THE USE OF LIPOSOMES FOR THE DELIVERY OF THERAPEUTIC AGENTS TO WOUNDS, CUTS AND ABRASIONS

Abstract: Methods for delivering therapeutic agents to wounds in liposomes which preferentially bind to the wounds are disclosed. Methods for delivering therapeutic agents in liposomes to wounds on the ocular surface of the eye are included.

Description: THE USE OF LIPOSOMES FOR THE DELIVERY OF THERAPEUTIC A6ENTS TO MOUNDS, CUTS AND ABRASIONS Background of the Invention This nvention 1s directed to methods for treating wounds, cuts, and abrasions caused by Injury, trauma, surgery or disease through the delivery of therapeutic healing agents to such sites. More particularly, the nvention 1s directed to the delivery of therapeutic agents to wounds, cuts and abrasions on the cornea or ocular surface of the eye via llposome and Uposome-polymer compositions which specifically adsorb or adhere to the wound, cut or abrasion. Liposomes are sac or vesicle-H e bllayer structures made of phosphol1p1ds. For an in-depth discussion of the preparation and properties of iposomes, ncorporation of drugs and proteins therein and targeted drug delivery; see, G. Gregor1ad1s, Lloosome Technology. Vols. I,II and III, CRC Press Inc., Boca Raton, Florida, 1964, the contents of which are incorporated herein by reference. The use of iposomes as delivery vehicles for drugs or other therapeutic agents 1s known. Liposomes as delivery vehicles of therapeutic agents to the eye 1s also known; see for example, U.S. Patent No. 4,649,047 Issued to R. aswan, March 10, 1987 disclosing the treatment of traumatic or surgical phacoanaphy actlc endophthalinltls or uveltls occurring throughout the eye globe by topical administration of cyclosporln 1n suitable exdplents, Including l posomes. U.S. Patent No. 4,588,578 ssued to Fountain et al., May 13, 1986 discloses the preparation of bloactlve agent carrying Hp1d vesicles which can be applied topically for the treatment of ocular disease such as glaucoma. Skuta et al., 1n American Journal of O p hthalmolo g y. Vol.103, No.5, May, 1987 disclose the delivery of 5-fluoro- uracil In a Hposome via subconjunct val Injection as a method for inhibiting flbroblast proliferation following posterior sclerectomles. Skuta et al. characterize their delivery system as an advantageous, sustained release delivery system; prior methods of treatment with the free drug required frequent subconjunctlval Injections. The use of liposomes for certain types of targeted drug delivery 1n the,eye has been described previously. Norley et al., In J. Immunol.. Vol.136, No.2 (January 15, 1986) disclose the targeting of drug-loaded liposomes to Herpes Simplex virus (HSV) Infected comeal cells, 1n vitro. The targeting moiety Incorporated 1n the liposomes 1s an antiviral monoclonal antibody to glycoprotem D of HSV containing a covaenty-bound fatty acyl chain. This "Immunoliposome" was shown to bind specifically to HSV Infected rabbit comeal cells. In another example of targeted drug delivery, intravitreally Injected liposomes were used for delivery of drugs to the cells of the retinal membrane, see, Stern et al., Investigative Ophthalmology and Visual Science. Vol.28, No.5, (May, 1987). U.S. Patent No.4,755,388 Issued to Heath et al. (July 5, 1988) discloses the use of liposome- encapsulated agents such as 5-fluoropyr1m1d1nes useful in preventing scar formation by inhibiting cell contraction and cell growt in impaired tissue in the eye. Heath et al. describe a variety of covalent modifications of the liposo e using Hgands and receptors for targeting to specific cell surfaces or for ligand binding. The preparation and use of bioadheslve iposomes for topical application of therapeutic agents to the ocular surface has also been described. Positively-charged iposomes and polymers are known for their nonspecific bioadhesion to surface regions of the eye; see, Fitzgerald, et al., Journal of Pharmac y and Pharmacology. Vol.39, pp.487-490, (1987); and Shlota et al., Ocular Delivery of Vitamin A Using Positively Charged Hoosomes. ARVO Annual Meeting Abstract Issue, p.159, No.22 (1987). F gur The sole figure illustrates adsorption of liposomes of the present