JPS61501686A - Liposome↓-gel composition - 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] Liposome-gel composition 1. Field of invention The present invention provides biologically active agents that provide sustained release of biologically active agents in biological systems. Compositions and methods for maintaining and immobilizing reservoirs of sexual agents are described.

According to the present invention, the biologically active agent is contained in liposomes sequestered in a gel matrix. is captured inside. When used in biological systems, encapsulated in a gel matrix Chained liposomes provide extended release of agents entrapped in the liposomes. Furthermore, the gel matrix also provides immobilization of the liposomes.

According to one embodiment of the invention, the liposome-gel composition of the invention In order to provide an extended release of the captured biologically active agent to the organism, Can be transplanted in vivo. When administered in vivo, the gel matrix Rapid cleansing of liposomes without inhibiting the release of agents entrapped in the somes Gives some protection. According to another embodiment of the invention, the liposome-gel composition is used as a support or overlay for cells grown in a culture medium, and This provides extended release of the captured biologically active agent into the culture medium.

2. Background of the invention 2.1 Liposomes Liposomes are capable of entrapping a variety of agents and are capable of entrapping these agents in vivo. is a lipid endoplasmic reticulum that can be used for delivery to cells and tissues. large number of liposomes For example, small lamellar vesicles (SUV), large lamellar vesicles (L UV), oligolamellar vesicles (REV) prepared by reverse phase evaporation or composed of one or more lipids, such as thilamellar vesicles (MLVs) Ru. Liposomes, edited by Ostro, Marcel Detska Incorporated, Diemer and Astor, 1983, “Lipo” in New York, pp. 27-51. "Some Preparation: Methods and Mechanisms" [Deamer and Uster, 19B3. “” LipO3Ome Preparation: Method and “HeChaniSmS”, 1983. in LipO3OmeS, 0s tro, ed,, Marcel Dekker, Inc,, New Yo rk, pp. 27-51].

There have been numerous reports regarding the potential use of liposomes in drug delivery systems. example To name a few, U.S. Patent No. 3.993.754 and U.S. Patent No. 4,145,410. There is a discussion in . In liposome delivery systems, the drug is delivered to the patient being treated. entrapped in liposomes to be administered. U.S. Patent No. 4.224.179 and See US Pat. No. 4,235,871.

Aqueous suspensions of liposomes can be administered in any desired manner (e.g., intravenously, intramuscularly). intraperitoneally, etc.). However, after these inoculations , many of the liposomes are dispersed from the position of inoculation, and polymorphonuclear and mononuclear leukemia degenerated by phagocytic cells such as globules and macrophages, or taken into cells (Poste, 1983, Paiol Cell, 47: 1 9-38 [Po5te, 1983゜Biol, Cel! , 47: 19- 38]). Therefore, the release of entrapment agents from liposomes is dependent on inoculation and degeneration. or limited to a period of time between purification of liposomes from body fluids.

The sustained drug release properties are described in Sears [5earS] U.S. Patent No. 4.298. ．． Other types of lipid microvesicles (microreservoirs) described by No. 594 Attributable to drug microcarriers 2.2 Polymer matrix and gel polymer matrix Trix and gels localize drug delivery from the site of administration in vivo used to slow down or delay dispersion. Harris et al., 1980 , Jay Faram Sosai, 69: 1271-1273 [Harri s et al.

1980, J.P.harm, Sci., 69: 1271-127 [3] used a cross-linked starch gel for localized delivery of prostaglandin E2. there was. Cotes et al., 1980 [Cotes et Rainbow, 1980] subcutaneously into animals in an attempt to prolong the period of elevated plasma hormone concentrations. Incorporating human growth hormone into the injected 16% partially hydrolyzed gelatin solution (JE 303-312]). Sarah kmjii close, -E remoter, 19 In 1983 [t4orimoto et Dan,, 1983], various peptides were When incorporated into a polyacrylic acid aqueous gel matrix containing long-chain fatty acids, 7 rams that demonstrated enhanced absorption of insulin Tan: 149-157 [1nternat1, J, Pharm, 14: 149 - 157 pieces).

Several other polymerizable compounds have been utilized to provide sustained release of drug delivery systems. There are two types of silicone elastomers: solid phase elastomers. Matrix type and drug storage body with uniformly dispersed powdered drug in the stomer membrane type in which the membrane is surrounded by a layer of silicone elastomer (Wadsworth and Latts). Tonasriya, 1981.

JE Faramacol Men's 6:313-320 [Wadsworth and Ratnasooriya, 1981. J, Pharmacol , Methods 6: 313-320]: Chiman et al., 1982 Year, Fertil and Sterill 38: 475-481 [Cheesm an et al.

1982, Fertil, and 5ter1.38: 475-481] See also ); progesterone-saturated polymethacrylate or cylindrical Rustic [5ilastic, trade name] Polymer (Ainz Work and Walli) Neds, 1982, J Am Soci 54: 1120-1127 [A invorth and Wolynetz, 1982, J. Am, Sc. i, 54: 1120-1127]): 6 months or 1 year of levonol A copolymer of lactic and glycolic acids that provides controlled release of gestrel (Pitt et al. , 1981, Natol Instrumental Drug Abresse Monograph 28:232-253] [Pitt et al., 1981. Na tl, In5t, Drug Abuse Res, Monogr, Se r, 28: 232-25]: Weiss et al., 1980, J.F. centre? Ramacol 32:399-403 [Wise et Niji, 1980. J. , Pharm, Pharmacol, 32; 399-403]): Biochemistry fibrin vehicle (Brown et al. [Brow et al. et Niji, Ko, in U.S. Patent No. 4.393.041): Anti-inflammatory and painless Gel compositions for sensitization (Noda et al., U.S. Pat. No. 4,393. No. 076) and wound protection gel compositions (Mason et al. [HaSOn et Niji], No. 4,393,048).

5th day of stopping and serving By sequestering a reservoir of biologically active agents in a gel matrix, the present invention The present invention describes compositions and methods for maintaining the reservoir. More specifically, raw Liposomes containing a biologically active agent can be introduced in vivo [in ViVO] or in vitro. Sequestered in a gel matrix that is administered in vitro. Game The matrix is made by dispersing liposomes in vivo or in vitro. (1) Body fluids that interact with the liposome bilayer or diffusion of the culture medium into the gel; (2) lipolysis for release of the entrapment agent; (3) the ability of the released agent to pass through the gel into the surrounding environment. Suppresses dispersion without inhibiting it.

Direct incorporation of biologically active agents into the gel matrix can be sustained to some extent. Entrapment of biologically active agents into liposomes can provide release or entrapment of biologically active agents into liposomes. may be tailored or modified to further delay leakage of entrapped agents. Therefore, it is possible to provide a more prolonged release of the active substance. however , the liposome itself does not degenerate or be purified when administered in vivo. Prolonged release of liposome-entrapped agents in vivo to was difficult to achieve. The present invention is based on the preparation of liposomes into gel matrices. Encapsulation of the product slowly removes the contents of the liposomes, as detailed herein. protects liposomes from purification without compromising their ability to release It is based on the discovery that

4. Detailed description of the invention In one embodiment of the invention, a suspension of liposomes entrapped with a biologically active agent The liquid is mixed with a suspension of gel material. The resulting mixture is administered at the site of administration. The preparation may be administered in vivo to form a gel, or the preparation may be formed into a gel prior to administration. It is also possible to do so. Both methods of administration require gel matrix at the site of injection. sequestration of liposomes in a liquid, resistance of liposomes to purification or degradation; and the liposome-entrapped agent at the site of administration over a period of time. The result is liberation.

