Aqueous Systems For The Preparation Of Lipid Based Pharmaceutical Compounds; Compositions, Methods, And Uses Thereof

Abstract

The present invention relates to a methods of preparing active compounds complexed with lipids using aqueous systems that are free of organic solvents, and methods of using the complexes, e.g., in treating a disease in a subject. In some embodiments, the present invention comprises a composition comprising a complex comprising at least one active compound, e.g., a polyene antibiotic, an immunosuppressant agent such as tacrolimus or a taxane or taxane derivative, and one or more lipids. In some embodiments, the present invention provides a method comprising preparing a composition comprising a lipid complex comprising at least one active compound and at least one lipid and administering the composition to a subject. In certain embodiments the subject is a mammal. In certain preferred embodiments, the subject is human.

Claims

1. A method of treating a disease in a subject, comprising: a) using an aqueous system to prepare a composition comprising a complex, said complex comprising at least one active compound and at least one lipid; and b) administering said composition to a subject.

2. The method of claim 1, wherein said complex comprises a lipid compound suspension and wherein said aqueous system comprises a process comprising: a) preparing a suspension comprising said at least one active compound and said at least one lipid in a first aqueous medium at a pH between about pH 4.0 and pH 8.0; b) treating said suspension to form a lipid-compound suspension of defined particle size; c) lyophilizing the lipid-compound suspension of defined particle size to form lyophilized material; and d) reconstituting said lyophilized material with a second aqueous medium to obtain a suspension of lipid formulation of defined particle size, said defined particle size having a mean particle size of less than 5 microns.

3. The method of claim 1, wherein said at least one active compound is selected from the group consisting of amphotericin-B with deoxycholate, amphotericin B without deoxycholate, docetaxel, paclitaxel, tacrolimus, doxorubicin, Epirubicin, anthracyclines, and etoposide.

4. The method of claim 1, wherein said at least one lipid is selected from the group consisting of egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), soy phosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosohatidylcholine (DPPC), disteroylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DMPG), cholesterol (Chol), cholesterol sulfate and its salts (CS), cholesterol hemisuccinate and its salts (Chems), cholesterol phosphate and its salts (CP), cholesterylphosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol, monoglycerides, diglycerides, triglycerides, carbohydrate-based lipids selected from a group consisting of galactolipid, mannolipid, galactolecithin, -sitosterol, stigmasterol, stigmastanol, lanosterol, -spinasterol, lathosterol, campesterol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatdylinositol, phosphatidic acid, and pegylated derivatives of distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

5. The method of claim 1, wherein said at least one lipid comprises one or more of fatty acids selected from a group consisting of saturated or unsaturated fatty acids.

6. The method of claims 1, wherein said composition further comprises polyethylene glycol.

7. The method of claim 1, wherein said at least one lipid is selected from the group consisting of cholesterol or cholesterol sulfate and salts thereof, cholesterol hemisuccinate and salts thereof, cholesterol phosphate and salts thereof, and wherein said composition further comprises at least one phospholipid.

8. The method of claim 1, wherein said at least one lipid comprises a cholesterol or cholesterol derivative, wherein the mole ratio of active compound to cholesterol or cholesterol derivative is between about 1:1 and 1:10.

9. The method of claim 1, wherein said at least one lipid comprises hydrogenated soy phosphatidylcholine or soy phosphatidylcholine, wherein the mole ratio of active compound and hydrogenated soy phosphatidylcholine or soy phosphatidylcholine is between about 1:1 to about 1:90.

10. The method of claim 1, wherein said composition comprises active compound at a concentration of from about 0.5 mg/mL to about 25 mg/mL.

11. The method of claim 1, wherein said composition comprises a total lipid concentration of from 2.5% by weight to about 95% by weight.

12. The method of claim 1, wherein the molar ratio of active compound to lipid in said composition is between 1:10 to 1:100.

13. The method of claim 1, wherein the weight-to-weight ratio of total active compound to total lipid in said composition is between 1:10 to 1:60.

14. The method of claim 1, wherein said composition comprises a form selected from the group consisting of powder, solution, suspension, emulsion, micelle, liposome, lipidic particle, gel, and paste form.

15. The method of claim 14, wherein said composition comprises a plurality of micelles, wherein said micelles are in the form of monomeric, dimeric, polymeric or mixture of micelles and vesicles.

16. The method of claim 1, wherein said preparing of a composition comprising a complex comprises preparing said complex in a lyophilized form.

17. The method of claim 16, wherein said preparing said complex in a lyophilized form comprises using a cryoprotectant, wherein said cryoprotectant comprises one or more sugars selected from a group consisting of trehalose, maltose, lactose, sucrose, glucose, and dextran.

18. The method of claims 1, wherein, said composition comprises a tablet or a filled capsule, and optionally comprises an enteric coating material.

19. The method of claim 1, wherein said active compound is a partially water soluble or water insoluble drug.

20. The method of claim 1, wherein said administering comprises oral, intravenous, subcutaneous, parenteral, intraperitoneal, rectal, vaginal, and/or topical delivery of said lipidic composition to said subject.

21. A process for preparing a lipid formulation of an active compound, wherein said process comprises using an aqueous system to prepare a composition comprising a complex, said complex comprising at least one active compound and at least one lipid.

22. The process of claim 21, wherein said process is a process for preparing a lipid formulation of defined particle size, wherein said process comprises: a) preparing a suspension comprising at least one active compound and at least one lipid in a first aqueous medium at a pH between about pH 4.0 and pH 8.0; b) treating said suspension to form a lipid-compound suspension of defined particle size; c) lyophilizing the lipid-compound suspension of defined particle size to form lyophilized material; and d) reconstituting said lyophilized material with a second aqueous medium to obtain a suspension of lipid formulation of defined particle size, said defined particle size having a mean particle size of less than 5 microns.

23. The process of claim 22, wherein said first aqueous medium is water.

24. The process of claim 22, wherein said first aqueous medium and said second aqueous medium are different.

25. The process of claim 22, wherein said treating said suspension comprises extruding said suspension through a selected size aperture.

26. The process of claim 22, wherein said treating said suspension comprises high pressure split homogenization.

27. The process of claim 22 wherein said lyophilizing is in the presence of a cryoprotectant.

28. The process of claim 21, wherein said active compound comprises an active compound selected from the group consisting of a polyene antibiotic, a macrolide, an anti-cancer drug, and an immunosuppressant.

29. The process of claim 21, wherein said active compound comprises a compound selected from the group consisting of docetaxel, paclitaxel, doxorubicin, epirubicin, tamoxifen, endoxifen, etoposide, anthracyclines, amphotericin B, tacrolimus, and sacrolimus.

30. The process of claim 21, wherein said at least one lipid is selected from the group consisting of egg phosphatidylcholine, egg phosphatidylglycerol, soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dipalmitoylphosohatidylcholine, disteroylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, cholesterol, cholesterol sulfate and its salts, cholesterol hemisuccinate and its salts, cholesterol phosphate and its salts, cholesterylphosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl succinate, cholesteryl oleate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), coprostanol, cholestanol, cholestane, cholic acid, cortisol, corticosterone, hydrocortisone, and calciferol.

31. The process of claim 21, wherein said lipid formulation comprises cholesterol sulfate, and wherein the molar ratio of active compound to cholesterol sulfate in said suspension is in between about 1:1 to about 1:10.

32. The process of claim 22, wherein the composition mean particle size upon reconstitution is about 10-5000 nm.

33. The process of claim 21, wherein said at least one active compound exhibits poor solubility in water, alcohols, and halogenated hydrocarbon solvents.

34. The process of claim 22, wherein said suspension of lipid formulation of defined particle size comprises a suspension of liposomes and/or lipidic particles.

35. A method treating a cell with a lipidic composition comprising at least one active agent and at least one lipid, comprising: a) using an aqueous system to prepare a composition comprising a complex, said complex comprising at least one active compound and at least one lipid; and b) exposing said cell to said lipidic composition.

36. The method of claim 35, wherein said exposing said cell comprises exposing said cell to said lipidic composition in vivo.

37. The method of claim 35, wherein said subject is a mammal.

38. The method of claim 37, wherein said mammal is human.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0073] This invention relates to the preparation of suspension, liposomes, lipid complex, or micelles in an aqueous system. The inventive preparation comprises at least one phospholipid, such as Soya phosphatidylcholine, in aqueous media with therapeutically active insoluble or poorly soluble compound.