Invention to corneal wounds versus their relative nonadsorption to unwounded corneas. Summary of the Invention This Invention is directed to methods for treating wounds by the delivery of therapeutic agents in liposomes to the wound site. The liposomes preferentially adsorb to wound sites and not to healthy tissue surrounding the sites. More particularly, the Invention 1s directed to the delivery of therapeutic agents in liposomes to wounds on the surface of the eye, such as the cornea, conjunctiva and sclera. Because the Hposome carriers bioadsorb almost exclusively to the wound site on the ocular surface, the amount of therapeutic agent normally administered as a free agent can be significantly reduced. Consequently, the therapeutic Index or margin of safety of a drug delivered according to the method of this invention will be improved. Although topical ophthalmic application of drug containing liposomes 1s known, the prior delivery methods have been used as a means for Improving the transport of therapeutic agents Into and across tissue barriers of the eye, such as the cornea, conjunctiva and sclera. Targetable and bloadhesive liposomes such as those disclosed by Norley et al. (1986) and Shlota et al. (1987) have been formulated through either covalent modification of the phosphollpid or intercalation of ligand molecules into the Hposo e bϋayer. The use of complex receptor-recognition Hgands, or positively charged components have been devised as targeting agents or nonspecific bloadhesive components for tissue surfaces. However, prior methods have not recognized that liposomes will preferentia ly bioadsorb, with a high degree of specificity, to corneal wounds, such as cuts and abrasions. The present Invention is based on the surprising discovery that iposomes adhere to wound sites but not to the surrounding healthy tissue. As used herein the term "wound" Includes cuts, scrapes, abrasions or tissue damage caused by Injury, trauma, surgery or disease. Thus, therapeutic agents which are known to be effective in the treatment of wounds can, according to this Invention, be delivered specifically to wound sites with any nontoxic phosphollpid/cholesterol Hposome. In particular, the iposomes containing therapeutic agents are useful for treating wounds of the eye, particularly the cornea. Detailed Description of the Preferred Embodiment! Liposomes which can be used according to this Invention include any liposoaβ which bioadsorbs to a wound site, particularly a corneal wound site, and shows no toxic, Infl mmatory or antlhealing effects. These iposomes wi l comprise a llpld and will typically comprise 50-90 mol of one or more synthetic or naturally occurring phospholiplds and 10-50 molar percent (tnolλ) cholesterol. Phospholiplds which can be used in formulating liposomes for use according to the present invention include: phosphat1dylchol1ne, phosphatldylglycerol, phosρhat1dylethano am1ne or phosphatldylserlne with diacyl chains of typically 14 to 18 carbons of either the saturated or monoene variety. Other lipids, Including phospholiplds, which can be used in the formu ation of the liposomes include, but are not limited to: phosphat1dyl1nos1tol, sphlngoiηyeHn, phosphatidic add, cerebrosldes, cardlolipin and Iysophosphol1p1ds. The liposomes can be prepared by a number of methods, inc uding but not limited to, procedures for preparing multilamellar (MLV), small unllamellar (SUV), large un1lamellar (LUV), p urilamellar (SPLV) and reverse-phase evaporation (REV) liposomes; see, G. Gregoriadis, U p osome Technology: Preparation of liposomes, Vol.l. Also see U.S. Patent No. 4,235,871, Issued to Papahadjopoulos et al., Incorporated herein by reference to the extent that it teaches the preparation of liposomes useful according to the present Invention as described above. It has also been found, according to the present invention, that liposomes which are relatively small, that 1s, 10 to 500 nanometers (nm) in diameter, preferably less than 100 n in diameter exhibit maximal wound site coverage 1n comparison to the larger MLV-type liposomes of 500 to 10,000 nm 1n diameter. The preferred Uposome composition of the present invention