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Claims  |
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What is claimed is:
1. A liposome-gel composition comprising a bioactive agent entrapped in
liposomes sequestered in a gel matrix in which:
(a) the gel matrix has a pore size relative to the liposomes so that the
liposomes are sequestered in the gel matrix without blocking (i) diffusion
of fluids into the gel which interact with bilayers of the liposomes, (ii)
the ability of the sequestered liposomes to release the entrapped
bioactive agent or (iii) the diffusion of the bioactive agent released
from the liposomes through the gel to the surrounding environment; and
(b) the gel matrix is capable of forming or remaining gelled at
temperatures and conditions of the environment in which it is administered
or applied.
2. The lipsosome-gel composition according to claim 1 wherein said gel
comprises an inorganic polymer.
3. The liposome-gel composition according to claim 1 wherein said gel
comprises an organic polymer.
4. The liposome-gel composition according to claim 1 wherein said gel is in
a gelled form.
5. The liposome-gel composition according to claim 1 wherein said gel is in
an ungelled form.
6. The liposome-gel composition according to claim 1 wherein said gel
comprises a celluosic.
7. The liposome-gel composition according to claim 1 wherein said gel
comprises methylcellulose.
8. The liposome-gel composition according to claim 1 wherein said gel
comprises agarose.
9. The liposome-gel composition according to claim 1 wherein said gel
comprises collagen.
10. The liposome-gel composition according to claim 1 wherein said gel
comprises gumarabic, ghatti, karay, tragacanth, guar, locust bean gum,
tamarind, carageenan, alginate, xanthan or chickle.
11. The liposome-gel composition according to claim 1 wherein said gel
comprises polyacrylamide.
12. The liposome-gel composition according to claim 1 wherein said gel
comprises polysiloxane.
13. The liposome-gel composition according to claim 1 wherein said gel
comprises polyacrylate.
14. The liposome-gel composition according to claim 13 wherein said
polyacrylate comprises hydroxyethylpolymethycrylate.
15. The liposome-gel composition according to claim 1 wherein said gel
comprises polymethylmethacrylate.
16. The liposome-gel composition according to claim 1 wherein said gel
comprises polyethylethyacrylate.
17. The liposome-gel composition according to claim 1 wherein said gel
comprises polymethacrylate.
18. The liposome-gel composition according to claim 1 wherein said gel
comprises lactic acid-glycolic acid copolymer.
19. The liposome-gel composition according to claim 1 wherein said gel
comprises .epsilon.-caprolactone.
20. The liposome-gel composition according to claim 1 wherein said gel
comprises ethylenevinylacetate copolymer.
21. The liposome-gel composition according to claim 1 wherein said gel
comprises ethylenevinylalcohol copolymer.
22. The liposome-gel composition according to claim 1 wherein said gel
comprises a polyanhydride.
23. The liposome-gel composition according to claim 21 wherein said
polyanhydride comprises malic anhydride.
24. The lipsome-gel composition according to claim 1 wherein said gel
comprises polyorthoester.
25. The liposome-gel composition according to claim 1 wherein said gel
comprises an amino acid polymer or copolymer.
26. The liposome-gel composition according to claim 1 wherein said gel
comprises gelatin.
27. The liposome-gel composition according to claim 1 wherein said gel
comprises starch or modified starch.
28. The liposome-gel composition according to claim 1 wherein said
liposomes are multilamellar vesicles.
29. The liposome-gel composition according to claim 1 wherein said
liposomes are small unilamellar vesicles.
30. The liposome-gel composition according to claim 1 wherein said
liposomes are reverse phase evaporated vesicles.
31. The liposome-gel composition according to claim 1 wherein said
liposomes are large unilamellar vesicles.
32. The liposome gel-composition according to claim 1 wherein said
liposomes are stable plurilamellar vesicles.
33. The liposome-gel composition according to claim 1 wherein said
liposomes are monophasic vesicles.
34. The liposome-gel composition according to claim 1 wherein said
liposomes sequestered in the gel comprise a plurality of different types
of liposomes.
35. The liposome-gel composition according to claim 1 wherein a plurality
of bioactive agents are entrapped in said liposomes.
