Protein-stabilized liposomal formulations of pharmaceutical agents

1. A method of preparing protein-stabilized liposome nanoparticles containing at least one lipophilic pharmaceutical agent, the method comprising:

(a) preparing an oil-in-water emulsion by a process comprising

i) preparing an organic solution comprising one or more phospholipids;

ii) mixing the at least one lipophilic pharmaceutical agent into said organic solution;

iii) infusing said organic mixture into an aqueous solution comprising an emulsion-forming protein, wherein said emulsion-forming protein comprises from about 0.1% to about 20% (w/v) of the aqueous solution, to form a the oil-in-water emulsion; and

(b) removing organic solvent from said emulsion to form the protein-stabilized liposome nanoparticles, wherein said liposome nanoparticles have sizes below 400 nm and said emulsion-forming protein adsorbed onto their surface.

2. The method of claim 1 , wherein the organic solution further includes cholesterol.

3. The method of claim 1 , wherein the organic solution further includes a PEG-phospholipid.

4. The method of claim 3 , wherein said PEG-phospholipid comprises poly(ethylene glycol)-derivatized distearoylphosphatidylethanolamine (PEG-DSPE) and/or poly(ethylene glycol)-derivatized ceramides (PEG-CER).

5. The method of claim 1 , wherein the lipophilic pharmaceutical agent is further identified as a substantially water insoluble pharmaceutical agent.

6. The method of claim 1 , wherein the phospholipid and lipophilic pharmaceutical agent are included in the organic solution at a ratio ranging from about 0.01:25 to about 1:10 drug:lipid (w/w).

7. The method of claim 1 wherein said emulsion forming protein comprises an albumin, immunoglobulin, casein, insulin, hemoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, or enzyme.

8. The method of claim 7 , wherein the emulsion forming protein is an albumin.

9. The method of claim 1 , wherein said aqueous solution is at a pH of between about 6.0 and 7.6.

10. The method of claim 1 , wherein said emulsion-forming protein comprises from about 1% to about 10% (w/v) of the aqueous solution.

11. The method of claim 1 , wherein the emulsion is formed by steps that comprise:

(a) agitating said mixture at between about 1000 and about 24,000 revolutions per minute; and

(b) subjecting the agitated mixture to high pressure microfluidization or homogenization at 5,000 to 30,000 PSI.

12. The method of claim 11 , wherein the emulsion is formed by steps that comprise:

(a) agitating said mixture at between about 3000 and about 10,000 revolutions per minute; and

(b) subjecting the agitated mixture to high pressure microfluidization or homogenization at 10,000 to 30,000 PSI.

13. The method of claim 11 , wherein the mixture is agitated using a homogenizer.

14. The method of claim 13 , wherein the mixture is agitated using a rotary homogenizer.

15. The method of claim 1 , wherein the lipid formulation is filtered.

16. The method of claim 15 , wherein the lipid formulation is filtered through a 0.20 or 0.22 micron filter.

17. The method of claim 1 , wherein the lipid formulation is lyophilized to provide a powder suitable for reconstitution in an aqueous suspension.

18. The method of claim 17 , wherein the lipid formulation is lyophilized in the presence of one or more cryoprotectants.

19. The method of claim 18 wherein the cryoprotectant is sucrose, mannitol or trehalose.

20. The method of claim 1 wherein the pharmaceutical agent comprises a cardiovascular drug, respiratory drug, sympathomimetic drug, cholinomimetic drug, adrenergic or adrenergic neuron blocking drug, analgesic/antipyretic, anesthetic, antiasthamatic, antibiotic, antidepressant, antidiabetic, antifungal agent, antihypertensive agent, anti-inflammatory, antineoplastic, antianxiety agent, immunosuppressive agent, antimigraine agent, sedatives/hypnotic, antianginal agent, antipsychotic agent, antimanic agent, antiarrhythmic, antiarthritic agent, antigout agent, anticoagulant, thrombolytic agent, antifibrinolytic agent, hemorheologic agent, antiplatelet agent, anticonvulsant, antiparkinson agent, antihistamine/antipruritic, agent useful for calcium regulation, antibacterial agent, antiviral agent, antimicrobial, anti-infective, bronchodialator, hormone, hypoglycemic agent, hypolipidemic agent, protein, nucleic acid, agent useful for erythropoiesis stimulation, antiulcer/antireflux agent, antinauseant/antiemetic, oil-soluble vitamin, mitotane, visadine, halonitrosourea, anthrocycline or ellipticine.

21. The method of claim 20 , wherein the pharmaceutical agent is Proscillaridin-A, oleandrin, digitoxin, digoxin, odoroside-A, colchicine, colchicine derivatives, ara-C derivatives, diphyllin, justicidin-A, diphyllin derivatives, cleistanthin A, chloroquine, amphotericin B, paclitaxel, docetaxel, cyclosporine, camptothecin, 7-ethyl-10-hydroxy-camptothecin, 10-hydroxy-camptothecin, a camptothecin derivative, podophyllotoxin, podophyllotoxin derivatives, vinblastine, vincristine, dihydroartemisinin, mitoxantrone, amphotericin B, epothilone-A, epothilone-B, epothilone-C, epothilone-D, an epothilone derivative, a benz[c]-phenanthridine alkaloid derivative, a chelerythrine alkaloid derivative or cisplatin.

22. The method of claim 1 , wherein said one or more phospholipids comprise one or more of hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinsitol (PI), monosialogangolioside, spingomyelin (SPM), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or dimyristoylphosphatidylglycerol (DMPG).

23. The method of claim 1 , wherein the ratio of pharmaceutical agent to lipid-protein ranges from about 0.0005 to about 1 (w/w).

24. The method of claim 23 , wherein the ratio of pharmaceutical agent to lipid-protein ranges from about 0.0005 to about 0.5 (w/w).

25. The method of claim 24 , wherein the ratio of pharmaceutical agent to lipid-protein ranges from about 0.001 to about 0.1 (w/w).

26. The method of claim 1 , wherein the lipid formulation is subjected to high shear stress to reduce particle size.

27. The method of claim 1 , wherein the lipid formulation is defined further as comprising nanoparticles having size less than about 220 nm.

28. The method of claim 27 , wherein the nanoparticles have a size of between about 80–160 nm.

29. The method of claim 28 , wherein the nanoparticles have a size of between about 100–120 nm.

30. A protein-stabilized lipid formulation comprising protein-stabilized liposome nanoparticles and a lipophilic pharmaceutical agent, the formulation prepared by a process selected from the group of processes consisting of those processes set forth in any one of claims 1 , 2 – 9 and 10 – 29 .