JPH04505319A - Use of liposomes as delivery of therapeutic agents for wounds, cuts and abrasions - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Use of liposomes as delivery of therapeutic agents for wounds, cuts and abrasions Background of the invention The present invention addresses injuries, trauma, surgery, or disease through the delivery of therapeutic and healing agents. methods for treating wounds, cuts and abrasions caused by Regarding. In particular, the present invention provides for corneal or ocular surface wounds with liposomes and liposome-polymer compositions. Relating to the delivery of therapeutic agents for wounds, cuts or abrasions.

Liposomes are sac- or sac-like bilayered structures that make up phospholipids. of liposomes Preparation and properties, drugs and proteins incorporated therein, and desired drug For an in-depth discussion of the delivery of , GregoriadisXLiposomeTechnolog7. Vo l I, If and Ill 5CRCPress Inc,, Bocx Rml on, Florida % 1984.

The use of liposomes as delivery vehicles for drugs or other therapeutic agents is Are known. Delivery of therapeutic agents to the eye - liposomes as vehicles It is known. For example, cyclospores contained in suitable excipients, including liposomes. Traumatic or surgical lens hypersensitivity occurring in the eye due to topical administration of phosphorus. R, K15vxn, March 1987 disclosing the treatment of inflammation or uveitis. See U.S. Pat. No. 4,649,047, issued on the 10th. FounHi No. 4, issued May 13, 1986, by N. et al.　588゜578 The issue contains a list of drugs that can be applied topically to treat eye diseases such as glaucoma. The preparation of bioactive agents that carry poid sacs is disclosed. 5kula other Ameri canJournal of Ophtba1molog7. Mo1. 10 3. No. 5. In May 1987, membrane ablation inhibited subsequent fibroblast proliferation. As a method to prevent this, 5-fluorouracil is injected subconjunctivally into liposomes. A delivery including delivery is disclosed. 5kDta and others have their delivery systems Characterized as an advantageous sustained-release delivery system, treatment with free drug Conventional methods require frequent subconjunctival injections.

The use of liposomes for certain types of targeted drug delivery in the eye has not been done before. Are listed. NoeLey et al., J. Immunol, Vol. I36. , No. 2 (January 15, 1986), sensitized corneal cells in vitro. infected Harpes simplex virus (HSV) (HupesSimp It has been disclosed to target drug-loaded liposomes to . Target special table Hei 4-505319 (3) incorporated into liposomes The component is linked to H8V glycoprotein D, which contains a covalently linked fatty acyl chain. It is an antiviral monoclonal antibody against. This “immunoliposome” is It was shown that it specifically binds to H3V infected rabbit corneal cells. Targeted drug delivery In another example of Bali, liposomes injected through a glass tube target cells in the retina. It was used for the delivery of medicines. 5tern other Inwes order ga order v e　Ophjhx1molog7　and　Visual　5science.

See Mol, 28, No. 5 (May 1987). By He5th et al. U.S. Pat. No. 4,755,388 (July 5, 1988), which describes Prevents scar formation by inhibiting cell shrinkage and cell growth in the attached tissue. of liposome encapsulating agents such as 5-fluoropyrimidines useful for Use is disclosed.

Heath targets ligands and specific cell surfaces, or describes various covalent modifications of liposomes using glue septa for binding to It is listed.

Preparation and preparation of bioadhesive liposomes for topical application of therapeutic agents to the ocular surface. and uses are also described. Positively charged liposomes and polymers are are known to form non-specific biological deposits on surface areas of FHtger Jo℃rntl of Phttmse7 and 5hiola outside of xld ularDeliver7 of Vitamin A UsingPosif ive17 Charged Liposomes, ^RVO^nnua1M e! jing Abstract l5sue, page 159, No. 22 (1 9117).

layer plane The only drawing shows the adsorption of the liposomes of the present invention to a corneal wound. compared to their relative non-adsorption to the cornea.

Summary of the invention The present invention aims to treat wounds by delivering liposomes containing therapeutic agents at the site of injury. Concerning methods of treatment.

Liposomes preferentially adsorb to the injured site and then attach to healthy tissue surrounding the site. Does not absorb.