comprises: phosphatldylchollne (50 mol%); the negatively charged phosphatldylglycerol (30 molX), with both phospholiplds containing acyl chains of palmitic add in the 1- positlon, and olelc add 1n the 2- position; and cholesterol (20 molft). For maximum wound coverage, the preferred iposomes are 20 to 200 nm 1n diameter, preferably less than 100 nm. The production of iposomes of less than 100 nm is accomplished by passing MLV liposomes through a MicrofluldizerR (M1croflu1d1cs, Corp., Newton, Mass.) and their size will typically vary less than about 30%. When composed of phosphatldylchollne with saturated acyl chains, the liposomes of the present Invention have an aqueous liquid-crystalline phase transition of between about 35 and 42 degrees centigrade, preferably close to the human physiological temperature of 37 degrees centigrade. The liposomes of the present invention which are composed of phosphatldylchollne with onoene containing acyl chains, exhibit phase transitions well below zero degrees centigrade, and a so show good bioadsorptlve properties to wound sites. This invention is not limited to only phospho lpld or phospholipid/cholesterol containing iposomes, but also comprises phospho ipid/cholesterol liposomes containing polymers. These liposome-polymer combinations provide for formulations with increased viscosity. Any polymer which will provide for increased viscosity around the iposomes, and 1s compatible with the tissues of the eye can be used. The polymer(s) are added to provide for a composition with a viscosity of between about 5 cps. and 1000 cps., preferably between about 15 cps. and 200 cps. Examples of polymers which can be used according to the methods of this invention Include natural polymers such as polysaccharldes (e.g. alglnate, starch, modified celluloses such as hydroxypropy ethylcellulose and hydroxyethy ce lulose), gelatins, albumin, casein, chitosan and collagen. In addition, synthetic polymers which can be used Include polylactideglycolide copolymer, polylactlde, polyhydroxyethylmethacryl te, polyesteramides, polyorthoesters, polymethacryl te, polyvinyl lcohol, polyvinylpyrrol1done, polyacryllc add, poyethyleneglycol and polymeric amino adds. The liposomes in their viscous matrix provide for longer retention of the formulation in the eye and therefore bloadsorptlon of the liposomes to the wound site over longer time periods. The percentage of polymer 1n the Uposome formulation will depend on the polymer Itself, but in general the concentration of polymer can range from about 0.25 - 3.0 wt.%. Therapeutic agents which can be delivered to wound sites in uposome or Hposome/polymer formulations which bioadsorb to such sites comprise all therapeutic agents useful for the treatment of wounds, cuts and abrasions caused by Injury, trauma, surgery and/or disease. Such therapeutic agents will typically comprise, but are not limited to: growth factors, steroids, antloxldants, nonsteroldal ant1Inflammatory agents, antibiotics, Immunomodul tors, antiallerglcs and compounds which make up basement membranes, such as fibronectln and amlnln. Further agents include "biological response modifiers" (I.e. agents whose mechanisms of action Involve the individual's own biological response); see, Oldha , Biological Response Modifiers. Chapter 1, Torrence (Ed.) Academic Press, 1985, Incorporated herein by reference. According to the present invention, the liposomes containing the therapeutic agent or drug for the treatment of wounds are applied topically to the eye. As will be understood by those skilled in the art, the administration, sequence of administration when more than one therapeutic agent 1s used, and the concentrations of the therapeutic agents 1n the liposomes of the present invention depends on numerous factors. These factors can Include: the specific therapeutic agent or agents to be used, the nature of the wound, and various clinical factors, Including the extent and type of wound being treated, the medical history of the patient and symptoms associated with the wound, such as Inf ammation or edema, etc. Selection of the specific therapeutic agent 1n the liposomes, their concentration and sequence of