Steroidal liposomes

Details Steroidal liposomes Steroidal liposomes

1993-08-18

2002-03-05

Lovering, Richard D. (Department: 1712)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C264S004100, C264S004600, C424S001210, C424S009100, C436S829000, C514S078000, C514S182000, C514S887000, C514S967000

Reexamination Certificate

active

06352716

ABSTRACT:

TABLE OF CONTENTS
1. Field of the Invention
2. Background of the Invention
2.1 Liposomes
2.2 Water-Soluble Sterols
3. Summary of the Invention
4. Brief Description of the Figures
5. Detailed Description of the Invention
6. Example: Cholesterol Hemisuccinate Liposomes Entrapping Water-soluble Compounds
6.1 Liposomes Prepared Using Various Salts Forms of Cholesterol Hemisuccinate
6.1.1 Tris-Salt Cholesterol Hemisuccinate MLVs
6.1.2 2-Amino-2-Methyl-1, 3-Propanediol Cholesterol Hemisuccinate-MLVs
6.1.3 2-Aminoethanol Cholesterol Hemisuccinate-MLVs
6.1.4 Bis-Tris-Propane Cholesterol Hemisuccinate-MLVs
6.1.5 Triethanolamine Cholesterol Hemisuccinate-MLVs
6.1.6 Miconazole Cholesterol Hemisuccinate-MLVs
6.1.7 Cholesterol Hemisuccinate-SUVs Prepared by Sonication
6.1.8 Cholesterol Hemisuccinate-SUVs Prepared by Extrusion Techniques
6.1.9 Miconazole—CHS-Tris Cream
6.1.10 Terconazole—CHS-Tris Cream
6.1.11 Miconazole—CHS-Tris Suppository
6.1.12 In Vivo Activity for Vaginal Candida Infections
6
.
2
Entrapment of Inulin in Cholesterol Hemisuccinate MLVs
6.2.1 Encapsulation Efficiency of Inulin in Cholesterol Hemisuccinate-MLVs and Egg Phosphatidylcholine-MLVs
6.3 Entrapment of Inulin in Cholesterol Hemisuccinate SUVs
6.4 Entrapment of Chromium in Cholesterol Hemisuccinate MLVs
6.4.1 Encapsulation Efficiency of Chromium in Cholesterol Hemisuccinate-MLVs
6.4.2 Captured Volume in Cholesterol Hemisuccinate MLVs: Chromium Entrapment Cholesterol Hemisuccinate Concentration
6.5 Ultrastructure of Cholesterol Hemisuccinate Liposomes
6.6 X-Ray Diffraction Analysis of Cholesterol Hemisuccinate Liposomes
6.7 Electron Spin Resonance Analysis of Cholesterol Hemisuccinate Liposomes
6.8 Isotonic Swelling of Cholesterol Hemisuccinate Liposomes
7. Example: Cholesterol Hemisuccinate Liposomes Entrapping Sparingly Soluble Compounds
7.1 Bovine Growth Hormone Entrapped in Cholesterol Hemisuccinate-SUVs
7.2 Insulin Entrapped in Cholesterol Hemisuccinate SUVs
7.3 Tylosin Entrapped in Cholesterol Hemisuccinate-SUVs
8. Example: The use of Cholesterol Hemisuccinate Liposomes to Entrap Lipid Soluble Compounds
8.1 Indomethacin Entrapped in Cholesterol Hemisuccinate-MLVs
8.1.2 Ultrastructure of Cholesterol Hemisuccinate Vesicles Containing Indomethacin
8.2 Diazepam Entrapped in Cholesterol Hemisuccinate-SUVs
9. Example: The Use of Cholesterol Hemisuccinate Liposomes to Determine Aminoglycoside Concentration in Serum
10. Example: In Vivo Administration of Cholesterol Hemisuccinate Liposomes
10.1 Treatment of Joint Arthritis Using Indomethacin Entrapped in Cholesterol Hemisuccinate-MLVs
10.2 In Vivo Administration of Diazepam Entrapped in Cholesterol Hemisuccinate SUVs
10.2.1 organ Distribution After Intravenous Innoculation
10.3 In Vivo Administration of Chromium Entrapped in Cholesterol hemisuccinate-MUVs
10.4 In Vivo Administration of Human Growth Hormone Entrapped in Cholesterol Hemisuccinate MLVs
1. FIELD OF THE INVENTION
The present invention relates to methods and compositions for the entrapment of compounds in liposomes composed of salt forms of organic acid derivatives of sterols that are capable of forming bilayers.
Sterols such as cholesterol or other lipids, to which a hydrophilic moiety such as a salt form of an organic acid is attached, can be used to prepare suspensions of multilamellar or small unilamellar vesicles. The sterol liposomes of the present invention may be prepared with or without the use of organic solvents. These vesicles may entrap water-soluble compounds, partially water-soluble compounds, and water-insoluble compounds.
The sterol vesicles described herein are particularly useful for the entrapment of biologically active compounds or pharmaceutical compounds which can be administered in vivo. Alternatively, the sterol liposomes of the present invention may be used in vitro. For instance, the cholesterol hemisuccinate liposomes described herein may be used in vitro in divalent cation-dependent assay systems.