36. The liposome-gel composition according to claim 34 wherein a plurality
of bioactive agents are entrapped in said liposomes.
37. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: antibacterial, antifugal, antiviral, and antiparasitic
compounds.
38. The liposome-gel composition according to claim 37 wherein said
antibacterial compound is gentamicin or a derivative thereof.
39. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is a cell receptor binding
compound.
40. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: hormones, neurotransmitters, tumoricidal compounds, growth
factors, and toxins.
41. The liposome-gel composition according to claim 40 wherein said
bioactive agent is growth hormone.
42. The liposome-gel composition according to claim 41 wherein said growth
hormone is human growth hormone.
43. The liposome-gel composition according to claim 40 wherein said
bioactive agent is insulin.
44. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: proteins, glycoproteins, and lipoproteins.
45. The liposome-gel composition according to claim 44 wherein said
glycoprotein is an immunoglobulin.
46. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is an immunomodulating compound.
47. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of catalysts and enzymes.
48. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: dyes, radiolabels, radioopaque compounds and fluorescent
compounds.
49. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: anti-inflammatory, antiglaucomic, mydriatic, analgesic and
anaesthetic compounds.
50. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: nucleic acids and polynucleotides.
51. The liposome-gel composition according to claim 1 wherein said
bioactive agent entrapped in liposomes is selected from the group
consisting of: monosaccharides, disaccharides, and polysaccharides.
52. The liposome-gel composition according to claim 1 further comprising a
bioactive agent sequestered in the gel.
53. The liposome-gel composition according to claim 52 wherein the
bioactive agent sequestered in the gel is different from the bioactive
agent entrapped in the liposomes.
54. The liposome-gel composition according to claim 52 wherein the
bioactive agent sequestered in the gel is the same as the bioactive agent
entrapped in the liposomes.
55. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: antibacterial, antifungal, antiviral, and antiparasitic
compounds.
56. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is a cell receptor binding
compound.
57. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: hormones, neurotransmitters, tumoricidal compounds, growth
factors and toxins.
58. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: proteins, glycoproteins, and lipoproteins.
59. The liposome-gel composition according to claim 52 wherein said
glycoprotein sequestered in the gel is an immunoglobulin.
60. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is an immunomodulating compound.
61. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of catalysts and enzymes.
62. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: dyes, radiolabels, radioopaque compounds and fluorescent
compounds.
63. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: anti-inflammatory, antiglaucomic, mydriatic, analgesic and
anaesthetic compounds.
64. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: nucleic acids and polynucleotides.
65. The liposome-gel composition according to claim 52 wherein said
bioactive agent sequestered in the gel is selected from the group
consisting of: monosaccharides, disaccharides, and polysaccharides.
66. A method for delivery in vivo of a bioactive agent comprising:
administering to a host in vivo a liposome composition comprising a
bioactive agent entrapped in liposomes sequestered in a gel matrix in
which:
(a) the gel matrix has a pore size relative to the liposomes so that the
liposomes are sequestered in the gel matrix without blocking (i) diffusion
of fluids into the gel which interact with bilayers of the liposomes, (ii)
the ability of the sequestered liposomes to release the entrapped
bioactive agent or (iii) the diffusion of the bioactive agent released
from the liposomes through the gel to the surrounding environment; and
(b) the gel matrix is capable of forming or remaining gelled at
temperatures and conditions of the environment in which it is
administrered or applied
wherein the gel of said composition is compatible with the host and is
capable of maintaining its gelled form in the host environment when
administered and of degrading over time after administration.
67. A method for delivery in vivo of a bioactive agent comprising:
administering to a host in vivo a liposome-gel composition comprising a
bioactive agent entrapped in liposomes sequestered in a gel matrix in
which:
(a) the gel matrix has a pore size relative to the liposomes so that the
liposomes are sequestered in the gel matrix without blocking (i) diffusion
of fluids into the gel which interact with bilayers of the liposomes, (ii)
the ability of the sequestered liposomes to release the entrapped
bioactive agent or (iii) the diffusion of the bioactive agent released
from the liposomes through the gel to the surrounding environment; and
(b) the gel matrix is capable of forming or remaining gelled at
temperatures and conditions of the environment in which it is administered
or applied
wherein the gel of said composition is compatible with the host and is
capable of maintaining its gelled form in the host environment when
administered and of degrading over time after administration and wherein
said liposomes sequestered in the gel comprise a plurality of different
types of liposomes.