In other embodiments of the invention, the liposome-gel preparation is grown into a culture medium. in cell or tissue culture systems to provide extended release of biologically active agents. can be used. Liposome-gel preparations serve as supports for cell adhesion and growth. Alternatively, the liposome-gel preparation may be suitable as a top layer for cell doubling. can be used.

The rate of release of entrapped biologically active agents depends on the type of liposome used and the Depends on the composition of the posome membrane. In fact, a population of different liposomes can be sequestered in the box.

Any biologically active agent that can be entrapped in liposomes can be used in accordance with the present invention. obtain. Examples of these are listed below. In fact, the same or different liposomes The two or more biologically active agents captured in the population can be It can be sequestered in a gel matrix for further use. After all, one biology a biologically active agent is entrapped in the liposome, and the same or a different biologically active agent may be included in the gel matrix. This liposome-gel preparation was administered If the biologically active agent contained in the gel matrix is rapidly released, On the other hand, biologically active agents entrapped in sequestered liposomes are slowly released. It will be done. Therefore, one biologically active agent can be found in sequestered liposomes and gelatin. When trapped in both the agent and the provides both sustained release of the substance, thereby eliminating the need to administer maintenance doses. It becomes. Alternatively, one biologically active agent can be trapped in sequestered liposomes. and other biologically active agents are entrapped in the gel matrix. Competitive therapy with any mixture of biologically active agents is possible. The following segment is , liposomes, gels, and biologically active agents that may be used in the practice of the present invention. This is an example of a type.

4.1 Preparation of liposomes The liposomes used in the present invention include, but are not limited to, M LV; Papahajopoulos and Miller [PapahajOpO1jlO3and 14i11er] (1967, Biochem Iofiz Acta” = 624-638 [1967, Biochem, Bio phys, Acta, 135: 624-638]): Papahajopuro REVs discussed in U.S. Pat. No. 4,235,871 by Soz; 1980 by Ka [5zoka] and Papahadjopoulos, 7 responses by The original method of Bangham et al. 1965, JE Mol Biol 13: 238-252 [1965, J, Mo1. Biol, shellfish: 238-252]); and stable protein Luli lamella vesicle [5table pluri lamel far yes icle] class (hereinafter referred to as 5PLV class) was issued on March 24, 1983. U.S. Patent Application Serial Number by Lenk et al. 476, 496@; and monophasic vesicles [monophas ic vesicles (hereinafter referred to as MPVs) in August 1983. U.S. patent of Fontaine et al., filed on the 8th Can be prepared by several methods, including those discussed in Application Serial No. 521,176. can be manufactured. The procedure for the preparation of 5PLVs and MPVs is described below.

5PLVs are prepared as follows. That is, first, there are no amphipathic lipids. The lipid mixture is then dissolved in an organic solvent. Although many organic solvents are suitable, Diethyl ether fluorinated hydrocarbons and mixtures of fluorinated hydrocarbons and ethers are preferred. Delicious. To this solution is added the aqueous phase and the active ingredient to be captured.

This biphasic mixture emulsifies the aqueous substance in the solvent and evaporates the solvent. It is converted into 5PLVs. Evaporation is an optional evaporation technique during or after sonication. methods, e.g. by passing vapors of inert gas through the mixture, by heating, Alternatively, the evaporation may be carried out by evaporation under reduced pressure. The volume of solvent used is Sufficiently exceeds the aqueous capacity so that the aqueous substance can be completely emulsified in the product It has to be something.

In practice, a minimum of about 3 volumes of solvent is used for about 1 volume of aqueous phase. fruit In this case, the ratio of solvent to aqueous phase is 1 volume of aqueous phase to 100 volumes of solvent or so. It can be changed to even more than that. The amount of lipid that coats the emulsion droplets is It must be sufficient to exceed the amount required (40 mg of lipid per volume of aqueous phase). Must be. This upper limit is limited only by cost-effective practicality, but 5PLVs can be prepared with 15 C] of lipid per 1 d of aqueous phase.

Many amphipathic lipids can be constituents of 5PLVs. Suitable hydrophilic groups are also Of course, these include, but are not limited to, phosphoric acid, carboxyl include. Suitable hydrophobic groups include, but are not limited to, saturated and and unsaturated aliphatic hydrocarbon groups and at least one aromatic and/or alicyclic group. aliphatic hydrocarbon groups substituted with Preferred amphiphilic compounds are It is a lipid and its chemical structure is closely related. Examples of this are, of course, these including, but not limited to, lecithin, phosphatidylethanolamine, solecithin, phosphotidylethanolamine, phosphatidylserine, phosph Atidylinositol, sphingomyelin, calciolipin, phosphatidyl Includes acids and cerebrosides. Suitable materials useful in the production of SPLVs Specific examples of such lipids include neutral lecithins (e.g. egg lecithin or soybean lecithin). ), as well as saturated synthetic lecithins (e.g. dimyristoylphosph?tidylic) Phosphorus, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine lucorin> and unsaturated synthetic lecithins (e.g. dioleoylphosphatidyl Synthetic lecithins such as choline or silyloylphosphatidylcholine) There are phospholipids containing The SPLV bilayer contains cholesterol, cholestanol, It may contain steroidal components such as cholestanol, cholestane, etc. acidic hydrophilic When a compound having a functional group (phosphoric acid, sulfuric acid, etc.) is used, the obtained SPLVs are When using a compound that is ionic and has a basic group such as an amine, , cationic liposomes have polyethyleneoxy or glycol groups. When a compound is used, neutral liposomes are obtained.

The size of SPLVs varies considerably. This range is from about 100 nm to about 10 ,000 nm (10 microns) and typically from about 100 nm to about 1 ,5000m. SPLVs consist of a few to more than 100 cells surrounding an aqueous compartment. Characterized by a lipid bilayer.

The following is an example of a ratio that can be used in SPLV synthesis. SPLV is spotty Phosphate in diethyl ether containing 5μq of hydroxytoluene (BHT) Add 50 μmol of lipid and then add 0 to the aqueous phase containing the active substance to be encapsulated. ．． 3rd was added and formed. From the substances to be captured and the lipids to be captured. The mixture is made by passing a vapor of an inert gas through the mixture to remove much of the solvent. subjected to sonication.

Other preferred liposome preparations that can be used are those prepared in a monophasic solvent system. These lipid endoplasmic reticulum are called monophasic vesicles or MPVs hereinafter. M.P. Class V is particularly stable and has a high capture ability. MPV is as follows Prepared using a unique manufacturing method. The lipid or lipid mixture and the aqueous component are - added to an organic solvent or combination of organic solvents in sufficient quantity to form a phase; Ru.

The organic solvent or organic solvents are evaporated until a thin film is formed. the next appropriate amount of aqueous component is added and the film is resuspended and agitated to dissolve the MPVs. Form.

The organic solvent or combination of organic solvents used in this manufacturing method is (1) water and (2) to form MPVs once mixed with water; It must be able to dissolve the lipids used.

For example, for organic solvents or mixtures of organic solvents that satisfy the following criteria, the manufacturing method (1) Forming a single phase with 0.2 d of aqueous component in 5 d of organic solvent; and (2) the lipid or lipid mixture is soluble in this single phase.

Of course, solvents that can be used include, but are not limited to, ethanol. alcohol, acetone, 2-propanol, methanol, tetrahydrofuran, graphite im [qlyme], dioxane, pyridine, diglyme [d i g l y111e, 1-methyl-2-pyrrolidone, butanol-2, butanol- 1. Isoamyl alcohol, isopropanol, 2-methoxyethanol or includes a combination of chloroform and methanol (eg, in a 1:1 ratio).