[0074] Particular embodiments of the invention are described in the Summary, and in this Detailed Description of the Invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. For example, the compositions and methods of the present invention are described in connection with particular polyene antibiotics, such as amphotericin B with or without deoxycholate. It should be understood that the present invention is not limited to methods or compositions using or comprising amphotericin B. In particular, the present invention relates to composition and method of preparing organic solvent-free formulation comprising one or more active compounds.

[0075] The present invention also relates to compositions and methods of delivering anticancer drugs, for example, docetaxel and paclitaxel, and immunosuppressant agents, such as tacrolimus and sacrolimus.

[0076] The present invention relates to compositions and methods for delivering polyene antibiotics that reduce the toxicity of the antibiotic to the host being treated. Several formulation strategies have been used to reduce the nephrotoxicity of amphotericin B. For example, certain lipid based formulations of amphotericin B have been found to reduce toxicity and to increase tolerance and therapeutic efficacy (Janoff, A. et al. U.S. Pat. No. 6,406,713, 2002, which is incorporated herein by reference in its entirety)

[0077] Amphotericin B is insoluble in aqueous solution and before it can be used clinically as an antifungal agent, a vehicle (carrier) has to be added to form dispersion. The commercial preparation of amphotericin B, FUNGIZONE is a mixture of amphotericin B, a detergent deoxycholate, and a buffer. When suspended in a glucose solution, FUNGIZONE forms colloidal dispersion suitable for intravenous injection. (Brajtburg, J. et al. 1990). FUNGIZONE the first marketed formulation of amphotericin B with deoxycholate remains the gold standard in spite of its renal toxicity. FUNGIZONE is currently marketed as lyophilized cake providing 50 mg amphotericin B and 41 mg of deoxycholate with 20.2 mg of sodium phosphates as a buffer.

[0078] In an effort to improve the delivery of amphotericin B in the treatment of fungal diseases, several liposome formulations have been designed. Liposomal composition containing egg phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, and cholesterol in molar ratio of 6:1:3 were more efficient in improving the therapeutic index as compared to free drug. Further, amphotericin B intercalated into mannosylated liposomes is less toxic and more effective as fungal killer (Ahmad, I. et al., 1989, 1990, 1991).

[0079] AMBISOME is a lyophilized formulation of amphotericin B incorporated into unilamellar liposomes formed from soy phosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol. AMBISOME binds to the fungal cells, resulting in death of the fungus. (Adler-Moore, Jill P. et al., 1994; Adler-Moore et al. 1993). AMBISOME formulation has greatly reduced the toxicity of amphotericin B, and high plasma concentrations and tissue accumulations of drug can be achieved with non-toxic doses of AMBISOME (Proffitt et al, U.S. Pat. No. 5,965,156, 1999; Proffitt, R. T. 1991).

[0080] ABELCET is liposome formulation consists of a 1:1 ratio of amphotericin B in combination with a 7:3 ratio of dimyristoyl phosphatidylcholine to dimyristoyl phosphatidylglycerol. The resulting complex forms a tightly packed ribbon structure, approximately 250 nm diameter. The safety and efficacy of ABELCET have been extensively evaluated in clinical studies and have shown that ABELCET is, in general less toxic than amphotericin B deoxycholate (Lister, J. 1996; Walsh, T. J. et al 1997).

[0081] In order to reduce the toxicity of amphotericin B, a new formulation has been developed consisting of a cholesteryl sulfate complex with amphotericin B, the amphotericin B colloidal dispersion (AMPHOTEC). AMPHOTEC is a stable complex of amphotericin B and cholesteryl sulfate in a 1:1 molar ratio. In vitro studies with fresh human blood have shown that the drug-lipid complex does not result in hemolysis of erythrocytes and that binding to plasma lipoproteins is less than that observed with FUNGIZONE However, the pharmacokinetics of amphotericin B following infusions of ABCD does not differ significantly from those of FUNGIZONE. (Szoka, F. C. Jr. U.S. Pat. No. 5,277,914 A 1994; Abra, R. and Guo, L. S. U.S. Pat. No. 5,194,266, 1993; Abra, R. U.S. Pat. No. 5,032,582, 1991; Abra, R. U.S. Pat. No. 4,822,777, 1989; Abra, R. et al. PCT Appl WO8701933, 1987; Sanders, S. et al. 1991).

[0082] The various lipid formulations of amphotericin B described above, however, are still capable of producing all of the toxicities associated with amphotericin B alone, although nephrotoxicity is reduced to some extent with all these formulations.

[0083] The present invention provides formulations using new lipid compositions that reduce the toxicities associated with active compounds such as amphotericin B deoxycholate.

[0084] The present invention provides compositions and methods for delivering active compounds such as polyene antibiotics, e.g., to a mammalian host. Examples of polyene antibiotics that find use in the present invention include but are not limited to amphotericin B deoxycholate (FUNGIZONE), Nystatin (Nys), Natamycin, Candicidin, Aureofungin A, Aureofungin B, Hamycin A, Hamycin B, Trienin, Pimaricin, Etruscomycin, Chainin, Dermostatin, Filipin, and Lymphosarcin. In some preferred embodiments, the present invention comprises compositions and methods for the delivery of amphotericin B deoxycholate (FUNGIZONE) to a mammalian host. Any suitable amount of an active compound, e.g., polyene antibiotics such as amphotericin B deoxycholate, can be used. Suitable amounts of polyene antibiotic are those amounts that can be stably incorporated into the complexes of the present invention.

[0085] The present invention provides compositions and methods of delivering anticancer drugs, e.g., to a mammalian host. Examples of anticancer drugs that find use in the present invention include but are not limited to paclitaxel, docetaxel, doxorubicin, daunomycin, epirubicin, etoposide, tamoxifen, endoxifen, vincristine anthracycline, and the like. Any suitable amount of anticancer drugs can be used. Suitable amounts of anticancer drugs are those amounts that can be stably incorporated into the complexes of the present invention.

[0086] The present invention provides compositions and method of delivering immunosuppressant agents. Examples of immunosuppressant agents that find use in the present invention include but not limited to tacrolimus and sacrolimus. Any suitable amount of immunosuppressant agents can be used. Suitable amounts of immunosuppressant agents are those amounts that can be incorporated into the complexes of the present invention.

[0087] The present inventions provide compositions and method for treating rejection reactions caused by the transplantations organs and tissues. Examples of organs and tissue transplantation include but not limited to heart, kidney, liver, lung, bone marrow, skin, cornea, pancreas, small intestine, muscle, limb, myoblast, intervertebral disc, cartilage, bone, blood vessel, nervous system, esophagus and the like.

[0088] In some embodiments, the present invention comprises a lipid complex with active compound (for example, amphotericin B with or without deoxycholate) in which the complex contains lipid or a mixture of lipids. In some embodiments, the complexes are in the form of micelles, emulsions or mixture of micelles and vesicles. The micelles of the present invention can be in the form, e.g., of monomeric, dimeric, polymeric or mixed micelles. In some embodiments, the complexes including micelles, emulsions or mixture of micelles and vesicles are predominately in the size range of 50 nm-20 micron, while in some preferred embodiments, the micelles and emulsions are in the size range of 50 nm-5 micron. In the complexes of the present invention, the antibiotic can be bound to the lipid by covalent, hydrophobic, electrostatic, hydrogen, or other bonds, and is considered bound even where the antibiotic is simply entrapped within the interior of lipid.

[0089] In some embodiments, active agent-lipid complexes (for example, amphotericin B-lipid complexes with or without deoxycholate) contain cholesterol or cholesterol derivatives. Examples of cholesterol derivatives that find use in the present invention include but are not limited to cholesteryl sulfate, cholesteryl hemisuccinate, cholesteryl succinate, cholesteryl oleate, cholesteryl linoleate, cholesteryl eicosapentenoate, cholesteryl linolenate, cholesteryl arachidonate, cholesteryl palmitate, cholesteryl stearate, cholesteryl myristate, polyethylene glycol derivatives of cholesterol (cholesterol-PEG), water soluble cholesterol (for example, cholesterol methyl--cyclodextrin), coprostanol, cholestanol, or cholestane, cholic acid, cortisol, corticosterone or hydrocortisone and 7-dehydrocholesterol. In some preferred embodiments, the cholesterol or cholesterol derivatives are complexed with an active compound at low pH (e.g., in the range of about pH 1.0 to pH 4.0).