68. A method for delivery in vivo of a bioactive agent comprising:
administering to a host in vivo a liposome-gel composition comprising a
bioactive agent entrapped in liposomes sequestered in a gel matrix in
which:
(a) the gel matrix has a pore size relative to the liposomes so that the
liposomes are sequestered in the gel matrix without blocking (i) diffusion
of fluids into the gel which interact with bilayers of the liposomes, (ii)
the ability of the sequestered liposomes to release the entrapped
bioactive agent or (iii) the diffusion of the bioactive agent released
from the liposomes through the gel to the surrounding environment; and
(b) the gel matrix is capable of forming or remaining gelled at
temperatures and conditions of the environment in which it is administered
or applied
wherein the gel or said composition is compatible with the host and is
capable of maintaining its gelled form in the host environment when
adminstered and of degrading over time after administration and wherein
said liposome further comprises a bioactive agent sequestered in the gel,
and wherein the gel of said composition is compatible with the host and
capable of maintaining its gelled from in the host environment when
administered and of degrading over time after administration.
69. The method according to claim 66 wherein the liposome-gel composition
is administered in its gelled form.
70. The method according to claim 66 wherein the liposome-gel composition
attains its gelled form after administration in vivo.
71. The method according to claim 66 wherein said route of administration
is intraperitoneal.
72. The method according to claim 66 wherein said route of administration
is intramuscular.
73. The method according to claim 66 wherein said route of administration
is subcutaneous.
74. The method according to claim 66 wherein said route of administration
is intra-articular.
75. The method according to claim 66 wherein said route of administration
is intra-aural.
76. The method according to claim 66 wherein said route of administration
is ocular. .
77. The method according to claim 66 wherein said route of administration
is topical.
78. The method according to claim 66 wherein said route of administration
is oral. |
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Claims |
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Description |
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TABLE OF CONTENTS
1. Field of the Invention
2. Background of the Invention
2.1. Liposomes
2.2. Polymer Matrices and Gels
3. Summary of the Invention
4. Detailed Description of the Invention
4.1. Preparation of Liposomes
4.2. Gel Matrices
4.3. Bioactive Agents
4.4. Release of Bioactive Agent
4.5. Use of the Liposome-gel Preparation in Living Systems
5. Example: Liposomes in Methylcellulose
5.1. Preparation of SPLVs and Methylcellulose Gel
5.2. Subcutaneous Administration of the SPLV-Methylcellulose Preparation
5.3. Intramuscular Administration of the SPLV-Methylcellulose Preparation
6. Example: Liposomes in Agarose
6.1. Preparation of SPLVs and Agarose Gel
6.2. Intraperitoneal Administration of the SPLV Agarose Preparation
6.3. Intramuscular Administration of the SPLV-Agarose Preparation
7. Example: Liposomes in Collagen
7.1. Preparation of SPLVs and Collagen Gel
7.2. Intramuscular Administration of the SPLV-Collagen Preparation
7.3. Release of SPLV-entrapped Agent from the Site of Inoculation
7.4. Subcutaneous Administration and Release of SPLV-entrapped Agent from
the Site of Inoculation
1. FIELD OF THE INVENTION
The invention describes compositions and methods for maintaining and
immobilizing a reservoir of a biologically active agent which provides for
the sustained release of the biologically active agent in living systems.
According to the present invention, a biologically active agent is
entrapped in liposomes which are sequestered in a gel matrix. When used in
living systems the liposomes sequestered in the gel matrix provide for
prolonged release of liposome entrapped agents and the gel matrix provides
for immobilization of the liposomes.
According to one embodiment of the present invention, the liposome-gel
compositions of the present invention can be implanted in vivo to provide
for the prolonged release of the entrapped bioactive agent to the host
organism. When administered in vivo, the gel matrix provides for
protection of the liposomes from rapid clearance without interfering with
release of the liposome entrapped agent. In another embodiment of the
present invention, the liposome-gel composition may be used as a support
or overlay for cells grown in culture and thus provide for the prolonged
release of the entrapped bioactive agent into the culture medium.