Evaporation should be carried out at a suitable temperature and pressure that maintains a single phase and promotes evaporation of the solvent. Must be. In fact, the temperatures and pressures selected are the same as those used to form MPVs. It does not depend on the phase transition temperature of the lipid used. The advantage of this latter point is that Although thermolabile products with desirable properties exist, well-known liposomes have Rises such as distearoyl phosphatidylcholine, which can only be formed at temperatures above This means that it can be incorporated into MPVs formed from lipids. This manufacturing method , typically 30 to 40% or more of the available water-soluble material is captured during evaporation and resuspended. 2-15% of the active water-soluble substance is trapped between the turbidity and if the lipid:drug ratio is Captures up to 70-80% of active fat-soluble substances when significantly increased can be done. The trapping of the aqueous phase in MLVs is such that the aqueous phase is trapped during the drying phase. Absolutely absent, only a small amount occurs during the rehydration stage, usually not exceeding 10%. It is.

Many lipids can be constituents of MPVs. Suitable hydrophilic groups are of course these including, but not limited to, phosphate, carboxyl, sulfate and amino groups. nothing. Suitable hydrophobic groups include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon group and at least one aromatic and/or alicyclic group aliphatic hydrocarbon groups substituted with Preferred amphiphilic compounds are phospholipids It is also closely related to chemical structure.

Specific examples of suitable lipids useful in the production of MPVs include, of course, These include, but are not limited to, neutral reticins or phosphatidyl coli. lecithins (e.g. egg lecithin or soybean lecithin), as well as saturated synthetic lecithins (e.g. egg lecithin or soybean lecithin). For example, simyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine phosphorus or distearoyl phosphatidylcholine) and unsaturated synthetic lecithins (e.g. dioleoylphosphatidylcholine, or silyloylphosphatidylcholine) There are phospholipids, including synthetic lecithins such as zircholine. Other phosphorus Lipids include, of course, but not limited to, phosphatidyl esters. Tanolamine, lysolecithin, phosphosphatidylethanolamine, phosph Atidylserine, phosphatidylinositol Includes phosphatidylic acid, ceramides and ceresdosides. MPV double layer such as cholesterol, cholestanol, cholestanol, cholestane etc. May contain steroid components. Compounds with acidic hydrophilic groups (phosphoric acid, sulfuric acid, etc.) When using When a compound having a functional group is used, a cationic liposome can be obtained.

MPVs are characterized in that the biologically active agent is entrapped within the MPV (where the biologically active agent is ntrapped ] J is defined as the trapping within the aqueous component or within the membrane duplex 1. be justified. ) can be advantageously used in donor systems. 1 or more in MPV class In order to capture the active substance above, the active substance or active substances are evaporated and diluted. It is added to the single phase before film formation takes place. Alternatively, there is no agent The active agents are the aqueous agents used to resuspend the thin film and form the MPVs. added into the ingredients. In fact, in order to obtain a high capture capacity, it is necessary to The group of substances used both in the single phase and in the aqueous component used to resuspend the film. added to. Adding two or more active substances, one active substance to a single phase addition or other agents to the aqueous component used to resuspend the film. It is also possible to capture them in one MPV by doing so.

4,2 Gel matrix Any type of gel matrix can be used in the present invention. Mere bondage is (1) The gel matrix must be able to sequester the liposomes, i.e. The pores of the gel matrix allow the liposomes to sequester in the gel. The size of the membrane must be appropriate; (2) Used in vivo; When exposed, the gel matrix must be compatible with the host organism ( That is, the level of toxicity is determined by the administration of the biologically active agent in the liposome-gel preparation. should be kept to a minimum so as not to outweigh its beneficial effects. ): (3) The gel is administered or administered by the gel. gel at the applied environmental temperature and conditions. It must be able to form a gel or maintain a gelatinous state. That is. For example, gels can be stored in body fluids and exposed to the body's temperature. The gelatinous state must be maintained at a certain temperature. Similarly, cells or When used in tissue culture, culture medium and used It must remain in a gelatinous state at certain temperatures. Those skilled in the art will understand that the gel degenerate over time, especially when applied in vivo You will be able to recognize that. However, once the biologically active agent is Once supply is substantially complete, degeneration of the gel matrix by the host organism and Metabolism is the desired outcome.

Any gel may be used in the practice of this invention. to form a gel like this Materials used for this include, but are not limited to, cellulose. carbohydrates such as methylcellulose, starch and modified starch, agarose gum arabic, gum gatsuchi, gum karaya, gum tragacanth, guar gum, Locust bean gum, tamarind gum, carrageenan, alginate, xantango mu, chicle, collagen, polyacrylamide, polysiloxanes, e.g. anhydrous Anhydride polymers such as malic acid copolymers, e.g. hydroxyethyl polymers Tacrylate, polymethyl methacrylate, polymethyl methacrylate, polymer Polyacrylates such as taclerite, ethylene-vinyl acetate copolymers ethylene-vinyl alcohol copolymers, polyorthoesters, ε-cap Rolactones, such as gelled albumin, amino acid polymers and copolymers What kind of amino acid polymers, gelatins, and liposomes can be used in vitro? Other organic or inorganic polymers that may be mixed are included.

After the mixture forms a gel, the resulting liposome-gel matrix is deposited into the tissue. Can be transplanted. In a particularly useful embodiment of the invention, sequestered liposomes A soft gel matrix such as agarose or collagen containing It will be done.

Alternatively, gels such as methylcellulose contain a gel matrix. They can be formed in tissues after mixing liposomes into suspension. After mixing, The suspension forms a gel, and the liposomes are dispersed and purified. It remains sequestered in the white gel matrix.

Regarding the method used to prepare and implant this gel matrix , the release of the liposome-entrapped agent is prolonged and the action at the site of inoculation is The relative concentration of substances increases.

4.3 Biologically active agents Virtually any biologically active agent can be used in liposomes for use in accordance with the present invention. can be trapped inside. Of course, such active substances are not limited to these. but not anti-tumor compounds, anti-viral compounds, anti-fungal compounds, and anti-inflammatory compounds. Tumor compounds, anti-tumor compounds, proteins, toxins, enzymes, hormones , neurotransmitters, lipoproteins, glycoproteins, immunoglobulins, immunostimulation Animals, dyes, radiolabels, radiopaque compounds, fluorescent compounds, polysaccharides classes, cell receptor binding molecules, anti-inflammatory drugs, anti-glaucoma drugs, mydriatic compounds , anesthetics, nucleic acids, polynucleotides, etc.

Indeed, if a competitive therapy is desired, one sequestered in a gel matrix Two or more biologically active agents are entrapped within a population of liposomes. Alright may also contain two or more molecules, each capturing the same or different biologically active agents. The above liposome populations (the same type of liposomes and different types of liposomes, such as 5PLVs, etc.) A mixture of MPVs, SUVs, LUVs, REVs, etc.) is present in the gel matrix. will be locked down.

In still other embodiments of the invention, the gel is entrapped by liposomes. They can be used as carriers for the same or different biologically active agents.

In some therapeutic applications, relatively large doses of drug compounds (i.e., compound A) followed by a maintenance dose of the same or different compound (i.e. compound B). It is hoped that they will be distributed. According to the invention, this means that compound B can be added to liposomes. and sequestering the liposomes in a gel matrix containing Compound A. , and is easily achieved by administering it in vivo with a single inoculation. to this Rapid delivery of Compound A by diffusion from the gel and release from the liposomes A slow delivery of Compound B results.

4.4 Release of biologically active agents The release of biologically active agents depends on the type of liposomes used and the liposome bilayer. can be controlled by the membrane composition as well as by the type and porosity of the gel used.