[0090] In some preferred embodiments, the compositions also include -, -, -tocopherols, vitamin E, calciferol, organic acid derivatives of -, -, -tocopherols, such as -tocopherol hemisuccinate (THS), -tocopherol succinate, or mixtures thereof.

[0091] In some preferred embodiments, active agent-lipid complexes (for example, amphotericin B-lipid complexes, with or without deoxycholate) contain sterols. Examples of sterols that find use in the present invention include -sitosterol, stigmasterol, stigmastanol, lanosterol, -spinasterol, lathosterol, campesterol and/or mixtures thereof.

[0092] Compositions of the present invention also include active compounds (for example, amphotericin B complexes with or without deoxycholate) with free and/or salts or esters of fatty acid. In some preferred embodiments, fatty acids range from carbon chain lengths of about C.sub.2 to C.sub.34, preferably between about C.sub.4 and about C.sub.24, and include tetranoic acid (C.sub.4:0), pentanoic acid (C.sub.5:0), hexanoic acid (C.sub.6:0), heptanoic acid (C.sub.7:0), octanoic acid (C.sub.8:0), nonanoic acid (C.sub.9:0), decanoic acid (C.sub.10:0), undecanoic acid (C.sub.11:0), dodecanoic acid (C.sub.12:0), tridecanoic acid (C.sub.13:0), tetradecanoic (myristic) acid (C.sub.14:0), pentadecanoic acid (C.sub.15:0), hexadecanoic (palmatic) acid (C.sub.16:0), heptadecanoic acid (C.sub.17:0), octadecanoic (stearic) acid (C.sub.18:0), nonadecanoic acid (C.sub.19:0), eicosanoic (arachidic) acid (C.sub.20:0), heneicosanoic acid (C.sub.21:0), docosanoic (behenic) acid (C.sub.22:0), tricosanoic acid (C.sub.23:0), tetracosanoic acid (C.sub.24:0), 10-undecenoic acid (C.sub.11:1), 11-dodecenoic acid (C.sub.12:1), 12-tridecenoic acid (C.sub.13:1), myristoleic acid (C.sub.14:1), 10-pentadecenoic acid (C.sub.15:1), palmitoleic acid (C.sub.16:1), oleic acid (C.sub.18:1), linoleic acid (C.sub.18:2), linolenic acid (C.sub.18:3), eicosenoic acid (C.sub.20:1), eicosdienoic acid (C.sub.20:2), eicosatrienoic acid (C.sub.20:3), arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid), and cis-5,8,11,14,17-eicosapentaenoic acid, among others. Other fatty acid chains also can be employed in the compositions. Examples of such include saturated fatty acids such as ethanoic (or acetic) acid, propanoic (or propionic) acid, butanoic (or butyric) acid, hexacosanoic (or cerotic) acid, octacosanoic (or montanic) acid, triacontanoic (or melissic) acid, dotriacontanoic (or lacceroic) acid, tetratriacontanoic (or gheddic) acid, pentatriacontanoic (or ceroplastic) acid, and the like; monoethenoic unsaturated fatty acids such as trans-2-butenoic (or crotonic) acid, cis-2-butenoic (or isocrotonoic) acid, 2-hexenoic (or isohydrosorbic) acid, 4-decanoic (or obtusilic) acid, 9-decanoic (or caproleic) acid, 4-dodecenoic (or linderic) acid, 5-dodecenoic (or denticetic) acid, 9-dodecenoic (or lauroleic) acid, 4-tetradecenoic (or tsuzuic) acid, 5-tetradecenoic (or physeteric) acid, 6-octadecenoic (or petroselenic) acid, trans-9-octadecenoic (or elaidic) acid, trans-11-octadecenoic (or vaccinic) acid, 9-eicosenoic (or gadoleic) acid, 11-eicosenoic (or gondoic) acid, 11-docosenoic (or cetoleic) acid, 13-decosenoic (or erucic) acid, 15-tetracosenoic (or nervonic) acid, 17-hexacosenoic (or ximenic) acid, 21-triacontenoic (or lumequeic) acid, and the like; dienoic unsaturated fatty acids such as 2,4-pentadienoic (or -vinylacrylic) acid, 2,4-hexadienoic (or sorbic) acid, 2,4-decadienoic (or stillingic) acid, 2,4-dodecadienoic acid, 9,1 2-hexadecadienoic acid, cis-9, cis-12-octadecadienoic (or -linoleic) acid, trans-9, trans-12-octadecadienoic (or linlolelaidic) acid, trans-10,trans-12-octadecadienoic acid, 11,14-eicosadienoic acid, 13,16-docosadienoic acid, 17,20-hexacosadienoic acid and the like; trienoic unsaturated fatty acids such as 6,10,14-hexadecatrienoic (or hiragonic) acid, 7,10,13-hexadecatrienoic acid, cis-6, cis-9-cis-12-octadecatrienoic (or -linoleic) acid, trans-8, trans-10-trans-12-octadecatrienoic (or -calendic) acid, cis-8, trans-10-cis-12-octadecatrienoic acid, cis-9, cis-12-cis-1 5-octadecatrienoic (or -linolenic) acid, trans-9, trans-12-trans-15-octadecatrienoic (or -linolenelaidic) acid, cis-9, trans-11-trans-13-octadecatrienoic (or -eleostearic) acid, trans-9, trans-11-trans-13-octadecatrienoic (or -eleostearic) acid, cis-9, trans-11-cis-1 3-octadecatrienoic (or punicic) acid, 5,8,11-eicosatrienoic acid, 8,11,14-eicosatrienoic acid and the like; tetraenoic unsaturated fatty acids such as 4,8,11,14-hexadecatetraenoic acid, 6,9,12,15-hexadecatetraenoic acid, 4,8,12,15-octadecatetraenoic (or moroctic) acid, 6,9,12,15-octadecatetraenoic acid, 9,11,13,15-octadecatetraenoic (or - or -parinaric) acid, 9,12,15,18-octadecatetraenoic acid, 4,8,12,16-eicosatetraenoic acid, 6,10,14,18-eicosatetraenoic acid, 4,7,10,13-docasatetraenoic acid, 7,10,13,16-docosatetraenoic acid, 8,12,16,19-docosatetraenoic acid and the like; penta- and hexa-enoic unsaturated fatty acids such as 4,8,12,15,18-eicosapentaenoic (or timnodonic) acid, 4,7,10,13,16-docosapentaenoic acid, 4,8,12,15,19-docosapentaenoic (or clupanodonic) acid, 7,10,13,16,19-docosapentaenoic, 4,7,10, 13,16,19-docosahexaenoic acid, 4,8,12,15,18,21-tetracosahexaenoic (or nisinic) acid and the like; branched-chain fatty acids such as 3-methylbutanoic (or isovaleric) acid, 8-methyldodecanoic acid, 10-methylundecanoic (or isolauric) acid, 11-methyldodecanoic (or isoundecylic) acid, 12-methyltridecanoic (or isomyristic) acid, 13-methyltetradecanoic (or isopentadecylic) acid, 14-methylpentadecanoic (or isopalmitic) acid, 15-methylhexadecanoic, 10-methylheptadecanoic acid, 16-methylheptadecanoic (or isostearic) acid, 18-methylnonadecanoic (or isoarachidic) acid, 20-methylheneicosanoic (or isobehenic) acid, 22-methyltricosanoic (or isolignoceric) acid, 24-methylpentacosanoic (or isocerotic) acid, 26-methylheptacosanoic (or isomonatonic) acid, 2,4,6-trimethyloctacosanoic (or mycoceranic or mycoserosic) acid, 2-methyl-cis-2-butenoic(angelic)acid, 2-methyl-trans-2-butenoic (or tiglic) acid, 4-methyl-3-pentenoic (or pyroterebic) acid and the like.

