Claims for Patent: 7,320,963

Summary for Patent: 7,320,963

Title:	Methods and compositions for delivery of pharmaceutical agents
Abstract:	Methods and compositions for delivering pharmaceutical agents into cells, in particular urothelial cells of the bladder, are provided. In the methods and compositions of the invention, a solubilized cholesterol composition is used to facilitate delivery of pharmaceutical agents. Preferably, the cholesterol is solubilized by a cyclodextrin (e.g., methyl-.beta.-cyclodextrin) and the pharmaceutical agent comprises a polynucleotide and either a cationic lipid, a cationic polymer or a dendrimer. Improved methods for transfecting polynucleotides into cells thus are also provided, using cationic lipids, cationic polymers or dendrimers and solubilized cholesterol, wherein the transfection efficiency is enhanced compared to use of cationic lipids, cationic polymers or dendrimers alone. Preferred methods of the invention involve transfecting polynucleotides into urothelial cells, preferably for therapeutic treatment of bladder cancer using, for example, a polynucleotide(s) encoding an interleukin(s), an interferon(s), a colony stimulating factor(s) and/or a tumor suppressor(s).
Inventor(s):	Esuvaranathan; Kesavan (Singapore, SG), Mahendran; Ratha (Singapore, SG), Lawrencia; Carmel (Singapore, SG)
Assignee:	Genecure Pte Ltd (Singapore, SG)
Application Number:	10/086,973
Patent Claims:	1. A transfection composition comprising: (i) a polynucleotide; (ii) a cationic lipid, a cationic polymer or a dendrimer, or combinations thereof; and (iii) a solubilized cholesterol preparation, wherein the solubilized cholesterol preparation comprises cholesterol solubilized with a cyclodextrin. 2. The transfection composition of claim 1, wherein the cyclodextrin is methyl-.beta.-cyclodextrin. 3. The transfection composition of claim 1, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, sulfated beta-cyclodextrin, tertiary amine beta-cyclodextrin, quaternary amine beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, 2,6-di-O-methyl-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose, sulfobutylether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin and maltosyl-beta-cyclodextrin. 4. The transfection composition of claim 1, wherein the cationic lipid, cationic polymer or dendrimer is selected from the group consisting of dioleoyl phosphatidylethanolamine (DOPE), [2,3-bis(oleoyl)propyl] trimethyl ammonium chloride (DOTMA), dioctadecyl amido glycyl spermine (DOGS), dioctadecyl diammonium bromide (DODAB), dioctadecyl diammonium chloride (DODAC), 2,3 dioleoyloxy-N-[sperminecarboxaminoethyl]-N-N-dimethyl-1-propanaminium (DOSPA), 3.beta.[N-(n', N'-dimethylaminoethane)-carbamoyl]cholesterol, dioleoyl (DC-Chol), 1-2-(oleoyloxy)-ethyl]-2-oleoyl]-3-(2-hydroxyethyl) imidazolinium chloride (DOIC), dioleoyl phosphatidyicholine (DOPC), dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide (DMRIE), poly amino amide (PAMAM), polylysine, polyhistidine, polyarginine, polyethyleneimine, poly(4-vinylpyridine), poly(vinylamine), poly(4-vinyl-N-alkyl pyridinium halide), or combinations thereof. 5. The transfection composition of claim 1, wherein (ii) is a cationic lipid which is DOTAP. 6. The transfection composition of claim 1, wherein (ii) is a dendrimer which is cationic dendrimer. 7. The transfection composition of claim 1, wherein the polynucleotide is plasmid DNA. 8. The transfection composition of claim 1, wherein the polynucleotide is selected from the group consisting of viral DNA, chromosomal fragments, antisense oligonucleotides, antisense phosphorothioate oligonucleotides, RNA molecules and ribozymes, or combinations thereof. 9. A method for transfecting a polynucleotide into cells, the method comprising: applying the transfection composition of claim 1 to cells, such that the cells are transfected with the polynucleotide. 10. The method of claim 1, wherein the cyclodextrin is methyl-.beta.-cyclodextrin. 11. The method of claim 1, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, sulfated beta-cyclodextrin, tertiary amine beta-cyclodextrin, quaternary amine beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, 2,6-di-O-methyl-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose, sulfobutylether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin and maltosyl-beta-cyclodextrin. 