The rate of release also depends on the size and composition of the biologically active agent itself.

The liposome itself is the primary rate controlling factor in the release of entrapped biologically active agents. It is a child. The rate of release depends on the number of bilayers, the liposome diameter and most importantly the bilayer It depends on the composition of the overlay.

Some researchers have suggested that "stabilizers" such as sterols, cholesterol, etc. It is added to the bilayer to change the permeability of liposomes (Papahajopoo). Ross, D., Kimilberg, H.K., 1974, Progress Insa. - Faith Science, edited by S.G. Davison, pp. 141-232, Oxford [Papahadjopoulos, D.

imilberg, H., 1974. in progress i n 5surface 5Cience, ed, S, G, Davison, pp, 141-232.0xfordl: Pergamon; Demel, R.A. Blackdorf, K.R., Pan Dman, L.L., 1972, By. Okem Pie Off-1 Suff') Evening, 255: 331-347 [Pergamo n; Demel, R, A,, Bruckdorf, K, R,, Van Deeman, L.L., 1972. Biochem, Biophis, Acta, 255: 331-347] Somes release their contents when they come into contact with body fluids or culture media. This is important. 7, 1983 for particularly useful liposome preparations. Fontaine U.S. Patent Application Serial No. 516,268, filed on May 22nd. , which involves the controlled release of liposome-entrapped agents. Liposomal preparations containing titratable substances incorporated into membranes are described. It is. The rate of release is thus controlled by appropriate denaturation of the liposome membrane. can be controlled.

The gel matrix is the second rate controlling factor in the release of biologically active agents.

Typically, low molecular weight biologically active agents (e.g., about 2000 Daltons or less) These agents, which are often of low molecular weight, are not suitable for any gel The porosity of the gel matrix allows biologically active agents to diffuse freely through the It is not related to the speed of release. For example, many wavy fungal compounds are suitable for use in the present invention. Diffuses freely through the gel matrix. In this relationship, liposomes The composition of the membrane is more important in determining the rate of release of entrapped agents. Become.

On the other hand, if the biologically active agent has a larger molecular weight, the pore size of the gel will be blocked. Evaluation of limiting factors in the diffusion of biologically active agents released from liposomes Become. Generally, polyacrylamide gels have a molecular weight of 106 daltons or higher. Eliminate the molecules above. The pore size of polyacrylamide gels is determined by the pore size used to prepare the gel. The concentration of acrylamide present (typically 4-20% acrylamide is It is used to prepare liquids. > depends on. The pore size also depends on the degree of gel crosslinking. It can be changed depending on the degree. fraction of biologically active agent entrapped in liposomes Knowing the molecular weight, one skilled in the art can control the concentration of acrylamide and the crosslinking of the gel. By controlling the rate of diffusion, gels can be prepared to obtain the exact diffusion rate desired.

Soft gels such as methylcellulose, collagen and agarose are removed by the lyacrylamide gel (e.g. molecule 1106 Dalt) molecules larger than or equal to the molecule), used to control the diffusion of larger molecules. and thus significantly limit the release of biologically active agents of larger molecular weight. Used for control. As explained earlier, the upper limit of the gel's pore size limits the encapsulation of liposomes. It should be decided in terms of chaining. The use of soft gels allows for greater It should be noted that there is no limitation to the release of molecular weight agents. Soft Gels can also be used to control the diffusion of smaller molecules as well. Soft The porosity of Kana gel is also controlled by its concentration as well as other factors. Ru.

to control the rate of diffusion of biologically active agents released from sequestered liposomes. parameters used, such as the size of the biologically active agent and the porosity of the gel. In addition to the agent, the biologically active agent itself and the state of the gel also influence the rate of diffusion. give. This is why the biologically active agent has an affinity for the gel matrix (e.g. , affinity based on electric charge, hydrogen bonding, van der Waals forces, etc.) The biologically active agent released from the sequestered liposomes then passes through the gel. Diffusion will be gradual.

Finally, the gel used to sequester the liposomes containing the entrapped biologically active agent. Whatever the gel matrix is, the matrix must be larger than the permeability limit of the gel. Molecules other than those having a molecular weight are The lix is freely permeable to the aqueous fluid.

Therefore, liposomes in a gel matrix can spread through the gel matrix. It interacts only with molecules that can be dispersed.

This means that the interaction of body fluids or culture media with the liposome membrane is This is important because it is an important factor that changes membrane permeability.

4.5 Use of liposome-gel preparations in biological systems The fluid composition may be bioactive for cells and/or fluids in vivo and in vitro. It can be used for sustained delivery of biologically active agents. Some of the implementations are described below. Bell.

When used in vivo, the liposome-gel composition of the present invention can be used before gel formation. Or is administered later. Routes of administration are, of course, not limited to these. However, inoculations or injections (e.g., intraperitoneal, myotropic, subcutaneous, intraauricular, intraarticular) , intramammary, etc.), topical application (e.g., on areas such as eyes, ears, skin, etc.) or over afflictions such as wounds, burns, etc.) and through the epithelial or mucocutaneous lining. (e.g., vaginal and other mucocutaneous linings, gastrointestinal mucosa, etc.).

For example, the liposome-gel preparations of the invention can be inoculated in vivo and exhibit biological activity. Provides sustained systemic release of the agent. Such applications are particularly applicable to hormones (e.g. (e.g., to control growth, fertility, glucose metabolism, etc.) or to control and treat infections. It is useful for systemic release of drugs such as wavy agents.

In other examples, liposome-gel preparations are applied topically. Topical application is Sustained release of antibacterial and/or coagulant factors during the healing process It is particularly useful in treating wounds (surgical or non-surgical). similar In addition, liposome-gel preparations may contain antibacterial and/or cell growth factors. Applied topically to burns for relief. Liposome-gel preparation is also used to treat infections by providing sustained release of wavy agents. applied to the eye, and this also refers to the repeated application of biologically active agents in the form of eye drops. This reduces the need for

In still other embodiments, the liposome-gel preparation is used for sustained release. Administered orally. Such applications may cause persistent damage to the oral epithelium and other oral tissues. It is useful for repeated and sustained applications to the epithelium of the gastrointestinal tract.

The liposomal gel preparations of the present invention also provide biological support into cell or tissue culture media. Used in vitro to provide sustained release of active agent. This kind of life Physically active agents include, but are not limited to, nutrients, drugs, , hormones, growth factors, etc., such as bosome-collagen gel, It is particularly useful for culturing muscle cells, nerve cells or liver cells. liposome-ge If the liquid preparation is used as an overlay, liposome-agarose gels are preferred. It is useful for

The specific embodiments described above and below are given by way of example only. and the invention is limited only by the scope of the appended claims.

5 Example: Liposomes in methylcellulose 5.1 8PLV and methylcellulose Preparation of Lulose Gel 5PL containing radiolabeled gentamicin sulfate V (125I-GS/5PLV) prepared as described in Section 4.1 Contained: egg phosph 1-tidylcholine (egg PC>100mg or 1251-p-hydro xyphenylfuropionic acid-derivatized gentamicin sulfate (125■-G Phosphate buffered saline (PBS) containing diethyl ether to which 0.3% Tell 57! added to. 5PLV is the evaporation of diethyl ether under nitrogen vapor conditions. It was formed by sonicating the resulting mixture while irradiating. 5 PLVs were resuspended in 31 ml of PB.

Methyl cellulose in PBS pH 7.2, usually 400-2000 centipoise Mix the 2% solution until homogeneous at 4°C and autoclave at 120°C. It was obtained by cooling at 4° C. for 24 hours.