In particular, the present invention provides liposomal treatment for ocular surface wounds such as the cornea, conjunctiva, and electromembrane. The present invention relates to the delivery of a therapeutic agent in a therapeutic agent. Liposomal carriers can cause damage to the ocular surface. Usually administered as a free drug because it bioadsorbs almost exclusively to the wound site The amount of therapeutic agent can be greatly reduced. As a result, according to the method of the present invention The therapeutic index or safety margin of drugs delivered by

Although topical application of drug-loaded liposomes to the eye is known, conventional delivery methods The method involves introducing therapeutic agents into and through the tissue barriers of the eye, such as the cornea, conjunctiva, and electromembrane. It is used as a means of improving cross-country transportation. Norle7 et al. (1986) and 5biolj et al. (1987). and bioadhesive liposomes are produced by covalent modification or bioadhesive modification of phospholipids. have been formulated either by insertion of a ligand molecule between the liposome bilayers. . The use of complex receptor-recognizing ligands, or positively charged moieties, It has been devised as a targeting agent or non-specific bioadhesive moiety for. but However, conventional technology has shown that liposomes have a highly specific ability to treat corneal wounds such as cuts and abrasions. Preferential biological adsorption with isomerism is not recognized. The present invention The surprising finding that somes attach at the site of injury but not to surrounding healthy tissue It is based on discovery. As used herein, the term "wound" refers to cuts, scratches, Scratches or tissue damage caused by injury, trauma, surgery, or disease. Including scratches. Therefore, therapeutic agents known to be effective in treating wounds are The invention uses non-toxic phospholipids/cholesterol liposomes to target damaged areas. Can be specifically delivered. In particular, liposomes containing therapeutic agents can be used to It is useful in treating corneal wounds.

Detailed description of preferred embodiments The liposomes that can be used in the present invention are suitable for use at sites of injury, particularly corneal injuries. A substance that is bioadsorbed to the site and exhibits no toxic, inflammatory or antihealing effects. It also includes liposomes. These liposomes contain lipoids and typically Typically 50-90 mol% of one or more synthetic or naturally occurring phospholipids and 10 to 50 mol% (mol%) of cholesterol. For the use of the present invention The phospholipids that can be used to formulate liposomes can be saturated or monophospholipids. any of the enes, typically having a diacyl chain of 14 to 18 carbons; lucorin, phosphatidylglycerin, phosphatidylethanolamine or Includes phosphatidylserine. Phosphorus that can be used in liposome formulations Other lipoids containing lipids include, but are not limited to, phosphatidylinos citol, sphingomyelin, phosphatidic acid, cerebroside, calcioli Includes pins and lysophospholipoids.

Liposomes include, but are not limited to, multilamellar (MLV), small unilamellar (SUV) , large unilamellar (LUV), multilamellar (SPLV) and reversed phase evaporation (REV) liposomes can be produced by a number of methods including processing to produce. G, G +ego+1adis, Lip++someTechnolog7:Pre See paration oj liposomes, Vol. Also, As mentioned above, liposomes useful in the present invention Special Table 1,4-505319 (4) Papahxdjopo, as embodied by reference to the extent that it teaches the manufacture of See U.S. Pat. No. 4,235,871 to Ulos et al.

The invention also provides for relatively small diameters, i.e. 10 to 500 nanometres (nm), preferably 1100 nm in diameter, liposomes with a diameter of 500 nm to 1 nm 0.000 nm, maximum damage site compared to larger MLV-type liposomes. It was found that it shows good coverage.

Preferred liposome compositions of the invention include phosphatidylcholine (50 mol%); Negatively charged phosphatidylglycerol (30 mol%); both phospholipids are in the 1-position contains palmitic acid at the 2-position and an oleic acid acyl group at the 2-position; and and cholesterol (20 mol%). Preferred for maximum damage coverage The liposomes have a diameter of 20 to 200 nm, preferably less than 1100 nm. be. For the production of liposomes smaller than 1100n, MLV liposomes are Fluidizer R (Mic+olluidixer) (Micro-11ui dics, Corp, Newton, Mass) and their size is typically changed by less than about 30%.