2. BACKGROUND OF THE INVENTION
2.1. Liposomes
Liposomes are lipid vesicles which can entrap a variety of pharmaceutical
agents and can be used for delivery of these agents to cells and tissues
in vivo. A multitude of liposomes can be constructed from one or more
lipids such that they are small unilamellar vesicles (SUV), large
unilamellar vesicles (LUV), oligolamellar vesicles prepared by reverse
phase evaporation (REV), or multilamellar vesicles (MLV). See review by
Deamer and Uster, 1983, "Liposome Preparation: Methods and Mechanisms,"
1983, in Liposomes, Ostro, ed., Marcel Dekker, Inc., New York, pp. 27-51.
Much has been written regarding the possibilities of using liposomes for
drug delivery systems. See for example, the disclosures in U.S. Pat. No.
3,993,754, U.S. Pat. No. 4,145,410. In liposome delivery systems the
medicament is entrapped in the liposome which is administered to the
patient to be treated. See U.S. Pat. No. 4,224,179 and U.S. Pat. No.
4,235,871.
Aqueous suspensions of liposomes may be inoculated in any desired way
(e.g., intravenously, intramuscularly, intraperitoneally, etc.). However,
after their inoculation, most of the liposomes are dispersed from the site
of inoculation, and either degraded or endocytosed by phagocytic cells
such as polymorphonuclear and mononuclear leucocytes, and macrophages
(Poste, 1983, Biol. Cell, 47: 19-38). Thus, the release of entrapped drug
from liposomes is limited to the period of time between inoculation and
degradation or clearance of liposomes from body fluids.
Sustained drug release characteristics can be ascribed to other types of
drug microcarriers such as lipid microvesicles (microreservoirs) described
by Sears, U.S. Pat. No. 4,298,594.
2.2. Polymer Matrices and Gels
Polymer matrices and gels have been used to localize delivery or retard
dispersion of drugs from the site of administration in vivo. Harris et
al., 1980, J. Pharm. Sci., 69: 1271-1273, used cross-linked starch gel for
localized delivery of prostaglandin E2. Cotes et al., 1980, incorporated
human growth hormone into a 16% partially hydrolysed gelatin solution
which was subcutaneously injected into animals in an attempt to extend the
period of elevated plasma hormone concentration (J. Endocrinol.
87:303-312). More recently, Morimoto et al., 1983, demonstrated enhanced
absorption of insulin when the peptide was incorporated into polyacrylic
acid aqueous gel bases containing various long chain fatty acids
(Internatl. J. Pharm. 14:149-157).
A variety of other polymeric compounds have been utilized to provide
sustained-release drug delivery systems, including: silicone elastomers of
two types, i.e., the matrix type wherein a powdered drug is dispersed
uniformly in a solid phase elastomer, and a membrane type wherein a
reservoir of drug is enclosed within a layer of silicone elastomer
(Wadsworth and Ratnasooriya, 1981, J. Pharmacol. Methods 6:313-320; see
also Cheesman et al., 1982, Fertil. and Sterl. 38:475-481);
polymethacrylate or silastic polymers impregnated with progesterone
(Ainsworth and Wolynetz, 1982, J. Am. Sci. 54:1120-1127); co-polymers of
lactic acid and glycolic acids which provide controlled release of
levonorgestrel for six months to one year (Pitt et al., 1981, Natl. Inst.
Drug Abuse Res. Monogr. Ser., 28:232-253; Wise et al., 1980, J. Pharm.
Pharmacol. 32: 399-403); a fibrin excipient that enables controlled
release of biochemical agents (Brown et al. in U.S. Pat. No. 4,393,041);
anti-inflammatory and analgesic gel compositions (Noda et al. in U.S Pat.
No. 4,393,076; and protective gel compositions for wounds (Mason et al. in
U.S. Pat. No. 4,393,048).