[0093] In certain preferred embodiments, active compounds (for example, amphotericin B-lipid complexes with or without deoxycholate) comprise phospholipids. Any suitable phospholipids can be used. For example, phospholipids can be obtained from natural sources or chemically synthesized. Examples of phospholipids that find use in the present invention include phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), sphingomyelin and the like, either used separately or in combination. Phosphatidylglycerols may be having short chain or long chain, saturated or unsaturated such as dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, diarachidonoylphosphatidylglycerol, short chain phosphatidylglycerol (C.sub.6-C.sub.8), and mixtures thereof. Examples of phosphatidylcholines includes dimyristoylphophatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, diarachidonoylphosphatidylcholine, egg phosphatidylcholine, soy phosphatidylcholine or hydrogenated soy phosphatidylcholine can be used, as can mixtures thereof.

[0094] In some embodiments, the present invention provides compositions comprising at least one active compound (for example, amphotericin B with or without deoxycholate) and derivatives of mono-, di- and tri-glycerides. Examples of the glycerides that find use in the present invention include but are not limited to 1-oleoyl-glycerol (monoolein) and 1,2-dioctanoyl-sn-glycerol.

[0095] Another aspect of the invention is to complex at least one active compound (for example, amphotericin B with or without deoxycholate) with at least one functionalized phospholipid, including but not limited to phosphatidylethanolamine, phosphatidylthioethanol, N-biotinylphosphatidylethanolamine, and phosphatidylethylene glycol. In some preferred embodiments, amphotericin B with or without deoxycholate is complexed with dioleoylphosphatidylethanolamine.

[0096] Another aspect of the invention is to complex at least one active compound (for example, amphotericin B with or without deoxycholate) with at least one carbohydrate-based lipid. Examples of carbohydrate-based lipids that find use in the present invention include but are not limited to galactolipids, mannolipids, galactolecithin and the like.

[0097] Yet another aspect of the invention is to complex at least one active compound (for example, amphotericin B with or without deoxycholate) with derivatives of phospholipids such as pegylated phospholipids. Examples include but not limited to the polyethylene glycol (Pegylated, PEG) derivatives of distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dioleoylphosphatidylglycerol and the like.

[0098] Another further aspect of the present invention provides compositions comprising at least one active compound (for example, amphotericin B with or without deoxycholate) and polyethylene glycol (PEG) and one or more lipids.

[0099] According to another aspect, the present invention provides compositions comprising at least one active compound (for example, amphotericin B with or without deoxycholate) complexed with one or more lipids. Example includes compositions comprising amphotericin B with or without deoxycholate, cholesterol or cholesterol derivatives and one or more phospholipids. Other examples of compositions according to the invention include amphotericin B with or without deoxycholate, -sitosterol, and one or more phospholipids. In some preferred embodiments, the composition of the present invention comprises amphotericin B, with or without deoxycholate, cholesteryl sulfate and hydrogenated soy phosphatidylcholine or soy phosphatidylcholine.

[0100] The composition of the present invention can be made by dissolving an active compound, for example, amphotericin B deoxycholate (e.g., Fungizone) in water at a concentration of about 0.5 mg/mL to about 25 mg/mL. In some embodiments, the antibiotic is dissolved at a concentration between 1 mg/mL and about 20 mg/mL. In certain preferred embodiments, the antibiotic is dissolved at a concentration of between 1 mg/mL and 10 mg/mL. In particularly preferred embodiments, the antibiotic is dissolved at a concentration of between 1 mg/mL and 5 mg/mL.

[0101] In some embodiments, compositions of the present invention contain about 2.5% to about 95% by weight of total lipid, preferably about 10% to about 90% by weight of total lipid or more, preferably about 20% to about 90% by weight of total lipid.

[0102] In some embodiments, compositions of the present invention contain at least one active compound (for example, amphotericin B, with or without sodium deoxycholate) and lipid(s) in mole ratio between 1:1 to 1:100, e.g., in between 1:1 and 1:20 molar ratio or in between 1:1 and 1:30 molar ratio or in between 1:1 and 1:40 molar ratio or in between 1:1 and 1:50 molar ratio, in between 1:1 and 1:60 molar ratio, in between 1:1 and 1:70 molar ratios, and in between 1:1 and 1:80 molar ratios. As used herein, the term in between is inclusive of the limits of a recited range. For example, a mole ratio in between 1:1 and 1:20 molar ratio includes ratios of 1:1 and 1:20.

[0103] In certain preferred embodiments, compositions of the present invention contain at least one active compound (for example, amphotericin B, with or without sodium deoxycholate), cholesteryl sulfate and hydrogenated soy phosphatidylcholine. Such compositions include amphotericin B and sodium deoxycholate in mole ratio of 1:2.

[0104] In certain preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and cholesteryl sulfate in a composition containing active compound (for example, amphotericin B), sodium deoxycholate, cholesteryl sulfate and hydrogenated soy phosphatidylcholine is in between 1:1 and 1:20, such as in between 1:1 and 1:10, or in between 1:1 and 1:5 or 1:land 1:2. In particularly preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and cholesteryl sulfate is in between 1:1 and 1:5.

[0105] In certain preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and hydrogenated soy phosphatidylcholine in a composition containing active compound (for example, amphotericin B, with or without sodium deoxycholate), cholesteryl sulfate and hydrogenated soy phosphatidylcholine is in between about 1:1 and 1:90, e.g., in between 1:1 and 1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and 1:40 and 1:1 and 1:30. In particularly preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and hydrogenated soy phosphatidylcholine is in between 1:5 and 1:60.

[0106] In certain preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and soy phosphatidylcholine in a composition containing active compound (for example, amphotericin B), with or without sodium deoxycholate, cholesteryl sulfate and soy phosphatidylcholine is in between 1:1 and 1:90, e.g., in between 1:1 and 1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and 1:40 and 1:1 and 1:30. In particularly preferred embodiments, the mole ratio of active compound (for example, amphotericin B) and soy phosphatidylcholine is in between 1:5 and 1:60.

[0107] In some embodiments, compositions of the present invention contain active compound (for example, amphotericin B) and total lipids having weight-to-weight ratio between 1:1 to 1:100 ratio such as in between 1:1 and 1:20 ratio or in between 1:1 and 1:30 ratio or in between 1:1 and 1:40 ratio or in between 1:1 and 1:50 ratio, or in between 1:1 and 1:60 ratio, or in between 1:1 and 1:70 ratio, and in between 1:1 and 1:80 ratio, or in between 1:1 and 1:90 ratio.

[0108] In some embodiments, the mole ratio of cholesterol or cholesteryl derivative (such as cholesteryl sulfate) and one or more phospholipids (for example, soy phosphatidylcholine) is in between 1:1 and 1:90, e.g., in between 1:1 and 1:70 or 1:1 and 1:60 or 1:1 and 1:50 or 1:1 and 1:40 and 1:1 and 1:30. In particularly preferred embodiments, the mole ratio of cholesterol derivative (for example, cholesteryl sulfate) and soy phosphatidylcholine is in between 1:1 and 1:20.

[0109] In some embodiments, the methods of the present invention involve dissolving active compound, e.g., amphotericin B (with or without deoxycholate), in water and mixing the dissolved antibiotic and the lipid(s) together. The active compound-lipid complex solution can be filtered through suitable filters to control the size distribution of the formed complexes.

[0110] In some embodiments, the method of the present invention involves mixing lipid(s) and sodium deoxycholate together in water and then adding active compound (for example, amphotericin B). The active compound-lipid complex solution can be filtered through suitable filters to control the size distribution of the formed complexes.

[0111] In some embodiments, the method comprises mixing amphotericin B and cholesteryl derivative, for example cholesteryl sulfate in water or buffer having pH in the range of 1 to 3.0 and can be heated if desired at temperature ranging from 25 C. to 60 C. The resulting suspension is then mixed with phospholipids, for example soy phoaphatidylcholine or hydrogenated soy phosphatidylcholine in water or buffer and the pH is adjusted with suitable base or buffer so the resulting suspension attains a pH ranging between 5.00 and 8.00. The acidic pH can be achieved by any suitable acid such as hydrochloric acid, phosphoric acid and the like. Examples of base or buffer includes but not limited to sodium succinate dibasic, sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, sodium hydroxide, and the like. The composition may further contain sugar. Examples of sugars includes but not limited to sucrose, lactose, dextrose, trehalose maltose, and the like. The percentage of sugar may range from 5% to about 25%. The resulting suspension can be homogenized or sonicated to reduce the particle size. In some embodiments, the hydrated suspension is filtered through suitable filters to control the size distribution of the formed complexes. In some embodiments, the hydrated composition can be lyophilized to obtain the composition in powder form. In some embodiments, the hydrated composition can be autoclaved.