When composed of phosphatidylcholine with a saturated acyl chain, the liposome of the present invention , an aqueous liquid at a temperature between about 35 and 42°C, preferably close to body temperature of 37°C. It has a crystal phase transition point. Composed of phosphatidylcholine with monoene-containing acyl group The liposomes of the present invention exhibit a phase transition below 0°C.

The invention is limited only to phospholipid or phospholipid/cholesterol containing liposomes. Phospholipid/cholesterol liposomes containing polymers are also include. These liposome-polymer combinations create formulations with high viscosity. provide. Surrounding liposomes gives high viscosity and is compatible with ocular tissues Any polymer can be used. The polymer(s) have approximately 5 cps and 1 00 cps, preferably between about 15 cps and 200 cps. added to provide a composition. Polymers that can be used in the method of the invention Examples include polysaccharides (e.g. alginate, starch, hydroxypropyl modified cellulose such as methylcellulose and hydroxyethylcellulose), Natural polymers such as latin, albumin, casein, chitosan and collagen can be lost. Furthermore, synthetic polymers that can be used include polylactide Lycolide copolymer, polylactide, polyhydroxyethyl methacrylate, poly Esteramide, polyorthoester, polymethacrylate, polyvinyl alcohol polyvinylpyrrolidone, polyacrylic acid, polyethylene glycol and Examples include synthetic amino acids. Liposomes in their viscous matrix are , retains the formula longer in the eye and therefore for a longer period of time at the injured site. Biosorption of liposomes to is imparted. Hundreds of polymers in liposome formulations The fraction is determined by the polymer itself, but generally the concentration of the polymer is about 0°25 -3.0% by weight.

In liposomes or liposomes/polymers that bioadsorb at the site of damage, Therapeutic agents that can be delivered to a wound site include injury, trauma, surgery, and/or disease. Includes all therapeutic agents useful in the treatment of wounds, cuts and abrasions caused by disease. So Typical therapeutic agents include, but are not limited to, growth factors and steroids. anti-oxidant, non-steroidal anti-inflammatory agent, antibiotic, immunomodulator, anti-allergy agents and basement membranes (bxsements) such as fibronectin and laminin. embranes). Other drugs are ``response modifiers'' (i.e., mechanisms of action with their own biological responses) (drugs that have symptoms). Old dham, embodied by the quotation.

Biologicxl Re5ponse Modifiers 5Chxpj er　I　.

Torrence (Ed, )^csdemtc Press 1985 ,checking.

According to the present invention, liposomes containing therapeutic agents or drugs for the treatment of wounds can be applied to the eyes. Applied locally. As will be understood by those skilled in the art, administration, one or more therapeutic agents may be When used, the concentration of therapeutic agent in the liposomes of the present invention is , depends on a number of factors. These factors include specific therapeutic agents or or drugs used, the nature of the wound, and the extent and type of wound being treated, patient medical history. and various clinical factors, including wound-related symptoms such as inflammation or edema. can include. Selection of specific therapeutic agents in liposomes, their concentration and Continuous delivery to the eyes and eyes can be performed by a skilled clinician guided by the above description. Probably. Furthermore, liposomal formulations can be bioadsorbed and/or or biofouling, the amount and quantity of therapeutic agents normally administered as free agents. and frequency decreases. Typically, the therapeutic agent applied to liposomes according to the present invention is The amount is about 2/2 of the dose administered by other methods such as aqueous drops, ointments or suspensions. It can be reduced to 3.

The following example demonstrates how a liposome and a liposome containing a therapeutic agent bind to a corneal wound. Regarding things. Although the examples illustrate preferential binding of liposomes to damaged sites, These are by no means limited.

Special Table Sen4-505319 (5) Ingredient concentration Phosphatidylcholine 2.28mg/ml Phosphatidylglycerin 1.3 5mg/ml cholesterol 0.46mg/ml Dioctadecyl-tetramethyl- Indocarbocyanine (dye) 0.11mg/ml preparation each of the phospholipids in chloroform, and cholesterol (phosphatidyl copolymer). Phosphorus 22.8mg, phosphatidylglycerin 13.5mg, cholesterol 4 ．． 6 mg) was added to the glass container. Fluorescent dye (0°11 mg) was then added to methane. It was added as an ingredient in the solution.