5.2SPLV-Methylcellulose Preparation Subcutaneous Administration Methylcellulose 2%) Volume One aliquot (divided) of the liquid was placed at air temperature into an equal volume of 125I-GS/5PLV. mixed with suspension.

An aliquot of this mixture (125I-GS/5PLV-methylcellulose preparation) (0,1d) is an adult Swiss Webster [5w1ss Webster] ? Mice were inoculated subcutaneously in the abdominal region. Two groups of control mice The treatment of the groups was as follows: one group contained l-G5. % methylcellulose (prepared as described in Section 5.1. >0.1 d( That is, l-G5 is not captured in 5PLV. ) in the abdominal area Inoculated subcutaneously: the second glue 1 was 125I-GS suspended in PBS. /5 PLV (in Section 5.1: Ai T was prepared. It was inoculated subcutaneously. Methylcellulose forms a gel at 37°C, so Shortly after (within a few seconds) subcutaneous inoculation of the chillulose preparation, a semi-solid rsemi-solid] "bumps" were formed. This bump is at least was visible or palpable for 24 hours. In contrast, mice in the control group Inoculation of 5PLV suspended in PBS subcutaneously inoculated over several minutes. No lasting visible or palpable evidence was formed.

Subcutaneous inoculation of 125I, as reduces the level of 125I-GS in the inoculated compartment. Confirmed by measurements. For this purpose, the skin around the location of the inoculation Skin and underlying tissues (1.5 oz. abdominal wall) were excised 24 hours after inoculation; Radioactivity was then measured using a gamma counter. The results (Table 1) are First, the liposomes were fixed at the inoculation location by a methylcellulose gel matrix. It was shown that

] to Lj! Radiolabeled Gentamicin Sulfate at the Location of Subcutaneous Inoculation in Mice Retention Remains after 24 hours In PBS (7) 125I-GS/5PLV 15゜0 in methylcellulose Muscular administration of 125I-GS3゜55.3 5PLV-methylcellulose preparation, etc. In a similar experiment, mice received 125I-GS/5PLV in the lower femoral region of the leg. - Methylcellulose preparation o.

5rII1. was inoculated with. Control mice were treated with 125 ml suspended in PBS. Equal amounts of I-GS/5PLV were inoculated. At intervals after inoculation, mice were sacrificed and The entire leg was incised and the radioactivity in the leg was measured. The results shown in Table 2 Radioactivity was detected in the legs of control mice that received liposome-gel inoculation. shows a more rapid decrease than in the mouse legs.

"Mino 1" Radiolabeled gentamicin sulfate at the location of intrafoil inoculation in mice Retention Remains after 88 hours Inoculation radiation level (%) 1251-GS/5PLV-Mef) I, -f: Ri I,, Loin 96.0125 I-GS/5PLV 46.8 Thus, the purification of 125i as is possible because liposomes containing this agent are When sequestered in a cellulose gel matrix and administered myotropically, reduced.

6. Example: Liposomes in agarose 6.1 8PLV and agarose gel The prepared SPLV was prepared using egg PC100111 () and l-G5 containing PB30.3d. or 125■-human growth hormone (125I-HGH, New England dog Claire [New England Nuclear])-containing hepes solution [HEP] ES buffer] as described in Section 5.1.

Agarose (Bio-Red Standard Low Mr [Bio-r ed 5 standard low + r ] > 0.5% to 2% solution is polymer – Melt the powder at 100’C and then sterilize it by autoclaving at 120°C. Prepared by. After cooling, the resulting gel was melted at 60'C. temperature was then lowered to 42°C.

To sequester the liposomes and inoculate the resulting gel-liposome preparation: procedure was used.

One volume of acarose solution was suspended in PBS buffer solution (pH 7,2). V1 volume was mixed. This suspension immediately has a large internal diameter (>1,5 mm). into the needle using a syringe that fits the needle. This syringe is agaro - The solution was placed horizontally at 4°C to gel. Gels containing liposomes are , a cylindrical body that can be easily extruded and inoculated (its capacity is smaller than that of the first needle) Depends on diameter and amount of solution sucked. ).

6.2 Intraperitoneal Administration of 5PLV-Agarose Preparation Adult Swiss Web Starma 125I-GS/SP LV sequestered in 1% agarose gel. Inoculated intraperitoneally (one gel cast per mouse). After 24 hours, Ma The mice were sacrificed, the gel was collected, and its radioactivity was measured. Result is, Liposomes are effective, showing that 95% of the initial radioactivity is in the recovered gel. was shown to be sealed off and maintained in position for inoculation.

6.3 125I-HGH/5PLV gives muscle orientation of 3PLV-7 galose preparation Sequestered in 0.5-2% agarose gel. Adult Swiss-Webster Mouse Inject 0.1 d of 12” I-HGH/5PLV-agarose gel preparation into the leg intramuscularly. Inoculated. A group of control mice were placed in Hepes dehydration solution (pH 7.2). Agarose gel containing suspended 1251GS/5PLV and I-HGH ( That is, 125■-HGH is not captured in 5PLV. ), or hepes 0°1rd of 1251-HGH in buffer solution was inoculated intramuscularly into the leg. inoculation At intervals, the mice were sacrificed, the inoculated legs were incised, and the residual radioactivity was measured. It was done. The results are shown in Table 3.

The results in Table 3 show that I-HGH retention in the body is due to the hormone being captured in 5PLV. This can be extended if the hormone is sequestered in a 7-gallose gel. However, the hormone-containing liposomes are sequestered in the gel matrix. When used, the hormone is optimally maintained at the site of inoculation.

(Margin below) Table 3 Retention of growth hormone at in-plane position of inoculation 125I-HGH5,00,7 not measured 0.2 0.3 0.1 Unmeasured 125I-HGH/5PLV 81.0 57 ．． O Not measured 16.0 0.7 0.4 Not measured 0.5% 26.0 20.0 1 4.0 16.0 14.0 14.0 8.01.0% 3B, 0 38.0 30.0 29.0 2B, 0 19.0 9.0125I-HGH/5PL V −Agarose 0.5% 59.0 55.0 47.0 45.0 2 &, 0 23.0 2 9.01.0% 74□0 63.0 54.0 37.0 24.0 20. 0 17.02.0% 100.0 100.0 Not measured 100.0 53. 0 3B, 0 unmeasured 1 percentage point agarose in boso-gel preparation Indicates the final concentration of the solution.

7. Example: Liposomes in collagen 7.1 5PLV and collagen gel Preparation of 125I-HGH/5PLV with egg PC100m() and 12”I-HGH/5PLV Using Hepes buffer solution 0.3rd containing HGH, as described in Section 5.1. It was prepared by

Acid-soluble rat tail collagen was developed by Michalopoulos and Pittot, 1975. Exbar Cell Res, 94: 70-78 [Hichalopoulou s and Pitot, 1975.　Exper, 　Ce1l　Res, 9 4nia 0-78] by a modification of the method described in [4. Simply put, 1 to 3 g of rat tail collagen fibers cut from the tails of two rats were mixed with glacial acetic acid. A diluted solution of (1:1000>300d) in water was stirred for 48 h at 4 °C. It was done. After allowing the mixture to settle for 24 hours, the solubilized rat A clear solution of tail collagen is decanted from the sedimented collagen fibers. It was. 0.3% or 0.9% (weight of fiber used per volume from acid solution) A stock concentration of acid-solubilized collagen (amount) was stored refrigerated until use.