3. SUMMARY OF THE INVENTION
This invention describes compositions and methods for maintaining
reservoirs of bioactive agents by sequestering the reservoir in a gel
matrix. More particularly, liposomes containing bioactive agents are
sequestered in a gel matrix which is administered in vivo or in vitro. The
gel matrix inhibits both dispersion of the liposomes in vivo or in vitro
and clearance of the liposomes in vivo without blocking (1) the diffusion
into the gel of body fluids or culture media which interact with the
liposome bilayer; (2) the ability of liposomes to release the entrapped
agent; or (3) the diffusion of the released agent through the gel to the
surrounding environment.
Although incorporation of a bioactive agent directly into a gel matrix may
provide for a certain degree of sustained-release, entrapment of a
bioactive agent in liposomes can provide for a more prolonged release of
the agent because the liposome membrane can be prepared or modified to
further retard the leak of the entrapped agent. However, because liposomes
themselves are degraded or cleared when administered in vivo, it is
difficult to achieve prolonged release of a liposome-entrapped agent in
vivo. The present invention is based upon the discovery that sequestering
a liposome preparation in a gel matrix, as described herein, protects the
liposomes from clearance but does not impair the ability of the liposomes
to release their contents slowly.
4. DETAILED DESCRIPTION OF THE INVENTION
According to one embodiment of the present invention, a suspension of
liposomes which entrap a biologically active agent is mixed with a
suspension of the gel material. The resulting mixture can be administered
in vivo to form a gel at the site of administration; alternatively, the
preparation can be allowed to gel before administration. Either method of
administration results in sequestering the liposomes in the gel matrix at
the site of injection; the resistance of the liposomes to clearance or
degradation; and the release over a period of time of the
liposome-entrapped agent at the site of administration.
In another embodiment of the present invention the liposome-gel preparation
may be used in cell or tissue culture systems to provide for the prolonged
release of the bioactive agent into the culture medium. The liposome-gel
preparation may serve as a support for cell adhesion and growth;
alternatively the liposome-gel preparation may be applied to the cell
culture as an overlay.
The rate of release of the entrapped bioactive agent is dependent on the
type of liposomes used and the composition of the liposome membranes. In
fact populations of different liposomes may be sequestered in the gel
matrix.
Any type of bioactive agent that can be entrapped in a liposome may be used
according to the present invention. Examples of these are listed infra. In
fact, two or more bioactive agents entrapped in the same or different
populations of liposomes may be sequestered in a gel matrix for use
according to the method of the present invention. Finally, one bioactive
agent may be entrapped in the liposomes, and the same or a different
bioactive agent may be contained in the gel matrix. When this liposome-gel
preparation is administered, the bioactive agent contained in the gel
matrix is released quickly whereas the bioactive agent entrapped in the
sequestered liposomes is released slowly. Thus, when one bioactive agent
is entrapped in both the sequestered liposomes and in the gel matrix one
dose may provide for both the initial dose of the agent and for its
sustained release, thereby avoiding the necessity of administering
maintenance doses. Alternatively, when one bioactive agent is entrapped in
the sequestered liposomes and another bioactive agent is entrapped within
the gel matrix, concurrent therapy using any mixture of bioactive agents
is possible. The subsections below are illustrative of the types of
liposomes, gels and bioactive agents which may be used in the practice of
the present invention.
4.1. Preparation of Liposomes
Liposomes used in the present invention can be prepared by a number of
methods, including but not limited to: the original methods of Bangham et
al. (1965, J. Mol. Biol. 13:238-252) which yield MLVs; SUVs as described
by Papahadjopoulos and Miller (1967, Biochem. Biophys. Acta. 135:624-638);
REVs as described by Papahadjopoulos in U.S. Pat. No. 4,235,871; and LUVs
as described by Szoka and Papahadjopoulos in 1980, Ann. Rev. Biophys.
Bioeng, 9:467-508; as well as methods described in U.S. patent application
Ser. No. 476,496 by Lenk et al., filed Mar. 24, 1983 which issued as U.S.
Pat. No. 4,522,803 yield stable plurilamellar vesicles (hereinafter
referred to as SPLVs); and methods described in U.S. patent application
Ser. No. 521,176 by Fountain et al. filed Aug. 8, 1983 which yield
monophasic vesicles (hereinafter referred to as MPVs). The procedures for
the preparation of SPLVs and MPVs are described below.