The solvent was then evaporated under nitrogen. Then, a 0.9 m thick lipoid fluorescent dye film was applied. Dulbecco's Phosphate with M Calcium and 0.49mM Magnesium Rehydrated with salt buffered saline (10ml). Adjust pH to 7. with diluted sodium hydroxide. Adjusted to 4. This hydration resulted in the spontaneous generation of multilamellar liposomes.

A newly dissected rabbit cornea was placed on BSS PlusR (Alcon Liboral). ories, Inc.). Place the cornea in a plastic culture dish, and , either by cutting through the epithelial surface with a scalpel blade or by simply scraping. went. Immediately after cutting or scraping, apply 1 μl of fluorescently labeled liposome formulation to the corneal surface. applied to the surface. After 5 minutes at 0°, each membrane was incubated at 37°C for 5 mR in a CO incubator. Washed three times with BSS Plus No. 1 using a rotary shaker.

on two glass cover pieces attached to a mechanized plastic flow cell. Observing the cornea using a fluorescence microscope in the chamber created by this method. Each cornea was examined by The flow cell has four 1 cm holes inside to view the lower phase. A plastic block (11.5x11.5cm 1.2 cm). A recessed area of 2.5X2.5cm and a depth of 2.5mm is Mechanically create and fit a 18X18mm cover piece over and around each hole into the recess. Within the area, the block was glued with silicone grease. silicone green depth Q to accommodate a 25X25mm cover piece glued to the shelf using a A 3 mm thin and shallow inserter was mechanically made into a 2.5×2.5 cm recess area.

Machine four grooves on two opposite ends of the block, two on one end. 2.5 cm x 2.5 crn so that the needle can be placed. It was made to fit exactly into each one of the 2.5 mm x 2.5 mm recess channels. Each note The firing needle was held in place by a screw washer fixed to the block. Perfusion Note Enter the chamber through the firing pin.

In the center of the block, there are two long grooves (8.5 cm am and depth 0.5 cm) perpendicular to the groove that holds the syringe needle to collect the perfusion. made mechanically. During the test, the flow cell was fixed to the microscope stage with screws. .

Place each injured cornea in an 18x18mm cup in one chamber of the flow cell. Placed in turn on top of the bar pieces and covered with a 25X25 mm top cover piece.

During the test, the cornea was constantly sprinkled with BSS PlusR, and the temperature was kept at 37°C. maintained. A television image of fluorescently labeled liposomes on the corneal surface is magnified 1134 times. recorded in magnification. The image appears in black and white, with the white area representing the area where the labeled liposomes are located. represents the area. The black area in the image represented the area where labeled liposomes were not present. child By observing these images, the labeled liposomes are substantially cut or scraped. It was found that it was located only in the same area. areas of the cornea that are not cut or scraped Almost no labeled liposomes were attached to the sample.

An intact or "normal" cornea is treated with labeled liposomes and the above method All labeled liposomes were observed to adhere to the cornea when washed according to I couldn't.

The image then shows white areas (i.e. highlights) representing bioadsorbed liposomes. Highlighted pixels) were quantified. child These results are averaged over 5 different images, with mean +/- standard error. As shown, the corneal surface area (μm2) is shown.

In the figure, labeled liposomes are present in a normal uncut or abraded cornea. Labeled liposomes do not adhere to the cut cornea, but the size of the labeled liposomes is approximately 50 μm2, and It can be seen that 60 μm2 of the substance was attached to the rubbed cornea.

The liposomes used according to the invention therefore preferentially target the site of injury. to deliver wound treatment agents to the wound because it adheres and does not adhere to non-wounded areas. It can be used for.

Example 2 The compositions described below may be applied topically to the eye to enhance epithelial regeneration of damaged corneas. I can do it.