delivery to the eye will be made by the skilled clinician guided by the foregoing description. In addition, because the Uposome-type formulation bioadsorbs and/or bloadheres specifically to the wound site, the amount and frequency of therapeutic agent normally administered as the free agent can be reduced. Typically, the amount of therapeutic agent administered in liposomes according to the present invention can be reduced by up to about 2/3 of the dose administered by other methods such as in aqueous drops, ointments or suspensions. The following examples are directed to the binding of liposomes and liposomes comprising therapeutic agents to corneal wounds. The examples are illustrative of the preferential binding of the liposomes to wound sites, but they are 1n no way limiting. EXMPli 1 ng edi nt Concentration Phosphatldylchollne 2.28 mg/ml Phosphatldylglycerol 1.35 mg/ml Cholesterol 0.46 mg/ml Dioctadecyl-tetramethyl-lndocarbocyanine (dye) 0.11 mg/ml Preparation Aliquots of phospholiplds and cholesterol in chloroform (22.8 mg phosphatldylchollne, 13.5 mg phosphatldylglycerol, 4.6 mg cholesterol) were added to a glass container. The fluorescent dye (0.11 mg) was then added as an aliquot in methanol. The solvent was then evaporated under nitrogen. The lipld fluorescent dye film was then rehydrated with Dulbecco's phosphate buffered sa ine containing 0.9 M calcium and 0.49 πrtM magnesium (10 ml). The pH was adjusted to 7.4 with dilute sodium hydroxide. This rehydratlon resulted 1n the spontaneous formation of utilamellar iposomes. Freshly dissected rabbit corneas were washed in BSS Plus R (Alcon Laboratories, Inc.). The corneas were placed in plastic culture dishes and either cut or scraped medially through the epithelial surface with a scalpel blade. Immediately following the cutting or scraping, 1,1 * 1 of the fluorescent- labeled Uposome formulation was applied to the corneal surface. After 0.5 min the corneas were washed 3 times with 5 ml BSS Plus R at 37°C 1n a CO2 Incubator using a rotary shaker. Each cornea was examined using fluorescence microscopy by viewing the corneas in a chamber created by two glass coversHps fitted in a machined plastic flow cell. The flow cell consists of a plastic block (11.5 X 11.5 cm X 1.2 cm) 1n which four 1 cm holes were bored for viewing under phase. A recessed area -2.5 X 2.5 cm and -2.5 mm deep was machined over and around each hole and an 18 X 18 mm coverslip adhered with silicon grease to the block within the recessed area. A thin shallow Inset ledge -0.3 mm deep was machined within the 2.5 X 2.5 cm recessed area for accommodating a top coverslip of 25 X 25 mm adhered to the ledge using si icon grease. On two opposite edges of the block, four channels, 2 per side, were machined allowing for placement of syringe needles, each tip just entering one of each of the 2.5 cm X 2.5 cm X 2.5 mm recessed chambers. Each syringe needle was held in place with a screw-washer fixed in the block. Perfusate enters the chambers through the syringe needles. In the center of the block two long channels (-8.5 cm X 1 cm and 0.5 cm deep) in fluid connection with the chambers were machined perpendicular to the channels holding the syringe needles for collection of perfusate. During examination the flow cell 1s fixed to the stage of the microscope with screws. Each wounded cornea was in turn placed on an 18 X 18 mm coverslip in one chamber of the flow cell and covered with a 25 X 25 imπ top coverslip. During examination the corneas were constantly perfused with BSS Plus R and the temperature was maintained at 37°C, Video images of the fluorescent-labeled iposomes present on the corneal surface were recorded at a magni ication of 1134X. The Images appeared black and white with the white regions representing those areas In which the labeled liposomes were located. The black areas in the Images represented areas where no labeled liposomes were present. On observing these Images, it was found that the labeled liposomes were located substantially in only the cut or scraped sites. Little to virtually no labeled iposomes adhered to the uncut or unscraped areas of any of the corneas. Nor were any labeled