SPLVs are prepared as follows: an amphipathic lipid or mixture of lipids is
dissolved in an organic solvent. Many organic solvents are suitable, but
diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated
hydrocarbons and ether are preferred. To this solution are added an
aqueous phase and the active ingredient to be entrapped. This biphasic
mixture is converted to SPLVs by emulsifying the aqueous material within
the solvent and evaporating the solvent. Evaporation can be accomplished
during or after sonication by any evaporative technique, e.g., evaporation
by passing a stream of inert gas over the mixture, by heating, or by
vacuum. The volume of solvent used must exceed the aqueous volume by a
sufficient amount so that the aqueous material can be completely
emulsified in the mixture.
In practice, a minimum of about 3 volumes of solvent to about 1 volume of
aqueous phase may be used. In fact, the ratio of solvent to aqueous phase
can vary up to 100 or more volumes of solvent to 1 volume aqueous phase.
The amount of lipid must be sufficient so as to exceed that amount needed
to coat the emulsion droplets (about 40 mg of lipid per ml of aqueous
phase). The upper boundary is limited only by the practicality of
cost-effectiveness, but SPLVs can be made with 15 gm of lipid per ml of
aqueous phase.
Most amphipathic lipids may be constituents of SPLVs. Suitable hydrophilic
groups include but are not limited to: phosphato, carboxylic, sulphato and
amino groups. Suitable hydrophobic groups include but are not limited to:
saturated and unsaturated aliphatic hydro-carbon groups and aliphatic
hydrocarbon groups substituted by at least one aromatic and/or
cycloaliphatic group. The preferred amphipathic compounds are
phospholipids and closely related chemical structures. Examples of these
include but are not limited to: lecithin, phosphatidyl-ethanolamine,
lysolecithin, lysophatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and
the cerebrosides. Specific examples of suitable lipids useful in the
production of SPLVs are phospholipids which include the natural lecithins
(e.g., egg lecithin or soybean lecithin) and synthetic lecithins, such as
saturated synthetic lecithins (e.g., dimyristoylphosphatidylcholine, or
dipalmitoylphosphatidylcholine or distearoyl-phosphatidylcholine) and
unsaturated synthetic lecithins (e.g., dioloylphosphatidylcholine or
dilinoloyl-phosphatidylcholine). The SPLV bilayers can contain a steroid
component such as cholesterol, coprostanol, cholestanol, cholestane and
the like. When using compounds with acidic hydrophilic groups (phosphato,
sulfato, etc.) the obtained SPLVs will be anionic; with basic groups such
as amino, cationic liposomes will be obtained; and with polyethylenoxy or
glycol groups neutral liposomes will be obtained. The size of the SPLVs
varies widely. The range extends from about 100 nm to about 10,000 nm (10
microns) and usually about 100 nm to about 1,500 nm. The SPLVs are
characterized by a few to over 100 lipid bilayers enclosing aqueous
compartments.
The following is an example of the proportions that may be used in SPLV
synthesis: SPLVs may be formed by adding 50 micromoles of phospholipid to
5 ml of diethyl ether containing 5 micrograms of butylatedhydroxytoluene
(BHT) and then adding 0.3 ml of aqueous phase containing the active
substance to be encapsulated. The resultant mixture which comprises the
material to be entrapped and the entrapping lipid is sonicated while
streaming an inert gas over the mixture thus removing most of the solvent.
Another suitable liposome preparation which may be used is lipid vesicles
prepared in a monophasic solvent system, hereinafter referred to as
monophasic vesicles or MPVs. MPVs are particularly stable and have a high
entrapment efficiency. MPVs are prepared by a unique process as follows: a
lipid or a mixture of lipids and an aqueous component are added to an
organic solvent or a combination of organic solvents in amounts sufficient
to form a monophase. The solvent or solvents are evaporated until a film
forms. Then an appropriate amount of aqueous component is added, and the
film is resuspended and agitated in order to form the MPVs.
The organic solvent or combination of solvents used in the process must be
(1) miscible with water and (2) once mixed with water should solubilize
the lipids used to make the MPVs.
For example, an organic solvent or mixture of solvents which satifies the
following criteria may be used in the process: (1) 5 ml of the organic
solvent forms a monophase with 0.2 ml of aqueous component and (2) the
lipid or mixture of lipids is soluble in the monophase.