[0112] In some embodiments, the present invention comprises mixing amphotericin B, sodium deoxycholate, and one or more lipids in any suitable sequence such that the resulting composition of the present invention comprises amphotericin B, sodium deoxycholate and one or more lipids. For example, in some embodiments, the method comprises of mixing amphotericin B in a solution containing sodium deoxycholate in water and then adjusting the pH with sodium hydroxide until the amphotericin B is completely dissolved. Lipids such as soy phosphatidylcholine are then added to the amphotericin B-sodium deoxycholate solution, followed by one more lipid, such as cholesteryl sulfate. The amphotericin B-lipid complex solution can be filtered through suitable filters to control the size distribution of the formed complexes.

[0113] In some embodiments, the present invention comprises mixing active compound (for example, amphotericin B), and one or more lipids in any suitable sequence such that the resulting composition of the present invention comprises active compound (for example, amphotericin B), and one or more lipids. For example, in some embodiments, the method comprises of mixing amphotericin B in water and then adjusting the pH with sodium hydroxide until the amphotericin B is completely dissolved. Lipids such as soy phosphatidylcholine are then added to the amphotericin B solution, followed by one more lipid, such as cholesteryl sulfate. The amphotericin B-lipid complex solution can be filtered through suitable filters to control the size distribution of the formed complexes. In another embodiment the amphotericin B and cholesteryl sulfate is mixed at any desired pH such as at low pH for example pH in between 1.00 and 4.00 or at higher pH for example, pH in between 9.00 and 12.00. The pH is then adjusted with suitable base or buffer to attain the pH of the resulting suspension in the range between 4.00 to 8.00 and then mixed with phospholipids, for example soy phosphatidylcholine or hydrogenated phosphatidylcholine.

[0114] In some embodiments, the method of preparation of the present invention comprises heating a composition comprising active compound (for example, amphotericin B in water) with or without deoxycholate and one or more lipids. In some embodiments, heating is at temperatures ranging from 30-121 C. In some preferred embodiments, heating is at a temperature between 40-80 C., while in some particularly preferred embodiments, heating is at a temperature between 40-70 C. In some embodiments, the hydrated composition can be autoclaved.

[0115] In some embodiments, the method of preparation of present invention comprising mixing active compound (for example, Tacrolimus), cholesteryl derivative (for example, cholesteryl sulfate) and phosphatidylcholine such as soy phosphatidylcholine or hydrogenated soy phosphatidylcholine in water or buffer. The resulting suspension can be homogenized or sonicated at any desired temperature ranging from 20-60 C. Examples of base or buffer includes but not limited to sodium succinate dibasic, sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, sodium hydroxide, and the like. The composition may further contain sugar. Examples of sugars includes but not limited to sucrose, lactose, dextrose, trehalose, maltose, and the like. The percentage of sugar may range from 5% to about 25%. The resulting suspension can be homogenized or sonicated to reduce the particle size. In some embodiments, the hydrated suspension is filtered through suitable filters to control the size distribution of the formed complexes. In some composition, the hydrated suspension can be lyophilized to obtain the composition in powder form. In some embodiments, the hydrated composition can be autoclaved.

[0116] In some embodiments, the method of preparation of present invention comprising mixing active compound (for example, Docetaxel), cholesteryl derivative (for example, cholesteryl sulfate) and phosphatidylcholine such as soy phosphatidylcholine or hydrogenated soy phosphatidylcholine in water or buffer. The resulting suspension can be homogenized or sonicated at any desired temperature ranging from 20-120 C. Examples of base or buffer includes but not limited to sodium succinate dibasic, sodium acetate sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, sodium hydroxide, and the like. The composition may further contain sugar. Examples of sugars includes but not limited to sucrose, lactose, dextrose, trehalose, maltose, and the like. The percentage of sugar may range from 5% to about 25%. The resulting suspension can be homogenized or sonicated to reduce the particle size. In some embodiments, the hydrated suspension is filtered through suitable filters to control the size distribution of the formed complexes. In some composition, the hydrated suspension can be lyophilized to obtain the composition in powder form. In some embodiments, the hydrated composition can be autoclaved.

[0117] In some embodiments, the method of preparation of present invention comprising mixing active compound (for example, Paclitaxel), cholesteryl derivative (for example, cholesteryl sulfate) and phosphatidylcholine such as soy phosphatidylcholine or hydrogenated soy phosphatidylcholine in water or buffer. The resulting suspension can be homogenized or sonicated at any desired temperature ranging from 20-120 C. Examples of base or buffer includes but not limited to sodium succinate dibasic, sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, sodium hydroxide, and the like. The composition may further contain sugar. Examples of sugars includes but not limited to sucrose, lactose, dextrose, trehalose, maltose, and the like. The percentage of sugar may range from 5% to about 25%. The resulting suspension can be homogenized or sonicated to reduce the particle size. In some embodiments, the hydrated suspension is filtered through suitable filters to control the size distribution of the formed complexes. In some composition, the hydrated suspension can be lyophilized to obtain the composition in powder form. In some embodiments, the hydrated composition can be autoclaved.

[0118] In some embodiments, the pH of the composition of invention ranges from about 3 to about 11, preferably having a pH of about 3.5 to about 8, and more preferably having a pH of about 4.0 to pH 8.0. In some embodiments, aqueous solutions having suitable pH are prepared from water having appropriate amount of buffers dissolved in it. In some preferred embodiments, buffers comprise mixtures of monobasic sodium phosphate, dibasic sodium phosphate and tribasic sodium phosphate. In some preferred embodiments, buffers comprise sodium carbonate, sodium bicarbonate, sodium hydroxide, ammonium acetate, sodium succinate, sodium citrate, tris (hydroxy-methyl) aminoethane, sodium benzoate, sodium acetate, and the like.

[0119] In some embodiments, filters are used to obtain the desired size range of the complexes from the filtrate. For example, the complexes can be formed and thereafter filtered through a 5 micron filter to obtain complex having a diameter of about 5 micron or less. Alternatively, 1 m, 500 nm, 200 nm, 100 nm or other filters can be used to obtain complexes having diameters of about 1 m, 500 nm, 200 nm, 100 nm or any suitable size range, respectively.

[0120] In some embodiments, the composition of the present invention can be sterilized by filtering through 0.22 m or 0.45 m filter under aseptic conditions. In another embodiments, the composition of the present invention can be sterilized by autoclaving in the range of 120 C.-130 C. for a duration of 15-20 minutes.

[0121] In some embodiments, the active compound-lipid complex (for example, amphotericin B-lipid complex) with or without deoxycholate is dried, e.g., by evaporation or lyophilization. In certain embodiments of the invention, the active compound-lipid complex (for example, amphotericin B-lipid complex) with or without deoxycholate is lyophilized with one or more cryoprotectants, such as sugars. Examples of sugars that find use in the present invention include but are not limited to trehalose, maltose, lactose, sucrose, glucose, and dextran. In preferred embodiments, the compositions of the present invention comprise trehalose and/or sucrose. The lyophilization is generally accomplished under vacuum and can take place either with or without prior freezing of the active compound-lipid complex (for example, amphotericin B-lipid preparation) with or without deoxycholate. While not limiting the lyophilization of the present invention to any particular configuration, the lyophilization in the present invention can be done, e.g., in vials or other containers having desired volumes. The lyophilization can also be done as bulk in trays. When desired, the complexes can be resuspended in any desirable solvent including water, saline, dextrose and buffer.

[0122] Pharmaceutical preparations that find use in the present invention include but are not limited to tablets, capsules, pills, dragees, suppositories, solutions, suspensions, emulsions, ointments; gels can be suitable pharmaceutical preparations. In some embodiments, e.g., for the oral mode of administration, active compound-lipid complex (for example, amphotericin B-lipid complex, tacrolimus lipid complex, paclitaxel or docetaxel lipid complexes) with or without deoxycholate is used in the form of tablets, capsules, lozenges, powders, syrups, aqueous solutions, suspensions and the like. In some embodiments, e.g., for topical application and suppositories, active compound-lipid complex (for example, amphotericin B-lipid complex, with or without deoxycholate) is provided in the form of gels, oils, and emulsions, such as are known by the addition of suitable water-soluble or water-insoluble excipients, for example polyethylene glycols, certain fats, and esters, compounds having a higher content of polyunsaturated fatty acids and derivatives thereof. Derivatives include but are not limited to mono-, di-, and triglycerides and their aliphatic esters (for example, fish oils, vegetable oils etc.) or mixtures of these substances. In some embodiments, excipients that find use in conjunction with the compositions of the present invention comprise those in which the drug complexes are sufficiently stable to allow for therapeutic use.

