mg~kg/day 1.6 x 10 3 mg/day/cm2 tolmetin - 1.2 x 10 3 mg/kg/day 0.16 x 10-3 mg/day/cm2 These calculation~ are carried out as follow6 (using the ibuprofen numbers as illustrative): 0.5 microliter/hr. of golution containing 3 mg/ml equals 0.5 x 10 6 liter/hr.
x (3 mg/10 3 liter). This reduces to 0.5 x 3 x 10 6/10 3 3 1.5 x 10 3 mg/hr.
~his equals 36 x 10 3 mg/day or 18 x 10 3 mg/kg/day (each rabbit weighs about 2 kg).
The area of traumatized tissue i8 about 15 cm2, 80 the ~0 dose expressed in terms of ~g/day/cm2 = 36/15 x 10 3 mg/day/cm2, or 2.4 x 10 3 mg/day/cm2.
Compared with the minimum recommended dose of 2.5 mgikg/day (from the Binger patent cited above) for Z5 6ystemically ad~inistered ibuprofen, the effective dose for topically administered ibuprofen may be two to three orders of magnitude lower. Obviously, this greatly reduced do6age significantly reduces the chances for ; undesired side effects.
The minimu~ effective dose using an MLV or an a~ueous composition containing a surface active agent as a carrier has apparently not been approached. Howevèr, it is noted from Example 15, Rabbit Nos. 12-13, that a total dose of about 0.3 mg of ibuprofen in an MLV carrier ~as lZ9Z946 effective. This number was calculated as follows:
0.98 mg/ml x 0.3 ml/0.7 ml, eguals 0.0278 mg/ml. 10 ml of this preparation was admini~tered, 80 the total dosage was 0.3 mg or 0.15 mg/~g or 0.02 mg/cm2. It would appear that an even lower concentration would be effective in this ~odel.
In the Tween 80 solutions, the smallest amount of tolmetin applied was 10 mg (from the data presented in Table XIII). It would appear that an even smaller dosage administered in this manner would be effective.
A reasonable extrapolation of the data presented herein ifi that a minimum effective concentration for an NSAID
preparation applied in a ~ingle dose would be between about 0.025 mg and 5 mg of N5AID per ml of total vehicle.
(By "total vehicle" i6 meant an orqanic vehicle such as MLV or Tween 80 plus diluent ~uch as water or PB5.) 5imilarly, for an N5AID preparation that is administered continuou~ly, as by catheter, minimum effective concentrations of N5AID in total vehicle will usually be found within the range of from about 0.01 to about 10 mg~ml.
ExamDle 17 In this series, the uterine horn of New Zealand wbite female rabbits wa~ used as the model for adhesion development. It is believed that the trau~a induced in this type of surgical procedure i~ more apt to produce severe adhesion~ than any trau~a ordinarily associated with surgery, and therefore thi~ i~ a very severe te~t for evaluating the efficacy of a medica~ent in inhibiting the formation of po~t-~urgical adhe~ions.
12~Z946 The rabbits were anaesthesized using acelepromazine and ketamine, and then underwent a lower median laparotomy incision. Then, the 6erosal surface of both uterine horns were abraded by grasping them with a gauze surgical sponge and pulling them away from the uterus until punctate bleeding developed.
Immediately after the uterine horns were traumatized as described above, varying quantities of a MLV liposome Iprepared by a procedure analogous to that described above in ~xample 13) containing the sodium salt of tolmetin were dripped on the traumatized site, and the rabbits were then closed. Seven days post-operatively, the rabbits were sacrificed and the development of adhesions was evaluated, with the results discussed below.
The lipo~omes were ~u~pended in PBS, at a concentration of 40 mg. of liposome per ml of suspension and 1.46 mg. of tolmetin per ml of ~uspension.
The results of the evaluation were as follows, with the quantities indicated being the volume of liposome suspen~ion dripped on the site of surgical trauma:
10 ml - Scant adhesion 3 ml - Scant adhesion, but a few more than with the 10 ~1 dose. The adhesions were filmy 1 ml - Mild adhesions Control- Ino medicament) - Severe adhesions:
essentially unable to open the rabbit without tearing adhesions which developed between the uterus and the bowel and , 1~92~ ~6 between the uterus and the anterior peritoneal wall.
The above-described study was repeated several times with the liposome/tolmetin combination, with es6entially the same results.