Ingredient concentration Phosphatidylcholine 23mg/ml Phosphatidylglycerin 14mg/ ml cholesterol 5mg/ml Epidermal growth factor (EGF) 10 u g/m 1 preparation For the preparation of 10 ml of composition, phospholipids and cholesterol are frozen in a glass container. Weighed 230 mg of phosphatidylcholine as a vacuum-dried powder. Luglycerin 140mg, Cholesterol 50mg) Special table Sen4-505319 (6) Then add 1 ml of t-butanol. The mixture was then placed in a water bath at 40°C. Dissolve by gentle mixing in a water bath, sterile filter, and then lyophilize. Dry to remove t-butanol. The lyophilized lipoid powder was then 0.49 m Dulbecco containing 0.49mM of calcium and 0.49mM of magnesium. 100 μg of sterile EGF dissolved in phosphate buffered saline (10 ml) Rehydrate with water. This hydration results in the spontaneous generation of multilamellar liposomes containing EGF. Ru. To maximize the entrapment of EGF in liposomes, the formulation is lyophilized twice. and then rehydrated with 10 ml of sterile deionized water before use.

Example 3 The following are liposomes/polymers that can be loaded with therapeutic agents for the treatment of corneal wounds. This is an example of a prescription.

oleoyl-phosphati Zircoline 2.3mg/ml prescription 1-valmitoyl-2- oleoyl-phosphati Dilglycerin 1.35mg/ml Prescription cholesterol 0.46mg/ml Prescription gelatin 25mg/ml prescription preparation Freeze phospholipids and cholesterol in glass containers as vacuum-dried powders in glass containers and then add tert-butanol (approximately 4 ml/4 mg total lipoid) . At this point lipophilic drugs and therapeutic agents dissolved in tertiary-butanol are applied to the wound. It can be added at an effective concentration for. The mixture was then heated to 40°C. Thoroughly dissolve by gentle mixing in a terbath and then lyophilize. Remove tertiary butanol.

The lipophilic powder contains 0.9mM calcium and 0.49mM magnesium. Rehydrate with Dulbecco's phosphate buffered saline. Dilute the pH Adjust to 7.4 with sodium hydroxide. Water-soluble drugs and therapeutic agents such as growth factors can be added at this point in an effective concentration for wound treatment. Drugs Multilamellar liposomes containing or therapeutic agents were incubated at 40°C in a rotating water bath. Formed by culturing and mixing. Maximizes capture of water-soluble drugs and therapeutic agents The solution was lyophilized again, dried again, rehydrated with water, and then spun Further incubation and mixing was carried out at 40°C in a water bath. If smaller than If unilamellar liposomes are desired, multilamellar liposomes can be combined with the desired proportion of smaller Repeat Mic-o-Fluidizer R (Mic+o-Mount) until liposomes are obtained. 1 dics, Corp.). As recognized by those skilled in the art, some Some liposomal formulations, e.g., those containing proteins, are should not be passed through a microfluidizer. As the final step, powder Polymerized gelatin (Swine, 5kin!7pe 1, Sigma Chemi cxlCo, St, Louis, MO,) was added to give a concentration of 25 mg/ml. I made it a concentrated prescription. The mixture is then heated gently and gelatinized at 40°C. Mix until Chin is dissolved.

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Claims (28)