[0123] In some embodiments, preparations of active compound-lipid complex (for example, amphotericin B-lipid complex with or without deoxycholate or tacrolimus-lipid complex, paclitaxel or docetaxel lipid complexes) are prepared in enteric coated tablets or capsules, e.g., to protect it from acids in the stomach. Enteric refers to the small intestine, therefore enteric coating generally refers to a coating that substantially prevents release of a medication before it reaches the small intestine. While not limiting the invention to any particular mechanism of action, it is understood that most enteric coatings work by presenting a surface that is stable at acidic pH but breaks down rapidly at higher pH. Enteric coatings that find use in the present invention comprise capsules filled with active compound-lipid complex (for example, amphotericin B-lipid complex with or without deoxycholate, tacrolimus lipid complex, paclitaxel or docetaxel lipid complexes) as according to methods well known in the art.

[0124] Preparations of active compound-lipid complex (for example, amphotericin B-lipid complex) with or without deoxycholate of the present invention can comprise complexes of varying size, or can comprise complexes of substantially uniform size. For example, in some embodiments the complexes have a size range of about 1 mm or less, while in preferred embodiments, the complexes are in the micron or sub-micron range. In some embodiments, the complexes have a diameter of about 5 m or less, such as 0.2 m or less, or even 0.1 m or less.

[0125] Active compound-lipid complex (for example, amphotericin B-lipid complex, with or without deoxycholate) of the present invention may comprise or consist essentially of micelles, mixed micelles, liposomes and vesicles of different shape and sizes.

[0126] As noted above, the technology outlined in the present invention for the preparation of amphotericin B complexes is also suitable for use with any other water-insoluble drugs.

[0127] In some embodiments, the inventive amphotericin B-lipid complex (with or without deoxycholate) is employed to treat a fungal infection, e.g., in a mammal. In this regard, the invention provides a method of treating fungal infections comprising administering to a subject (e.g. a patient having a fungal infection) a composition comprising a complex of amphotericin B-with or without deoxycholate and lipid(s) in an amount sufficient to treat the fungal infection within the subject.

[0128] The composition of the present invention can be employed to treat infections caused by numerous fungi and parasites, including but not limited to, Acremonium sp., Aspergillus fumigatus, Aspergillus pneumonia, Blastomyces dermatitides, Candida albicans, Candida guillermondi, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans, Fusarium sp., Histoplasma capsulatum, Mucor mucedo, Rhodotorula sp., Sporothrix schenckii, Acanthamoeba polyphaga, Entomophthora sp., Histoplasma capsulatumm Leishmania brasiliensis, Rhizopus sp., Rhodotorula sp., Torulopsis glabrata, Paracoccidioides brasiliensis. Additional fungal pathogens include Trichosporon, Muco, Alternaria, Bipolaris, Curvularia, etc.

[0129] The composition of present invention can also be employed to treat Visceral Leishmaniasis also called as Kala-azar and infections caused by Leishmania donovani complex, L. d donovani, L. d infantum, L. d archibaldi, L. d chagasi, Phlebotomus sp. and Lutzomya logipalpis.

[0130] The composition of present invention can also be employed to treat viral infections such as those caused, e.g., by human immunodeficiency virus (HIV), herpes simplex viruses (HSV-1 and HSV2), hepatitis C virus (HCV) and cyotomegalovirus (CMV).

[0131] In some embodiments, the inventive active compound lipid-complex (for example, docetaxel-lipid complex or paclitaxel-lipid complex) is employed to treat a cancer, e.g., in a mammal. In this regard, the invention provides a method of treating cancer comprising administering to a subject (e.g. a patient having a cancer) a composition comprising a complex of active compound lipid-complex (for example, docetaxel-lipid complex or paclitaxel-lipid complex) and lipid(s) in an amount sufficient to treat the cancer within the subject. The cancer can be any type of cancer in a mammal. Examples include, but are not limited to cancers of the head, neck, brain, blood, (e.g. leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, lymphoma, myeloma), breast, lung, pancreas, bone, spleen, bladder, prostate, testes, colon, kidney, ovary and skin (e.g. Kaposi's sarcoma), bone marrow, liver, stomach, tongue, mouth and larynx. In addition, active compound-lipid complex of the present invention are useful in reducing the tendency of cancer cells to develop a resistance to other therapeutic agents such as anti-cancer agents, chemotherapy and radiation. Thus, other therapeutic agents can be advantageously employed with the present invention in the formation of an active combination or by separate administration.

[0132] In some embodiments, the inventive active compound lipid-complex (for example, tacrolimus-lipid complex) is employed to treat rejection reactions caused by organ transplantations and can be administered organ or tissue transplantation, e.g., in a mammal. In this regard, the invention provides a method of preventing organ or tissue rejection comprising administering to a subject (e.g. a patient having an organ or tissue transplantation) a composition comprising a complex of active compound lipid-complex (for example, tacrolimus-lipid complex) and lipid(s) in an amount sufficient to prevent an organ or tissue rejection within the subject.

[0133] The examples of the present invention are illustrated below but the invention is not limited to the following examples and modifications can be made without departing from the purports described in this application.

EXAMPLE 1

[0134] Amphotericin B (1 gm) was suspended in aqueous medium at pH 1.5 to 3.5 and mixed with 3 gm of Sodium Cholesteryl Sulfate. Soya Phosphatidylcholine (7 gm) was stirred and mixed with Amphotericin B and Sodium Cholesteryl Sulfate Complex for 30 min. The mixture was then subjected to high pressure homogenization. The formulation was lyophilized in the presence of 7.5-9.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume diameter amounted to less than 200 nm

EXAMPLE 2

[0135] Amphotericin B formulation with lipids as described in Example I was used to test the hemolysis of red blood cells (RBCs). At 0.16 mg/mL FUNGIZONE50% of the cells were lysed compared to Amphotericin B lipid suspension where no lysis occurred after incubation with RBCs. Toxicity study was also carried out in Balb/c mice. A total of 9 mice (7 weeks old) were subjected to intravenous administration of amphotericin B formulation at 20 mg/kg. The mice were monitored for 30 days. At the end of 30 days no mortality was observed. This indicated that maximum tolerated dose using this formulation exceeds 20 mg/kg.

TABLE-US-00001 Group Dose Survival I 20 mg/kg 9/9

EXAMPLE 3

[0136] Amphotericin B (1 gm) was suspended in aqueous medium at pH 1.5 to 3.5 and mixed with 3 gm of sodium cholesteryl sulfate. Hydrogenated soya phosphatidylcholine (7 gm) was stirred and mixed with amphotericin B and sodium cholesteryl sulfate complex for 30 min. The mixture was then subjected to high pressure homogenization. The formulation was lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm

EXAMPLE 4

[0137] Amphotericin B (20 mg) and sodium deoxycholate (6.56 mg) were dissolved in water (10 mL) at pH 11.00 to 12.5 using sodium hydroxide. The pH was then adjusted to pH 7.00-8.5 with suitable acid (for example, phosphoric acid). Hydrogenated soy phosphatidylcholine (930 mg) and cholesteryl sulfate (10.4 mg) was mixed in water (10 mL) and homogenized or sonicated for 30 minutes. The lipid suspension was then mixed with amphotericin B-deoxycholate solution and further homogenized or sonicated for 1 hr. The suspension can be heated if desired at temperature ranging from 25 C. to 60 C. The formulation was lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The formulation was tested for toxicity in Balb/c mice and compared with Deoxycholate formulation of Amphotericin B (FUNGIZONE). The animals were weighed and assigned to different groups randomly (5 animals/group). The results are reported in the table below as the number of mice surviving per total.

TABLE-US-00002 Treatment Dose (mg/kg) Survival/Total Fungizone 0.5 5/5 1.0 5/5 2.0 4/5 4.0 0/5 Amphotericin - B 12.0 5/5 Formulation 14.0 5/5 17.0 5/5 20.0 0/5
The data indicated that the liposome formulation of amphotericin B was significantly less toxic when compared to the marketed product (FUNGIZONE).