liposomes observed adhering to the cornea when unwounded or "normal"' corneas were treated with labeled liposomes and washed according to the above described procedure. The images were then quantitated for the area of white region (I.e. highlighted pixels) which represented the bloadsorbed liposomes. These results were expressed as cornea surface area (jm 2 ) as shown in the Figure with an average +/- standard error of the mean for five different images. In the Figure it can be seen that substantially no labeled iposomes adhered to the uncut or unscraped normal cornea, whereas the labeled liposomes adhered to about 50 Jim 2 in the cut corneas' and 60 μn~ 1n the scraped corneas. Therefore, iposomes used according to the methods of the present Invention can be used to deliver wound treating agents to wounds because they preferentially adhere to wound sites and not to unwounded sites. Example 2 The following composition can be applied topically to the eye to promote reepitheliatlon of a wounded cornea. Ingredient Concentration Phosphatldylchollne 23 mg/ml Phosphatldylglycerol 14 mg/ml Cholesterol 5 mg/ml Epidermal Growth Factor (EGF) 10 ug/ Preparation For the preparation of 10 ml of composition, the phospholiplds and cholesterol are weighed as dry lyophilized powders 1n a glass container (230 mg phosphatldylchollne, 140 mg phosphatldylglycerol, 50 mg cholesterol) followed by the addition of 1 ml t-butanol. The mixture 1s then solub111zed by gentle mixing 1n a 40°C water bath, sterile filtered and then freeze dried to remove the t-butanol. The lyophilized llpld powder is then rehydrated with 100 jig of sterile EGF dissolved in Dulbecco's phosphate buffered saline containing 0.9 m calcium and 0.49 mM magnesium (10 ml). This rehydratlon will result in the spontaneous formation of multilamellar liposomes containing EGF. To maximize entrapment of EGF in the liposomes, the formulation 1s freeze dried a second time then rehydrated with 10ml sterile delonized water before use. Example 3 The following is an example of a Hposome/polymer formulation to which therapeutic agents can be added for the treatment of comeal wounds. inoedient Concentration l-palmltoyl-2 oleoyl- phosphat1dylchol1ne 2.3 mg/ml formulation l-palm1toyl-2-oleoyl- phosphatldylglycerol 1.35 mg/ml formulation cholesterol 0.46 mg/ml formulation gelatin 25 mg/ml formulation Preparation The phospholiplds and cholesterol are weighed as dry lyophilized powders in a glass container followed by the addition of tertiary-butanol (about 4 ml/4 mg of total lipld). Llpophilic drugs and therapeutic agents which are soluble 1n tertiary- butanol can be added at this point at concentrations effective for the treatment of wounds. The mixture is then completely solubllized by gentle mixing 1n a 40°C water bath and then freeze dried to remove the tertiary-butanol. The lyophilized powder is rehydrated with Dulbecco's phosphate buffered saline containing 0.9 mM calcium and 0.49 mH magnesium. The pH is adjusted to 7.4 with di ute sodium hydroxide. Water soluble drugs and therapeutic agents, such as growth factors, can be added at thi point at concentrations effective for the treatment of wounds. Mul ilamellar liposomes containing drugs or therapeutic agents are formed by Incubation and mixing 1n a rotary water bath at 40°C. For maximum entrapment of water soluble drugs and therapeutic agents, the solution can be freeze dried again and dried again and rehydrated with water followed by further Incubation and mixing in a rotary water bath at 40°C. If smaller un1lamellar liposomes are desired the multil mellar liposomes can be passed through a M1crofluid1zer R (Microfluldlcs, Corp.) repeatedly until the desired proportion of smaller liposomes 1s obtained. As will be appreciated by those skilled in the art, some Uposome formulations, for example, some of those containing proteins, should not be passed through the m1crof1u1d1zer because of denaturation of the protein. As a last step, powdered gelatin (Swine, skin type 1, Sigma Chemical Co., St. Louis, HO.) 1s added at a concentration of 25 mg/ml formulation. The mixture 1s then gently heated and mixed at 40°C until the gelatin is solubllized.