When the free acid form of tolmetin contained in 15 milliliters of aqueous Tween 80 (5 wsight per cent Tween 80, 1 mg/ml of tolmetin) was substituted for the tolmetin/liposome suspension in the double uterine horn model study, the formation of post-surgical adhe6ions was substantially prevented.
Claims (26)
1. Use of an effective amount of a sterile composition including as an active ingredient a non-steroidal anti-inflammatory drug, for inhibiting post-surgical adhesion formation in mammals.
2. Use of Claim 1 wherein the active ingre-dient is ibuprofen or a pharmaceutically acceptable salt or ester thereof.
3. Use of Claim 1 wherein the active ingre-dient is suprofen or a pharmaceutically acceptable salt or ester thereof.
4. Use of Claim 1 wherein the active ingre-dient is tolmetin or a pharmaceutically acceptable salt or ester thereof.
5. Use of Claim 1 wherein the active ingre-dient is contained in a controlled release carrier that releases said active ingredient in an effective amount over said period of time.
6. Use of Claim 5 wherein said carrier is a phospholipid.
7. Use of Claim 6 wherein said phospholipid is a phospholipid vesicle.
8. Use of Claim 7 wherein the phospholipid vesicle is a multilamellar vesicle.
9. Use of Claim 8 wherein the vesicle has a size of from about 1 to about 10 microns.
10. Use of Claim 8 wherein the vesicle comprises phosphatidyl choline wherein the fatty acid moieties are derived from a single fatty acid.
11. Use of Claim 8 wherein the vesicle comprises a mixture of a phosphatidyl choline and cholesterol.
12. Use of Claim 5 wherein said carrier is an absorbable polymer.
13. Use of Claim 12 wherein said absorbable polymer is a homopolymer or copolymer of lactic acid, glycolic acid, their cyclic dimer esters, or p-diox-anone.
14. Use of Claim 13 wherein said polymer is in the form of microcapsules.
15. Use of Claim 14 wherein said micro-capsules include lecithin in an amount sufficient to enhance the ability of said microcapsules to adhere to the site of surgical trauma.
16. Use of Claim 1 wherein the active ingredient is contained in an aqueous composition including a surface active agent.
17. Use of Claim 16 wherein the aqueous composition comprises an aqueous solution of ethox-ylated sorbitan mono-oleate.
18. Use of Claim 1 wherein the concentration of active ingredient in said composition is at least about 0.025 to 5 milligrams per milliliter.
19. Use of Claim 1 wherein the concentration of active ingredient in said composition is at least about 0.01 to 10 milligrams per milliliter.
20. A sterile aqueous composition including a surface active agent and a non-steroidal anti-inflammatory drug.
21. The sterile composition of Claim 20 wherein the drug is ibuprofen, suprofen, tolmetin, or a pharmaceutically acceptable salt or ester thereof.
22. A sterile composition comprising a phospholipid vesicle containing a non-steroidal anti-inflammatory drug.
23. A sterile composition of Claim 22 wherein the drug is ibuprofen, tolmetin, suprofen, or a pharmaceutically acceptable salt or ester thereof.
24. A sterile composition comprising an absorbable polymer microcapsule containing a non-steroidal anti-inflammatory drug.
25. The composition of Claim 24 wherein the polymer is a homopolymer or copolymer of lactide, glycolide, or p-dioxanone.
26. The composition of Claim 25 wherein the drug is ibuprofen, suprofen, tolmetin, or a pharma-ceutically acceptable salt or ester thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000523793A CA1292946C (en) | 1986-11-25 | 1986-11-25 | Method for inhibiting post-surgical adhesion formation by the topical administration of non- steroidal anti-inflammatory drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000523793A CA1292946C (en) | 1986-11-25 | 1986-11-25 | Method for inhibiting post-surgical adhesion formation by the topical administration of non- steroidal anti-inflammatory drug |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1292946C true CA1292946C (en) | 1991-12-10 |
Family
ID=4134429
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000523793A Expired - Lifetime CA1292946C (en) | 1986-11-25 | 1986-11-25 | Method for inhibiting post-surgical adhesion formation by the topical administration of non- steroidal anti-inflammatory drug |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1292946C (en) |
-
1986
- 1986-11-25 CA CA000523793A patent/CA1292946C/en not_active Expired - Lifetime
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