12. The method of claim 1, wherein (ii) is a cationic lipid which is DOTAP. 13. The method of claim 1, wherein (ii) is a dendrimer which is cationic dendrimer. 14. The method of claim 1, wherein (ii) is selected from the group consisting of DOPE, DOTMA, DOGS, DODAB, DODAC, DOSPA, DC-Chol, DOIC, DOPC, DMRIE, PAMAM, polylysine, polyhistidine, polyarginine, polyethyleneimine, poly(4-vinyl pyridine), poly(vinylamine), poly(4-vinyl-N-alkyl pyridinium halide), or combinations thereof. 15. The method of claim 1, wherein the polynucleotide is plasmid DNA. 16. The method of claim 1, wherein the polynucleotide is selected from the group consisting of viral DNA, chromosomal fragments, antisense oligonucleotides, antisense phosphorothioate oligonucleotides, RNA molecules and ribozymes, or combinations thereof. 17. The method of claim 1, wherein the cells are eukaryotic cells. 18. The method of claim 17, wherein the cells are mammalian cells. 19. The method of claim 18, wherein the cells are urothelial cells. 20. The method of claim 1, wherein the transfection composition is applied to cells in culture. 21. The method of claim 1, wherein the transfection composition is applied to cells in vivo. 22. The method of claim 19, wherein the transfection composition is applied to urothelial cells in vivo by intravesical delivery to a bladder of a subject. 23. In a method for transfecting a polynucleotide into cells wherein the polynucleotide is complexed with a cationic lipid, a cationic polymer or a dendrimer, said method comprising applying the transfection composition of claim 1 to said cells, the improvement comprising formulating the polynucleotide and the cationic lipid, cationic polymer or dendrimer with a solubilized cholesterol preparation, wherein the solubilized cholesterol preparation comprises cholesterol solubilized with a cyclodextrin. 24. The method of claim 23, wherein the cyclodextrin is methyl-.beta.-cyclodextrin. 25. The method of claim 23, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, sulfated beta-cyclodextrin, tertiary amine beta-cyclodextrin, quatemary amine beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, 2,6-di-O-methyl-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose, sulfobutylether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin and maltosyl-beta-cyclodextrin. 26. The method of claim 23, wherein the cationic lipid is DOTAP. 27. The method of claim 23, wherein the dendrimer is cationic dendrimer. 28. The method of claim 23, wherein the cationic lipid, cationic polymer or dendrimer is selected from the group consisting of DOPE, DOTMA, DOGS, DODAB, DODAC, DOSPA, DC-Chol, DOIC, DOPC, DMRIE, PAMAM, polylysine, polyhistidine, polyarginine, polyethyleneimine, poly(4-vinylpyridine), poly(vinylamine), poly(4-vinyl-N-alkyl pyridinium halide), or combinations thereof. 29. The method of claim 23, wherein the polynucleotide is plasmid DNA. 30. The method of claim 23, wherein the polynucleotide is selected from the group consisting of viral DNA, chromosomal fragments, antisense oligonucleotides, antisense phosphorothioate oligonucleotides, RNA molecules and ribozymes, or combinations thereof. 31. The method of claim 23, wherein the cells are eukaryotic cells. 32. The method of claim 31, wherein the cells are mammalian cells. 33. The method of claim 32, wherein the cells are urothelial cells. 34. The method of claim 23, wherein the transfection composition is applied to cells in culture. 35. The method of claim 23, wherein the transfection composition is applied to cells in vivo. 36. The method of claim 33, wherein the transfection composition is applied to urothelial cells in vivo by intravesical delivery to a bladder of a subject. 37. A method for delivering a polynucleotide into urothelial cells of a subject, the method comprising: delivering a transfection composition according to claim 1 intravesicularly into the bladder of the subject, such that the polynucleotide is delivered into urothelial cells of the subject. 38. The method of claim 37, wherein the cyclodextrin is methyl-.beta.-cyclodextrin. 