Liposomes were sequestered in gels as follows: acetic acid solubilized rat tail collar. Gen (0.3-1%) 1.7d was added to 10 times concentrated Heves buffer solution (50In!! PES, 0.75M NaCΩ, 0.75M KCI)) and 0.28MNa0f -1 binary mixture. in the Hebbes buffer solution immediately after adding the buffer solution. An aliquot of 125I-HGH/SPLV (0.1d to 0.2 d) was added to the solution and mixed to obtain a homogeneous distribution. This suspension was gelled at 37°C for 1 hour.

Experiments investigated the relationship between ink and gel sequestering ability of lipids in gels (as liposomes) It was done in order to Therefore, an aliquot of 0.2 d of various liposome dilutions is Hebes I as mentioned above! added to rat tail collagen in solution at 37°C. It took an hour to form a gel. Liposomes that were not sequestered in the gel were placed under reduced pressure. Nylon mesh filter (pore size 81.2μ, Matsumaster Curl Supplement) Rye Company, Deaton, New Chassis [McMaster-Carr 5upply Co., Dayton, N.J.) I left. The radioactivity in the gel collected on the filter was measured. Table 4 is: 54-74% of I-HGH/5PLV could be sequestered in 0.3% collagen gel. It illustrates that.

Table 4 also shows that the amount of liposomes that can be sequestered in a collagen gel is It is shown that this is increased by increasing the concentration of -gen to 0.9%. .

Other liposome preparations (Table 4) contain fibrotin covalently crosslinked to lipids. Was. This glycoprotein fibronectic protein has a high affinity for collagen. Chin is Weiner et al., filed on September 19, 1983. Enzyme Catalysts Described in U.S. Patent Application Serial No. 533,583 of The liposomes were covalently cross-linked into double molecules by the conjugation method. To put it simply, I-H GH/5PLV1. Egg PC and phosphatidylethanolamine (8: 2 mg Tris (Tris(hydroxymethyl)) containing 1%) and 125I-HGH aminomethane) prepared as described in Section 5.1 using a saline buffer solution. Ta. To covalently bind fibronectin to liposomes, I-HGH/5P The LV suspension contains Ca(, Q220!n!!, Factor -Glutaminase, Alpha Therabiotic Corporation, Los Angeles Alpha Therapeutic Copr, California Los Anc+eles, CA]>1001;], thrombin (Sigma , St. Louis, Missouri [Sigma, St. Louis, so 3 ) Fibronectin in 1 volume of Tris saline buffer solution with 1 unit Incorporated, Boston, Massachusetts 1seragen Inc,, BO3tOn, MAI or Collaborative Research In Corporate 2 Lexington, Massachusetts [Col Iabor ative Re5search Inc, Lexington, MA] > 1 tnll and incubated for 1 hour at 37°C (one unit of thrombin is 37°C to solidify the fibronectin solution within 15 seconds. ) After culture, fipronectic Denatured-8PLV (I-HGH/FN-3PLV) was heated at 10,000Xq for 10 minutes. It was pelletized and washed three times.

week qax Lipid added to gel Collagen % Radiation level sequestered in gel % 125I-HGH/5PLV (0,2d) 100.0mg 0.3 54 33.3mg 0.3 57 12.3mc+ O0374 7,9+ng O,372 5.0mg 0.3 66 3.8n+g O, 371 10.0mg 0.3 81 125I-HGH/FN-3PLV (0,2d) 8.3m (] O0386 As shown in Table 4, fibronectin-denatured liposomes are attached to collagen gels. When added, a significantly enhanced sequestration of the liposomes is achieved.

7.2. 5PLV - Storage of growth hormone in the tissue after intracylinder administration of collagen preparation To determine the effect of liposome sequestration on adult Swiss-Webster Mice received 125I-HGH/5PLV sequestered in 0.3% collagen gel or 125I-HG) 71/FN-3PLV (as described in Section 7.1 above) It was prepared in this way. ) was inoculated intramuscularly into the leg. The comparison group is a buffer 125I-HGH/5PLV suspended in solution or 125I in buffer solution -HG　H was inoculated. At intervals after inoculation, mice are sacrificed and the inoculated leg were cut open and the residual radioactivity was measured.

This result (Table 5) shows that hormone-containing liposomes are Ensure that the hormone is optimally maintained at the site of inoculation when blocked during the It shows. Denaturation of liposome membranes due to the attachment of fibronectin is shown by Coller et al. Despite more sequestration of liposomes into Gengel (see Table 4), the contact This does not result in significantly increased retention of the biologically active agent at the seed location. But long , a more linear rate of 1251-growth hormone release in this group. observed.

Table 5 1251-human remains in the lungs 3~5243714 125I-HGH5,01,00,20,30,1125I-HGH/5PLV 81.0 57.0 1B, 0 1.0 0.4125I-HGH/5PLV 78.0 57.0 55.0 27.0 19.0-Collagen 125T-HGH/FN-82,077,054,044,07,03PLV- collagen a Average value of 3 mice per group.

In other experiments, commercially available pepsin and acid-digested bovine skin collagen were used to In a collagen gel prepared using a Vitrogen The sustained release of 125I-HGH from the sequestered liposomes is due to the gel becoming acidic. compared to that observed when prepared using solubilized rat collagen. .

Products from Collagen Corporation [Col Iagen Copr,] A certain Wittgen is from Flow Laboratories. s] (McLean, Virginia [+ctean, VA]) Ta. Vitrogen gel (0.3%) was prepared according to the manufacturer's instructions.

Liposomes containing entrapped 125I-HGH were prepared as described in Section 7.1. and was sequestered in the gel.

Adult Swiss-Webster mice were treated with rat tail acid-solubilized collagen or bovine (Vitrogen) sealed in 0.3% collagen gel prepared using collagen. Chained 12”I-HGH/5PLV or 125I-1-IGH/FN-8P LV was inoculated in the leg. The control group consisted of 125I- suspended in a buffer solution. Free 125 suspended in HGH/5PLV or rat tail acid-solubilized collagen I-HGH was inoculated. At various times after inoculation, mice are sacrificed and inoculated. The leg was incised and residual radioactivity was measured.

As shown in Table 6, the sequestration of liposomes in Vitrogen gel was performed 7 days after inoculation. Although this gel enhanced retention of the hormone at the site of eye inoculation, the acid-solubilized rat tail Ragen was not effective. Denaturation of sequestered liposomes with fibronectin demonstrated the retention of the hormone at the inoculation site on the first day after inoculation in both gel forms. significantly increased, but not for guitrogen gel at 7 days post-inoculation. .

Table 6 Retention of growth hormone after myotropic inoculation Two different glue bosome-collagen matrices The effect of 125 remaining in the lungs - humans 125I-HGH/Rat tail 1゜0　0゜2 collagen 125I-HGH/5PLV 57゜0 1.0125I-HGH/5PLV- 57,027,0 rat tail collagen 125T-HGH/FN-3PLV 77°0 40.0 Rat tail collagen 125I-HGH/5PLV-42゜6 14.3 Vitrogen 125I-HGH/FN-3PLV-75, 815, 4 Vitrogen a Average value of 5 mice per group.

7゜3 Release of the agent trapped in the 5PLV from the inoculation site Additional studies have shown that compounds released in vivo from liposomes sequestered in collagen carried out to assess whether the long hormone is in its active (functional) form. . Bovine Growth Hormone (BGH) is essentially the same as that described in Section 5.1. BGHl, 75 m (1/egg PC100 +++ c (BGH-3PLV), BGH-8PLV (BGH1, A 0.25d aliquot of 0.8% collagen gel (containing 75mg) This is similar to that described in . ) was locked down inside, then this was the 25th 39-day-old female Sprague Dawley who had her pituitary gland removed. awly] Rat (Charles River Incoholated [Charl e! S RiVerInc, ]) was inoculated intramuscularly. Control animals had ptosis. He received no treatment after his body was removed. Rat growth was measured daily and inoculated It was expressed in grams by subtracting the weight at the time of inoculation from the actual weight afterward.