[Claims] 1. Affected eyes with a composition containing an effective amount of a therapeutic agent in liposomes that adsorb to the wound A method of treating an eye wound with a therapeutic agent for treatment comprising topical application to the eye. 2. Ribosomes contain approximately 50-90 mol% of synthetic or naturally occurring phospholipids and The method of claim 1, comprising about 10 to 50 mole percent cholesterol. 3. The phospholipid is a 14- to 18-carbon acyl group containing saturated or one unsaturated bond. Phosphatidylcholine, phosphatidylglycerin, phosphatidyl ether with chain Claims selected from the group consisting of tanolamine and phosphatidylserine The method described in box 1. 4. The ribosome contains approximately 50 mol% phosphatidylcholine, approximately 30 mol% phosphatidylcholine, and approximately 30 mol% phosphatidylcholine. A claim consisting of fatidylglycerin and about 20 mole percent cholesterol. Method of Section 2. 5. Claim 4: The ribosome has a diameter of about 10 to 10,000 nm. the method of. 6. Claim 1, wherein the ribosome has a diameter of about 10 to 10,000 nm. the method of. 7. The method of claim 6, wherein the ribosomes have a diameter of about 20 to 500 nm. . 8. Sufficient that the composition further has a viscosity between about 5 cps and 1000 cps. 2. The method of claim 1, comprising: an amount of polymer. 9. In the composition, about 50 to 90 mole percent of ribosomes are synthetically or naturally produced. phospholipids and about 10-50 mol% cholesterol, and the concentration of the polymer is 9. The method of claim 8, between about 0.25 and 3.0% by weight. 10. Polymers include polysaccharides, gelatin, albumin, casein, chitosan, and colloid. Gen, polylactide, polylactide glycolide copolymer, polyorthoester, Polymethacrylate, polyvinyl alcohol, polyacrylic acid, polyvinylpyro Lydone, polyhydroxyethyl methacrylate, polyesteramide, polyethylene Claim 8 selected from the group consisting of lene glycol and polymerizable amino acids. Section method. 11. 11. The method of claim 10, wherein the polymer is gelatin. 12. Contains a sufficient amount of drug to promote wound healing, and the drug is absorbed into the wound by adsorption. Pharmaceutical compositions such as those contained in ribosomes adapted to selectively bind to for the treatment of surgically caused eye wounds by applying it topically to the wound. , a method to enhance wound healing. 13. Ribosomes contain about 50-90 mol% of synthetic or naturally occurring phospholipids and and about 10 to 50 mole percent cholesterol. 14. Phospholipids are saturated or 14- to 18-carbon acyl groups containing one unsaturated bond. Phosphatidylcholine, phosphatidylglycerin, phosphatidyl with chain of the claims selected from the group consisting of ethanolamine and phosphatidylserine. The method of scope item 13. 15. Claim 1, wherein the ribosome has a diameter of about 10 to 10,000 nm. Method 2. 16. Claim 12, wherein the ribosome has a diameter of about 20 to 500 nm. Method. 17. 13. The method of claim 12, wherein the drug is EGF. 18. Contains a sufficient amount of drug to achieve the desired therapeutic effect and absorbs into the eye through adsorption. An ophthalmic composition containing ribosomes that has an affinity for binding to the site of injury is administered to the eye. A method of selectively delivering drugs to the site of eye injury, comprising topical application. 19. Ribosomes contain about 50-90 mol% of synthetic or naturally occurring phospholipids and and about 10 to 50 mole percent cholesterol. 20. Phospholipids are saturated or 14- to 18-carbon acyl groups containing one unsaturated bond. Phosphatidylcholine, phosphatidylglycerin, phosphatidyl with chain of the claims selected from the group consisting of ethanolamine and phosphatidylserine. Method of scope item 19. 21. The ribosome contains approximately 50 mol% phosphatidylcholine and approximately 30 mol% phosphatidylcholine. A claim consisting of sphatidylglycerin and about 20 mole % cholesterol. The method of paragraph 19. 22. Claim 21, wherein the ribosome has a diameter of about 20 to 500 nm. Method. 23. Claim 1, wherein the ribosome has a diameter of about 10 to 10,000 nm. Method of Section 8. 24. Claim 23, wherein the ribosome has a diameter of about 20 to 500 nm. Method. 25. The topical ophthalmic composition of claim 1 which is selected. 26. In the composition, about 50 to 90 mole percent of ribosomes are synthetic or naturally occurring. Contains about 10-50 mol% of phospholipids and cholesterol, and the concentration of the polymer 26. The method of claim 25, wherein the amount is between about 0.25 and 3.0% by weight. 27. Polymers include polysaccharides, gelatin, albumin, casein, chitosan, and colloid. Gen, polylactide, polylactide glycolide copolymer, polyorthoester, Polymethacrylate, polyvinyl alcohol, polyacrylic acid, polyvinylpyro Lydone, polyhydroxyethyl methacrylate, polyesteramide, polyethylene Claim 2 selected from the group consisting of lene glycol and polymerizable amino acids. Method in Section 5. 28. 28. The method of claim 27, wherein the polymer is gelatin.