39. The method of claim 37, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, sulfated beta-cyclodextrin, tertiary amine beta-cyclodextrin, quatemary amine beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, 2,6-di-O-methyl-beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose, sulfobutylether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin and maltosyl-beta-cyclodextrin. 40. The method of claim 37, wherein (ii) is a cationic lipid which is DOTAP. 41. The method of claim 37, wherein (ii) is a dendrimer which is cationic dendrimer. 42. The method of claim 37, wherein the cationic lipid, cationic polymer or dendrimer is selected from the group consisting of DOPE, DOTMA, DOGS, DODAB, DODAC, DOSPA, DC-Chol, DOIC, DOPC, DMRIE, PAMAM, polylysine, polyhistidine, polyarginine, polyethyleneimine, poly(4-vinylpyridine), poly(vinylamine), poly(4-vinvl-N-alkyl pyridinium halide), or combinations thereof. 43. The method of claim 37, wherein the polynucleotide is plasmid DNA. 44. The method of claim 37, wherein the polynucleotide is selected from the group consisting of viral DNA, chromosomal fragments, antisense oligonucleotides, antisense phosphorothioate oligonucleotides, RNA molecules and ribozymes, or combinations thereof. 45. A method for treating superficial bladder cancer in a subject, the method comprising: delivering the transfection composition of claim 1 intravesicularly into the bladder of a subject, such that bladder epithelial cancer cells of the subject are transfected with the polynucleotide, wherein the polynucleotide imparts anti-cancer activity against bladder cancer cells. 46. The method of claim 45, wherein the cyclodextrin is methyl-.beta.-cyclodextrin. 47. The method of claim 45, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, sulfated beta-cyclodextrin, tertiary amine beta-cyclodextrin, quatemary amine beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, 2,6-di-O-methyl-beta-cyclodextrin, hydroxyethyl-beta-cyciodextrin, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose, sulfobutylether-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, glucosyl-beta-cyclodextrin and maltosyl-beta-cyclodextrin. 48. The method of claim 45, wherein (ii) is a cationic lipid which is DOTAP. 49. The method of claim 45, wherein (ii) is dendrimer which is cationic dendrimer. 50. The method of claim 45, wherein the cationic lipid, cationic polymer or dendrimer is selected from the group consisting of DOPE, DOTMA, DOGS, DODAB, DODAC, DOSPA, DC-Chol, DOIC, DOPC, DMRIE, PAMAM, polylysine, polyhistidine, polyarginine, polyethyleneimine, poly(4-vinylpyridine), poly(vinylamine), poly(4-vinvl-N-alkyl pyridinium halide), or combinations thereof. 51. The method of claim 45, wherein the polynucleotide comprises at least one expression vector encoding at least one protein selected from the group consisting of interleukins, interferons, colony stimulating factors, anti-angiogenic factors, anti-metastatic factors, membrane receptors and tumor suppressors. 52. The method of claim 45, wherein the polynucleotide comprises an expression vector encoding a protein selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-18 interferon-.alpha., interferon-.beta., interferon-.gamma., granulocyte-macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (GCSF), p53, and an antagonist of vascular endothelial cell growth factor (VEGF). 53. The method of claim 45, wherein the polynucleotide comprises an expression vector encoding interleukin-2 (IL-2). 54. The method of claim 45, wherein the polynucleotide comprises an expression vector encoding granulocyte macrophage colony stimulating factor (GMCSF). 55. The method of claim 45, wherein the polynucleotide comprises an expression vector encoding interferon-.gamma.. 56. The method of claim 45, wherein the polynucleotide comprises at least one expression vector encoding two or more of interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GMCSF) and interferon-.gamma.. 57. The method of claim 45, which further comprises performing an additional anti-bladder cancer treatment on the subject. 58. The method of claim 57, wherein the additional anti-bladder cancer treatment comprises Bacillus Calmette-Guerin (BCG) therapy.

Details for Patent 7,320,963

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Teknika Llc	TICE BCG	bcg live	For Injection	102821	06/21/1989		2019-09-01