EXAMPLE 5

[0138] Amphotericin B formulation with lipids as described in Example IV was prepared without deoxycholate. The resulting formulation was lyophilized in the presence of 7.5% sucrose or lactose. This formulation also showed similar characteristics as of Example 4.

EXAMPLE 6

[0139] Amphotericin B (50 mg) and Cholesteryl sulfate (50 mg) were mixed together in water at pH 2.5-3. SPC (500 mg) was suspended in water separately which was mixed with amphotericin B and cholesteryl sulfate suspension and homogenized using high pressure homogenizer. The formulation was lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The reconstituted formulation was tested for toxicity in Balb/c mice with single dose intravenous injection and no mortality was observed at 20 mg/kg dose level as found in Example II. The particle size was determined using Nicomp particle sizer 380. The particle size data is given in the table below.

TABLE-US-00003 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 128.4 nm 99% Distribution 401.8 nm 90% Distribution 224.6 nm 80% Distribution 175.9 nm 75% Distribution 160.3 nm 50% Distribution 110.2 nm 25% Distribution 75.9 nm

EXAMPLE 7

[0140] Amphotericin B (100 mg) and deoxycholate (33 mg) were dissolved in water at pH 9-12.00 and later adjusted to pH 7.5. The amphotericin B suspension was then mixed with cholesteryl sulfate (52 mg) and hydrogenated soyphosphatidylcholine (4.62 g) in water and sonicated at 60 minutes. The formulation was lyophilized both in vials and in bulk in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm

TABLE-US-00004 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 76.1 nm 99% Distribution 227.1 nm 90% Distribution 130.2 nm 80% Distribution 103.1 nm 75% Distribution 94.3 nm 50% Distribution 66.0 nm 25% Distribution 46.2 nm

EXAMPLE 8

[0141] Amphotericin B (50 mg) and Cholesteryl sulfate (50 mg) are mixed together in sodium succinate buffer at pH 2.5-3. SPC (500 mg) in sodium succinate buffer is suspended in water separately which is mixed with amphotericin B and cholesteryl sulfate suspension and homogenized using high pressure homogenizer. The formulation is lyophilized in the presence of 7.5-9.5% sucrose or 9.5% lactose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm

EXAMPLE 9

[0142] Amphotericin B (2 g) and Cholesteryl sulfate (1.04 g) were mixed together in succinate buffer at pH 2.5 and sonicated for 5 min at room temperature. Soy lecithin (18.96 g) in sodium succinate buffer (pH 2.5) was with Amphotericin-Cholesteryl sulfate suspension and homogenized using high pressure homogenizer. The formulation was then autoclaved at 121 C. for 15 minutes before it was mixed with 7.5-9.5% sucrose or 9.5% lactose solution under aseptic conditions. The particle size was determined using Nicomp particle sizer 380. The particle size data is shown in the table below.

TABLE-US-00005 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 693.3 nm 99% Distribution 1992.5 nm 90% Distribution 1169.7 nm 80% Distribution 934.6 nm 75% Distribution 858.2 nm 50% Distribution 608.4 nm 25% Distribution 431.3 nm
The HPLC analysis of the inventive formulation comprising amphotericin B, soy phosphatidylcholine, cholesteryl sulfate was done and the results are outlined in the table below.

TABLE-US-00006 Components Assay Results Amphotericin B 96.4% Cholesteryl Sulfate 95.0% Soy Phosphatidylcholine 87.5%
Systemic Adverse Events: A Comparison between FUNGIZONE and Amphotericin B Lipid Suspension in Healthy Human Volunteers.

[0143] The safety and tolerance of FUNGIZONE versus Amphotericin B Lipid Suspension was evaluated in Human male subjects. In this study a total 24 volunteers were enrolled. Out of this six (n=6) were given FUNGIZONE (0.6 mg/kg) intravenously and eighteen (n=18) of them received Amphotericin B Lipid Suspension (0.6 mg/kg-1.5 mg/kg).

[0144] In the Amphotericin B Lipid Suspension, mild adverse events were reported in 3/18 (17%) healthy male subjects and 4/6 (66%) who were infused FUNGIZONE. Overall, Amphotericin B Lipid Suspension is apparently safe and well tolerated up to 1.5 mg/kg.

EXAMPLE 10

[0145] Tacrolimus (20 mg) and Cholesteryl sulfate (20 mg) were mixed in water (10 mL) and sonicated for 30 min to form a suspension. SPC in water (10 mL) was mixed with Tacrolimus and Cholesteryl Sulfate suspension and homogenized using high pressure homogenizer. The formulation was lyophilized both in vials and in bulk in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume diameter amounted to less than 200 nm.

EXAMPLE 11

[0146] Deoxycholate (1 mg) and Cholesteryl sulfate (1 mg) were mixed in water and sonicated for 30 min to form a suspension. SPC in water was mixed with Tacrolimus and Cholesteryl Sulfate suspension and homogenized using high pressure homogenizer. The formulation was lyophilized in the presence of 7.5-% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume diameter amounted to less than 200 nm.

EXAMPLE 12

[0147] Tacrolimus (100 mg), Cholesteryl sulfate (60 mg), and Soy lecithin (3.94 g) were mixed together in water (70 mL) and homogenized using high pressure homogenizer. The resulting suspension was then filtered through 0.2 filter and then mixed with 7.5% sucrose solution (30 mL) and lyophilized both in vials and in bulk. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm.

TABLE-US-00007 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 42.9 nm 99% Distribution 141.0 nm 90% Distribution 76.5 nm 80% Distribution 59.2 nm 75% Distribution 53.8 nm 50% Distribution 36.5 nm 25% Distribution 25.2 nm

EXAMPLE 13

[0148] Tacrolimus (200 mg), Cholesteryl sulfate (120 mg), and Soy lecithin (7.88 g) were mixed together in water (70 mL) and homogenized using high pressure homogenizer. The resulting suspension was then filtered through 0.2 filter and then mixed with 7.5% sucrose (30 mL) and lyophilized both in vials and in bulk. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm.

TABLE-US-00008 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 76.4 nm 99% Distribution 240.8 nm 90% Distribution 134.3 nm 80% Distribution 105.1 nm 75% Distribution 95.8 nm 50% Distribution 65.9 nm 25% Distribution 45.5 nm

[0149] The Tacrolimus lipid suspension was tested for toxicity in Balb/c mice. The single test dose at 10 mg/kg and 20 mg/kg was intravenously administered to mice. All the mice survived with no significant loss of body weight. Similarly, repeat dose toxicity study was conducted with a dose of 10 mg/kg or 20 mg/kg for consecutively 5 days with accumulated dose of 50 mg/kg and 100 mg/kg respectively. All the animals in the group survived. The results are reported in the table below as the number of mice surviving per total.

TABLE-US-00009 Treatment Dose (mg/kg) Survival/Total Single dose 10 5/5 20 5/5 Repeat dose 10 5/5 20 5/5

EXAMPLE 14

[0150] Cholesteryl sulfate (2.08 mg) and hydrogenated soyphosphatidylcholine (185.92 mg) in 0.9% aq. Sodium chloride solution (2 mL) was sonicated at 65 C. for 30 minutes before Doxorubicin (40 mg) in 0.9% sodium chloride solution (2 mL) was added and further sonicated for 60 minutes. The formulation was lyophilized in the presence of 7.5% sucrose or and reconstituted in water for injection.

EXAMPLE 15

[0151] Cholesteryl sulfate (20 mg) and soy lecithin (156.8 mg) in 0.9% aq. sodium chloride solution was sonicated at 65 C. for 30 minutes before Doxorubicin (40 mg) in 0.9% sodium chloride solution (10 mL) was added and further sonicated for 60 minutes. The formulation is lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume diameter amounted to less than 200 nm.

EXAMPLE 16

[0152] Docetaxel (20 mg), Cholesteryl sulfate (12.0 mg), and Soy lecithin (788 mg) were mixed together in water (10 mL) and using high pressure homogenizer. The formulation is lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm.