Claims: W≤_Cliiffl: 1. A method for treating ophthalmic wounds with therapeutic agents for such treatment, which comprises: applying topically to the affected eye a composition comprising an effective amount of a therapeutic agent in liposomes which adsorb to the wounds. 2. The method of claim 1 wherein the liposomes comprise about 50-90 mol% synthetic or naturally occurring phospholiplds and about 10-50 mol cholesterol. 3. The method of claim 2 wherein the phosphol1p1d is selected from the group consisting of: phosphatldylchollne, phosphatldylglycerol, phosphat1dylethanolam1ne and phosphatidylserine with acyl chains of from 14 to 18 carbons, said chains being saturated or containing one unsaturated bond. 4. The method of claim 2 wherein the iposomes comprise about 50 mol% phosphatldylcho lne, about 30 mol% phosphatldylglycerol and about 20 mol% cholesterol. 5. The method of claim 4 wherein the liposomes are about 10 to 10,000 nm 1n diameter. 6 ~ . The method of claim 1 wherein the liposomes are about 10 to 10,000 nm in diameter. 7. The method of claim 6 wherein the liposomes are about 20 to 500 nm in diameter. 8. The method of claim 1 wherein the composition further comprises a polymer in an amount sufficient to provide the composition with a viscosity of between about 5 cps. and 1000 cps. 9. The method of claim 8 wherein in the composition the liposomes comprise about 50-90 molft synthetic or naturally occurring phospholiplds and about 10-50 moIX cholesterol and the polymer concentration 1s between about 0.25-3.0 wt.X. 10. The method of claim 8 wherein the polymer 1s selected IYUM the group consisting of polysaccharldes, gelatins, albumin, casein, chltosan, collagen, polylactlde, polylactideglycolide copolymer, polyorthoesters, polymethacr late, polyvlnylalcohol, polyacryHc acid, polyvinylpyrrol1done, polyhydroxyethyl- methacrylate, polyesteramides, polyethyleneglycol and polymeric amino adds. 11. The method of claim 10 wherein the polymer 1s a gelatin. 12. A method for treating an ocular wound resulting from surgery to promote the healing of the wound, which comprises: applying topically to the wound a pharmaceutical composition comprising an amount of a drug sufficient to promote healing of the wound, said drug being contained in liposomes adapted for selective binding to the wound by means of adsorption. 13. The method of claim 12 wherein the liposomes comprise about 50-90 mol% synthetic or naturally occurring phospholiplds and about 10-50 molX cholesterol. 14. The method of claim 13 wherein the phospholipld is selected from the group consisting of: phosphat1dyl hol1ne, phosphatidylglycero , phosphatldylethanolamine and phosphatidylserine with acyl chains of from 14 to 18 carbons, said chains being saturated or containing one unsaturated bond. 15. The method of claim 12 wherein the liposomes are about 10 to 10,000 nm in diameter. 16. The method of claim 12 wherein the liposomes are about 20 to 500 nm in diameter. 17. The method of claim 12 wherein the drug is EGF. 18. A method of selectively delivering a drug to an ophthalmic wound site, which comprises: applying topically to the eye an ophthalmic pharmaceutical composition comprising iposomes containing a drug in an amount sufficient to achieve the desired therapeutic result, said liposomes having an affinity for binding to the ophthalmic wound site by means of adsorption. 19. The method of claim 18 wherein the liposomes comprise about 50-90 mol% synthetic or naturally occurring phospholiplds and about 10-50 moIX cholesterol. 20. The method of claim 19 wherein the phospholipld is selected from the group consisting of: phosphatldyl ho lne, phosphatldylglycerol, phosphatldylethanolamine and phosphatidylserine with acyl chains of from 14 to 18 carbons, said chains being saturated or containing one unsaturated bond. 21. The method of claim 19 wherein the liposomes comprise about 50 molX phosphatldylchollne, about 30 molX phosphatldylglycerol and about 20 molK cholesterol. 22. The method of claim 21 wherein the liposomes are about 20 to 500 nm 1n diameter. 23. The method of claim 18 wherein the liposomes are about 10 to 10,000 nm in diameter. 24. The method of claim 23 wherein the liposomes are about 20 to 500 nm in diameter. 25. The method of claim 18 wherein the composition further comprises a polymer in an amount sufficient to provide the composition with a viscosity of between about 5 cps. and 1000 cps. 26. The method of claim 25 wherein in the composition the liposomes comprise about 50-90 molX synthetic or naturally occurring phospholiplds and about 10-50 molX cholesterol and the polymer concentration 1s between about 0.25-3.0 wt.%. 27. The method of claim 25 wherein the polymer Is selected from the group consisting of polysaccharides, gelatins, albumin, casein, chitosan, collagen, polylactlde, polylactideglycolide copolymer, polyorthoesters, polymethacrylate, polyvlnylalcohol, polyacrylic add, polyvinylpyrrolidone, polyhydroxyethyl- methacrylate, polyestera ldes, polyethyleneglycol and polymeric amlno adds. 28. The method of claim 27 wherein the polymer 1s a gelatin.

Inventors: CORDOVA DIANA MARIA, SHAW JACK MICHAEL, CORDOVA, DIANA, MARIA, SHAW, JACK, MICHAEL
Applicants: ALCON LAB INC, ALCON LABORATORIES, INC.
Application year: 1990
Main classification: A61K37
IPC codes: A61K9, A61K45, A61K47, A61P27