2. BACKGROUND OF THE INVENTION
2.1. LIPOSOMES
Liposomes are completely closed bilayer membranes containing an encapsulated aqueous phase. Liposomes may be any variety of multilamellar vesicles (onion-like structures characterized by concentric membrane bilayers each separated by an aqueous layer) or unilamellar vesicles (possessing a single membrane bilayer).
Two parameters of liposome preparations are functions of vesicle size and lipid concentration: (1) Captured volume, defined as the volume enclosed by a given amount of lipid, is expressed as units of liters entrapped per mole of total lipid (1mol
−1
). The captured volume depends upon the radius of the liposomes which in turn is affected by the lipid composition of the vesicles and the ionic composition of the medium. (2) Encapsulation efficiency, defined as the fraction of the aqueous compartment sequestered by the bilayers, is expressed as a percentage. The encapsulation efficiency is directly proportional to the lipid concentration; when more lipid is present, more solute can be sequestered within liposomes. (See Deamer and Uster, 1983, Liposome Preparation: Methods and Mechanisms, in Liposomes, ed. M. Ostro, Marcel Dekker, Inc., NY, pp. 27-51.)
The original method for liposome preparation (Bangham et al., 1965, J. Mol. Biol. 13: 238-252) involved suspending phospholipids in an organic solvent which was then evaporated to dryness leaving a waxy deposit of phospholipid on the reaction vessel. Then an appropriate amount of aqueous phase was added, the mixture was allowed to “swell,” and the resulting liposomes which consisted of multilamellar vesicles (hereinafter referred to as MLVs) were dispersed by mechanical means. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) “tails” of the lipid orient toward the center of the bilayer while the hydrophilic (polar) “heads” orient towards the aqueous phase. This technique provided the basis for the development of the small sonicated unilamellar vesicles (hereinafter referred to as SUVs) described by Papahadjopoulos and Miller (1967, Biochim. Biophys. Acta. 135: 624-638). Both MLVs and SUVs, however, have limitations as model systems.
In attempts to increase captured volume or encapsulation efficiency a number of methods for the preparation of liposomes comprising phospholipid bilayers have been developed; however, all methods require the use of organic solvents. Some of these methods are briefly described below.
An effort to increase the encapsulation efficiency involved first forming liposome precursors or micelles, i.e., vesicles containing an aqueous phase surrounded by a monolayer of lipid molecules oriented so that the polar head groups are directed towards the aqueous phase. Liposome precursors are formed by adding the aqueous solution to be encapsulated to a solution of polar lipid in an organic solvent and sonicating. The liposome precursors are then emulsified in a second aqueous phase in the presence of excess lipid and evaporated. The resultant liposomes, consisting of an aqueous phase encapsulated by a lipid bilayer are dispersed in aqueous phase (see U.S. Pat. No. 4,224,179 issued Sep. 23, 1980 to M. Schneider).
In another attempt to maximize the encapsulation efficiency, Papahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describes a “reverse-phase evaporation process” for making oligolamellar lipid vesicles also known as reverse-phase evaporation vesicles (hereinafter referred to as REVs). According to this procedure, the aqueous material to be encapsulated is added to a mixture of polar lipid in an organic solvent. Then a homogeneous water-in-oil type of emulsion is formed and the organic solvent is evaporated until a gel is formed. The gel is then converted to a suspension by dispersing the gel-like mixture in an aqueous media. The REVs produced consist mostly of unilamellar vesicles (large unilamellar vesicles, or LUVs) and some oligolamellar vesicles which are characterized by only a few concentric bilayers with a large internal aqueous space.
Much has been written regarding the possibilities of using liposomes f

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