Solvents which may be used include but are not limited to ethanol, acetone,
2-propanol, methanol, tetrahydrofuran, glyme, dioxane, pyridine, diglyme,
1-methyl-2-pyrrolidone, butanol-2, butanol-1, isoamyl alcohol,
isopropanol, 2-methoxyethanol, or a combination of chlorform methanol
(e.g., in a 1:1 ratio).
The evaporation should be accomplished at suitable temperatures and
pressures which maintain the monophase and facilitate the evaporation of
the solvents. In fact, the temperatures and pressures chosen are not
dependent upon the phase-transition temperature of the lipid used to form
the MPVs. The advantage of this latter point is that heat labile products
which have desirable properties can be incorporated in MPVs prepared from
phospholipids such as distearoylphosphatidylcholine, which can be formed
into conventional liposomes only at temperatures above the
phase-transition temperature of the phospholipids. The process usually
allows more than 30-40% of the available water-soluble material to be
entrapped during evaporation and 2-15% of the available water-soluble
material to be entrapped during the resuspension; and up to 70-80% of the
available lipid-soluble material can be entrapped if the lipid:drug ratio
is increased significantly. With MLVs the entrapment of aqueous phase,
which only occurs during the rehydration step since no aqueous phase is
present during the drying step, usually does not exceed 10%.
Most lipids may be constituents of MPVs. Suitable hydrophilic groups
include but are not limited to: phosphato, carboxylic, sulphato and amino
groups. Suitable hydrophobic groups include but are not limited to:
saturated and unsaturated aliphatic hydrocarbon groups and aliphatic
hydrocarbon groups substituted by at least one aromatic and/or
cycloaliphatic group. The preferred amphipathic compounds are
phospholipids and closely related chemical structures.
Specific examples of suitable lipids useful in the production of MPVs are
phospholipids which include but are not limited to the natural lecithins
or phosphatidylcholines (e.g., egg lecithin or soybean lecithin) and
synthetic lecithins, such as saturated synthetic lecithins (e.g.,
dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or
distearoylphosphatidylcholine) and unsaturated synthetic lecithins (e.g.,
dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine). Other
phospholipids include but are not limited to phosphatidylethonolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid,
ceramides and the cerebrosides. The MPV bilayers can contain a steroid
component such as cholesterol, coprostanol, cholestanol, cholestane and
the like. When using compounds with acidic hydrophilic groups (phosphato,
sulfato, etc.) the obtained MPVs will be anionic; with basic groups such
as amino, cationic liposomes will be obtained.
MPVs may advantageously be used in delivery systems wherein a bioactive
agent is entrapped within the MPV ("entrapped" is defined as entrapment
within the aqueous compartment or within the membrane bilayer). In order
to entrap one or more agents in MPVs, the agent or agents may be added to
the monophase prior to evaporation and formation of the film.
Alternatively, the agent or agents may be added with the aqueous component
used to resuspend the film and form the MPVs. In fact, to obtain a high
entrapment efficiency, the agent or agents may be added to both the
monophase and to the aqueous component used to resuspend the film. Two or
more agents can also be entrapped in one MPV preparation by adding one
agent to the monophase and the other to the aqueous component used to
resuspend the film.
4.2. Gel Matrices
Any type of gel matrix may be used in the present invention. The only
constraints are (1) the gel matrix must be capable of seqestering the
liposomes; i.e., the pores of the gel matrix must be of the appropriate
size relative to the size of the liposomes in order to sequester the
liposomes in the gel; (2) when used in vivo the gel matrix must be
compatible with the recipient organism (i.e., the level of toxicity should
be kept to a minimum so as not to outweigh the beneficial effects of
administering the bioactive agent in the liposome-gel preparation); (3)
the gel must be capable of forming a gel or of remaining gelled at the
temperatures and conditions of the environment in which it is administered
or applied. For example the gel must remain gelled in the body fluids and
at the temperatures to which it is exposed in vivo. Similarly, when used
in cell or tissue culture the gel must remain gelled in the culture media
and at the incubation temperatures used. Those skilled in the art can
appreciate that the gel will degrade with the passage of time, especially
when applied in vivo; ho | | |