The results (Table 7) are indicative of the group of animals treated with the BGH/5PLV-collagen preparation. Loop steadily gains weight, which is due to growth hormone sequestered in collagen. This indicates that the liposomes are released from the liposomes and retain their activity. be.

No increase in body weight was observed in the rats in the control group.

Table 7 Untreated 0 0 -0.8 -0.8 -0.2 0.5 -0.4 -0.9 8GH/5PLV-Collagen 1.2 3.9 4.7 7.3 B, 7 9 ．． 4 9.6 9.9 One injection a Average value of 8 rats per group.

7.4 Subcutaneous administration and release of agents entrapped in the 5PLV from the inoculation site 5PLV containing insulin (insulin/5PLV) is described in Section 4.1. Dipalmitoylphosphatidylcholine 100 mCl was prepared as follows: Dissolved in 5 ml of diethyl ether. Add to this bovine insulin (25 units/ m9) (Sigma Chemical Company, St. Louis, Missouri > 15m Aqueous buffer solution (PBS or 0.01M Tris) at pH 7.4 containing c+0 ．． 3m was added.

Sonic waves constructed from egg PC to solubilize insulin in aqueous buffer solution Hormone powder was added into the solution (50mM) of the treated minute unilamellar vesicles. It is rarely necessary to split it first. Following solubilization, this aqueous droplet is sonicate under the above conditions in nitrogen until the ether is completely evaporated. It was emulsified with. Lipid/insulin paste forms insulin/5PLV It was rehydrated in order to Insulin/5PLV contains 210mg of CaCΩ. Washed three times with buffer solution. CaCΩ2 pelleted insulin/5PLV It made it easier. The degree of insulin capture measured by 14C-insulin labeling is It was 20-30%.

Insulin/5PLV and insulin/5PLV sequestered in collagen gel Sustained release of insulin from the bloodstream was investigated in an animal model of diabetes.

Diabetes, Charles River Laboratories (Wilmington, Mass.) State) or Hilltop Laboratories Animals [Hilltop Laboratory Animals] (Scottozol, Pensylvani A state [5cottdale.

In Sprague-Dawley rats supplied by PA]), streptocytic Shin 50mCl/! It was expressed by intraperitoneal injection of II for 2 consecutive days. I set it. Two weeks after infusion, diabetics measure water consumption, urine output and urine glucose. It was evaluated by Urine glucose is measured using glucose test kit No. 15- UV (Sigma Chemical Company).

St. Louis, Missouri).

Insulin/SPLV is added to collagen as described in Section 7.1. Sequestered in gel (0.9% collagen). Experimental animals had a body weight of 1kc+ in their hind legs. Insulin/5PL in collagen gel corresponding to 4ml (l of insulin) received a single subcutaneous injection of V. Comparative control animals were treated with the same amount of buffer solution as the experimental animals. Free insulin, free insulin in collagen gel or free insulin/ received a single subcutaneous injection of either 5 PLV. At least 5 animals in each group used in the loop.

As shown in Table 8, urine glucose values are free insulin/5PLV or Collar Received a single subcutaneous injection of either insulin/5PLV sequestered in Gengel. was suppressed in diabetic rats. However, only insulin/5PLV In animals treated with (release) was visible in the mouth for 8 hours after treatment. Glucose in the urine is After 24 hours, it started to rise again. On the other hand, the particles sequestered in the collagen gel In animals treated with insulin/5PLV, maximal glucose depression was I saw it on the second day. A satisfactory and significant decrease in urinary glucose was observed after ffi placement. It was still evident in the first week. This means that lipo to collagen gel Some sequestration hypothesized that insulin release was simply a liposome-dependent phenomenon. interfere with the release of insulin into the systemic circulation, as no difference can be expected. This shows that.

Free insulin injected subcutaneously was rapidly cleared from the systemic circulation (data Didn't show it. ) should be kept in mind.

(Margin below) Table 8 Systemic release of insulin after subcutaneous administration Urine glucose from untreated diabetic animals Normal (non-diabetic) 5.8 10.3 10.4 5.3 24 Untreated diabetes 123.9144.5 128.4222.0 - ince 1,1 in/5 PLV 63.4 27.6 52.4104.8 69.6 Diabetes Insu IJ-n/5PLV-69,951,855,79,338,9 Collagen Diabetes a, Percent load from the value of urine glucose output (m9/old) at time Q shows.

b, Average value of 5 rats per group. Free insulin in 1 m solution Rats injected with a single injection cleared the hormone within 4 hours (data not shown). There wasn't. ) C1 Normal represents an untreated non-diabetic animal, which also serves as a control.

Procedural formalities (method) %formula% 1.Display of the incident PCT/US85100220 2. Name of the invention Liposome-gel composition 3. Person who makes corrections Relationship to the incident: Patent applicant Address: Princeton, New Jersey, United States 08540 Forestal Center Country: United States of America 6. Subject of correction (1) “Representative of patent applicant” in writing pursuant to Article 184-5, Paragraph 1 of the Patent Law No Jun (2) Translation of the description and claims 7, contents of amendments international search report 11m season”=”’ic=/assi/=o2t.

Claims (78)