TABLE-US-00010 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 93.9 nm 99% Distribution 264.1 nm 90% Distribution 157.0 nm 80% Distribution 126.1 nm 75% Distribution 116.0 nm 50% Distribution 83.0 nm 25% Distribution 59.4 nm

EXAMPLE 17

[0153] Docetaxel (40 mg), Cholesteryl sulfate (24.0 mg), and Soy lecithin (1.57 g) were mixed together in water (10 mL) using high pressure homogenizer. The formulation is lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume diameter amounted to less than 200 nm

EXAMPLE 18

[0154] Paclitaxel (20 mg), Cholesteryl sulfate (11.4 mg), and Soy lecithin (788.6 mg) were mixed together in water (10 mL) and homogenized using high pressure homogenizer. The formulation is lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The mean volume weighting diameter amounted to less than 200 nm

TABLE-US-00011 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 124.1 nm 99% Distribution 357.4 nm 90% Distribution 209.6 nm 80% Distribution 167.4 nm 75% Distribution 153.7 nm 50% Distribution 108.9 nm 25% Distribution 77.2 nm

[0155] The paclitaxel lipid suspension was tested for toxicity in Balb/c mice. The test dose (40 mg/kg) was intravenously administered to mice and the animals were monitored for 30 days. All the mice survived with no significant loss of body weight. Similarly, repeat dose toxicity study was conducted with a dose of 40 mg/kg for consecutively 5 days with accumulated dose of 200 mg/kg. All the animals in the group survived. The study was monitored for 30 days. The results are reported in the table below as the number of mice surviving per total

TABLE-US-00012 Treatment Dose (mg/kg) Survival/Total Single dose 40 3/3 Repeat dose 40 4/4

EXAMPLE 19

[0156] Paclitaxel (40 mg), Cholesteryl sulfate (22.8 mg), and Soy lecithin (1.58 g) were mixed together in water (10 mL) and homogenized using high pressure homogenizer. The formulation is lyophilized in the presence of 7.5% sucrose and reconstituted in water for injection. The particle size was determined using Nicomp particle sizer 380. The particle size data is shown in the table below.

TABLE-US-00013 Mean/Distributions Particle Size (Volume Weighting) Mean Volume Weighting Diameter 839.1 nm 99% Distribution 3425.8 nm 90% Distribution 1636.3 nm 80% Distribution 1185.7 nm 75% Distribution 1048.7 nm 50% Distribution 638.5 nm 25% Distribution 388.7 nm

REFERENCES

[0157] 1. Abra, R. and Szoka, F. C.; PCT Appl WO8701933, 1987; Sanders, S. et al. 1991

[0158] 2. Abra, R. U.S. Pat. No. 5,032,582, 1991.

[0159] 3. Abra, R. U.S. Pat. No. 4,822,777, 1989

[0160] 4. Abra, R. and Gua, L. S U.S. Pat. No. 5,194,266, 1993

[0161] 5. Adler-Moore, J.; Jill, P.; Proffitt, R. t. J. Liposome Res. 1993, 2, 429-450.

[0162] 6. Ahmad, I., Agarwal, A.; Pal, A.; Guru, P. Y.; Bachhawat, B. K. and Gupta, C. M. J. Biosciences, 1991, 14, 217-221.

[0163] 7. Ahmad, I.; Sarkar, A. K.; and Bachhawat, B. K. Mol. Cell. Biochem. 1989, 91, 85-90.

[0164] 8. Ahmad, I.; Sarkar, A. K.; and Bachhawat, B. K. Biot. Appl. Biochem. 1990, 12, 550-556.

[0165] 9. Bissery, M-C.; Laborie, M.; Vacu, J.; Verrecchia, T. U.S. Pat. No. 6,146,663, 2000.

[0166] 10. Brajtburg, J.; Powderly, W. G.; Kobayashi, G. S.; Medoff, G. Antimicrob. Agents and Chemother. 1990, 34, 381-384.

[0167] 11. De Kruijff, B. and Demel, R. A.; Biochim. Biophys. Acta, 1974, 339, 57-70.

[0168] 12. Deray G.; Mercadal, L.; Bagnis, C. Nephrologie, 2002, 23, 119-122.

[0169] 13. Dumont, F. J. In:Libermann, R. Mukherjee, A. Eds. Drug development in transplantation and autoimmunity, Austin, Tex.: RG Landes, 1996, 175.

[0170] 14. Hammarstrom, L.; and Smith, C. I. E. Acta Patho. Microbial. Scand. 1977, 85, 277-283.

[0171] 15. Hennenfent, K. L. and Govindan, R. Annals of Oncol. 2006, 17, 735-749.

[0172] 16. Janoff, A. S.; Madden, T. D.; Cullis, P. R.; Kearns, J. J.; Durning, A G.; U.S. Pat. No. 6,406,713, 2002.

[0173] 17. Ingle, G. R.; Sievers, T. M.; Holt, C. D. Ann Pharmacother, 2000, 34, 1044-1055.

[0174] 18. Knoll, G. A. and Bell, R. C. BMJ, 1999, 318, 1104-1107.

[0175] 19. Lister, J. Eur J. Haematol. 1996, 56 (suppl 57), 18-23.

[0176] 20. Little, J. R.; Plut E. J., Kotler-Brajtburg, J.; Mendoff, G. and Kobayashi, G. S. Immunochem., 1978, 15, 219-224.

[0177] 21. Medoff, G. and Kobayashi, G. S. J Am. Med. Assoc. 1975, 232, 619-620.

[0178] 22. Podder, H.; Podbielski, J.; Hussein, I.; Katz, S.; van Buren, C.; Kahan, B. D.; Transpl. Int. 2001, 14, 135-142.

[0179] 23. Proffitt, R. T.; Adler-Moore, J.; Fujii, G.; Satorius, A, Lee, M. J. A.; Bailey, A.; J. Controlled Release 1994, 28, 342-343.

[0180] 24. Otsubo, T.; Maesaki, S.; Hossain, M. A.; Yamamoto, Y.; Tomono, K.; Tashiro, T.; Seki, J.; Tomii, Y.; Sonoke, S.; Kohno, S. J. Antimicrob. Agents and Chemother. 1999, 43, 471-475.

[0181] 25. Proffitt, R. T.; Adler-Moore, J., Chiang, S-M U.S. Pat. No. 5,965,156 1999.

[0182] 26. Proffitt, R. T.; Satorius, A.; Chiang, S. M.; Sullivan. L.; Adler-Moore, J. P. J. Antimicrob. Agents Chemother. 1991, 28 (Suppl. B), 49-61.

[0183] 27. Sanders, S. W.; Buchi, K. N.; Goddard, M. S.; Lang, J. K.; Tolman, K. G. Antimicrob. Agents and Chemother. 1991, 35, 1029-1034.

[0184] 28. Ramos, H.; Brajtburg, J.; Marquez, V.; Cohen, B. E. Drugs Exptl. & Clin. Res. 1995, 21, 211-216.

[0185] 29. Ramos, H.; Valdivieso, E.; Gamargo, M.; Dagger, F.; Cohen, B. E. J. Memb. Biol. 1996, 152, 6575.

[0186] 30. Schiff, P. B.; Fant, J.; Horowitz, S. B. Nature, 1979, 227, 665-667.

[0187] 31. Straubinger, R. M.; Sharma, A.; Mayhew, E. U.S. Pat. No. 5,415,869, 1995.

[0188] 32. Szoka, F. C. Jr. U.S. Pat. No. 5,277,914 A, 1994

[0189] 33. Szoka, F. C. Jr,; Milholland, D.; Barza, M. Antimicrob. Agents and Chemother. 1987, 31, 421-429.

[0190] 34. ten Tije, A. J.; Verweij, J.; Loos, W. J.; Sparreboom, A. Clin Pharmacokinet, 2003, 42, 665-685.

[0191] 35. Valeriote, F.; Lynch, R.; Medoff, G.; and Kumar, B. V. J. Natl. Cancer. Inst. 1976, 56, 557-559.

[0192] 36. Walsh, T. J.; Whitcomb, P.; Piscitelli, S. Figg, W. D.; Hill, S.; Chanock, S. J.; Jarosinski, P.; Gupta, R.; Pizzo, P. A. J. Antimicrob. Agents Chemother. 1997, 41, 1944-1948.

[0193] All references, including publications, patent applications, and patents cited herein, including those in the preceding list and otherwise cited in this specification, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were set forth in the entirely herein.

[0194] Preferred embodiments of this invention are described, including the best mode known to the inventors for carrying out the invention. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention, and the inventors intend for the inventions to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Indeed, any modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.