[Claims] 1. at least one biological activity entrapped in a liposome sequestered in a gel; A liposome-gel composition composed of a sex agent. 2. The gel according to claim 1, wherein the gel is composed of an inorganic polymer. Posome-gel composition. 3. The gel according to claim 1, wherein the gel is composed of an organic polymer. Posome-gel composition. 4. Liposome-gel according to claim 1, wherein the gel is in gelled form. Composition. 5. The ribosome according to claim 1, wherein the gel is in an ungelled form. - Gel composition. 6. Lipo according to claim 1, wherein the gel is composed of cellulose. Some-gel composition. 7. Claim 1, wherein the gel is composed of methylcellulose. Liposome-gel composition. 8. The liposol according to claim 1, wherein the gel is composed of agarose. mu-gel composition. 9. The liposol according to claim 1, wherein the gel is composed of collagen. mu-gel composition. 10. Gels include gum arabic, gum ghatti, gum karaya, gum tragacanth, and guar. Rubber, locust bean gum, tamarind gum, carrageenan, alginate, xane The liposol according to claim 1, which is composed of tan rubber or chicle. mu-gel composition. 11. According to claim 1, the gel is composed of polyamylylamide. The liposome-gel composition described above. 12. Claim 1, wherein the gel is composed of polysiloxane. Liposome-gel composition. 13. According to claim 1, the gel is composed of polyacrylate. liposome-gel composition. 14. Claims in which the polyacrylate is hydroxyethyl polymethacrylate 14. Liposome-gel composition according to item 13. 15. Claim 1, wherein the gel is composed of polymethyl methacrylate. The liposome-gel composition described in Section 1. 16. Claim 1, wherein the gel is composed of polymethyl methacrylate. The liposome-gel composition described in Section 1. 17. Claim 1, wherein the gel is composed of polymethacrylate. The liposome-gel composition described above. 18. Claim No. 1, wherein the gel is composed of a lactic acid-glycolic acid copolymer. Liposome-gel composition according to item 1. 19. Claim 1, wherein the gel is composed of ε-caprolactone. The liposome-gel composition described above. 20. Claims in which the gel is composed of ethylene-vinyl acetate copolymer The liposome-gel composition according to item 1. 21. Claim that the gel is composed of ethylene-vinyl alcohol copolymer The liposome-gel composition according to item 1. 22. The gel according to claim 1, wherein the gel is composed of an anhydride polymer. Posome-gel composition. 23. Claim 22, wherein the anhydride polymer is composed of malic anhydride. The liposome-gel composition described in . 24. According to claim 1, the gel is composed of polyorthoester. The liposome-gel composition described above. 25. Claims in which the gel is composed of an amino acid polymer or copolymer The liposome-gel composition according to item 1. 26. The liposol according to claim 1, wherein the gel is composed of gelatin. mu-gel composition. 27. Claim 1, wherein the gel is composed of starch or modified starch. The liposome-gel composition described above. 28. Ribosomes are multilamellar vesicles. The liposome-gel composition according to claim 1, which is [sicle]. 29. Ribosomes are small unilamellar vesicles [smallunilamel The liposome-gel combination according to claim 1, which is a lar vesicle] A product. 30. Ribosomes are reverse phase evaporation vesicles [reverse phase evap The liposome according to claim 1, which is an orated vesicle] Gel composition. 31. Liposomes are large unilamel vesicles. The liposome-gel combination according to claim 1, which is a lar vesicle] A product. 32. Liposomes are stable plurilamellar vesicles [stabieplurilla The liposome according to claim 1, which is a mellar vesicle] Gel composition. 33. Ribosomes are monophasic vesicles The liposome-gel composition according to claim 1. 34. Liposomes sequestered in a gel can contain at least two different types of liposomes. The liposome-gel composition according to claim 1, which is composed of a liposome-gel composition. 35. at least two biologically active agents are entrapped within the liposome. A liposome-gel composition according to claim 1. 36. at least two biologically active agents are entrapped within the liposome. 35. The liposome-gel composition of claim 34. 37. Biologically active agents entrapped in liposomes have antibacterial, antifungal, and antifungal properties. Claims selected from the group consisting of viral and antiparasitic compounds The liposome-gel composition according to item 1. 38. Claim No. 1, wherein the antibacterial compound is gentamicin or a derivative thereof. Liposome-gel composition according to item 37. 39. The biologically active agent entrapped in the liposome is a cell receptor binding compound. The liposome-gel composition according to claim 1. 40. Biologically active agents entrapped in liposomes include hormones, neurotransmitters, The claimed compound is selected from the group consisting of anti-tumor compounds, growth factors and toxins. A liposome-gel composition according to scope 1. 41. 41. The liposol according to claim 40, wherein the biologically active agent is growth hormone. mu-gel composition. 42. Lipo according to claim 41, wherein the growth hormone is human growth hormone. Some-gel composition. 43. 41. The liposome of claim 40, wherein the biologically active agent is insulin. Mugel composition. 44. Biologically active agents entrapped in liposomes can contain proteins, glycoproteins and and the ribbon according to claim 1, which is selected from the group consisting of Posome-gel composition. 45. The liposome according to claim 44, wherein the glycoprotein is an immunoglobulin. Mugel composition. 46. The biologically active agent entrapped in the liposome is an immunoagonist compound. The liposome-gel composition according to item 1. 47. The biologically active agent entrapped in the liposomes is a group consisting of catalysts and enzymes. The liposome-gel composition according to claim 1, which is selected from the following. 48. Biologically active agents entrapped in liposomes may include dyes, radiolabels, radiation Claims selected from the group consisting of opaque compounds and fluorescent compounds The liposome-gel composition according to item 1. 49. Biologically active agents trapped in ribosomes have anti-inflammatory, anti-glaucoma, and anti-inflammatory properties. Claims selected from the group consisting of pupillary, analgesic and narcotic compounds The liposome-gel composition according to item 1. 50. Biologically active agents entrapped in liposomes include nucleic acids and polynucleotides. The liposome-gel according to claim 1, which is selected from the group consisting of le composition. 51. The biologically active agents entrapped in liposomes include monosaccharides, disaccharides and polysaccharides. The liposome-gel set according to claim 1, which is selected from the group similar to A product. 52. Claims further comprising a biologically active agent sequestered in the gel. The liposome-gel composition according to item 1. 53. Biologically active agents sequestered in gels are biologically active agents entrapped in liposomes. 53. The liposome-gel composition of claim 52, which is different from the active agent. thing. 54. Biologically active agents sequestered in gels are biologically active agents entrapped in liposomes. The liposome-gel composition of claim 52, which is the same as the active agent. . 55. Biologically active agents sequestered in the gel have antibacterial, antifungal, and antiviral properties. Claim 52 selected from the group consisting of anti-parasitic and anti-parasitic compounds. The liposome-gel composition described in Section 1. 56. Claims that the biologically active agent sequestered in the gel is a cell receptor binding compound The liposome-gel composition according to item 52. 57. Biologically active agents sequestered in the gel include hormones, neurotransmitters, and antitumor agents. Claim 1 selected from the group consisting of sexual compounds, growth factors and toxins. Liposome-gel composition according to item 52. 58. Biologically active agents that are sequestered in the gel contain proteins, glycoproteins and The liposome according to claim 52, which is selected from the group consisting of proteins. mu-gel composition. 59. Claim 52, wherein the glycoprotein sequestered in the gel is an immunoglobulin. The liposome-gel composition described in Section 1. 60. Claims wherein the biologically active agent sequestered in the gel is an immune agonist compound. 53. The liposome-gel composition according to item 52. 61. The biologically active agent sequestered in the gel is selected from the group consisting of catalysts and enzymes. 53. The liposome-gel composition of claim 52, which is disclosed. 62. The biologically active agent sequestered in the gel can be dyed, radiolabeled, or radiopaque. Claim 52, which is selected from the group consisting of fluorescent compounds and fluorescent compounds. The liposome-gel composition described in Section 1. 63. The biologically active agents sequestered in the gel have anti-inflammatory, anti-glaucoma, mydriatic, Claim 52 selected from the group consisting of analgesic and narcotic compounds. The liposome-gel composition described in Section 1. 64. Biologically active agents that are sequestered in the gel are separated from nucleic acids and polynucleotides. The liposome-gel set according to claim 52, which is selected from the group consisting of A product. 65. The biologically active agents sequestered in the gel are monosaccharides, disaccharides and polysaccharides. The liposome-gel composition of claim 52, which is selected from the group . 66. The gel of the liposome-gel composition is compatible with the host and administered. Claim 1 is capable of maintaining a gelled form in the host environment when 2. Biology by administering the liposome-gel composition described in section 1. Method for in vivo delivery of therapeutically active agents. 67. The gel of the liposome-gel composition is compatible with the host and administered. Claim 3 is capable of maintaining a gelled form in the host environment when An organism obtained by administering the liposome-gel composition according to item 4 into a host organism. Method for in vivo delivery of biologically active agents. 68. The gel of the liposome-gel composition is compatible with the host and administered. Claim 5 is capable of maintaining a gelled form in the host environment when An organism obtained by administering the liposome-gel composition according to item 2 into a host organism. Method for in vivo delivery of biologically active agents. 69. The liposome-gel composition is one that is administered in its gelled form. 67. The method of claim 66. 70. The liposome-gel composition achieves its gelled form after in vivo administration. 67. The method of claim 66. 71. 67. The method of claim 66, wherein the route of administration is intraperitoneal. 72. 67. The method of claim 66, wherein the route of administration is intramuscular. 73. 67. The method of claim 66, wherein the route of administration is subcutaneous. 74. 67. The method of claim 66, wherein the route of administration is intraarticular. 75. 67. The method of claim 66, wherein the route of administration is intraotic. 76. 67. The method of claim 66, wherein the route of administration is ocular. 77. 67. The method of claim 66, wherein the route of administration is local. 78. 67. The method of claim 66, wherein the route of administration is oral.