Saposin C-DOPS: A Novel Anti-Tumor Agent

• US 20090269373A1
• Filed: 12/11/2008
• Published: 10/29/2009
• Est. Priority Date: 04/28/2003
• Status: Active Grant

• Current Assignee: Children's Hospital & Medical Center
• Original Assignee: Children's Hospital & Medical Center
• Inventors: Qi, Xiaoyang
• Granted Patent: US 8,937,156 B2
• Time in Patent Office: Days
• US Class Current: 424/400
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/685 one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
• CPC Class Codes: A61K 38/17 from animals; from humans enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 9/4816 Wall or shell material
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

Compositions and methods for treating subjects with disorders characterized by hyper-proliferating cells such as tumors and cancers are provided. The compositions comprise agents that are combinations of saposin C (or prosaposin-related polypeptides) and phospholips (or inner leaflet components). This anti-tumor agent is administered in the methods of the invention according to a dosing regimen. Administering an agent of the invention results in a positive therapeutic response in a subject with a tumor.

Claims

1. A method for modulating the distribution of an inner leaflet component in a plasma membrane of a cell of a subject comprising administering to the subject a therapeutically effective amount of a phospholipid nanovesicle incorporating a polypeptide comprising:

a phospholipid selected from the group consisting of phosphatidylserine, phosphatidylethanolamine and structural analogs thereof, an isolated saposin C-related polypeptide, wherein the polypeptide is selected from the group consisting of;

(a) a polypeptide having an amino acid sequence at least 80 percent identical to SEQ ID NO;

2;

(b) a polypeptide having an amino acid sequence as set forth in SEQ ID NO;

2 and having one or more conservative substitutions; and

(c) a polypeptide having an amino acid sequence identical to SEQ ID NO;

2; and

a pharmaceutically acceptable carrier.

3. The method of claim 1, wherein the phosphatidylserine or structural analog thereof is dioleoylphosphatidylserine.

4. The method of claim 3, wherein the molar ratio of saposin C-related polypeptide to phospholipid is in the range from about 1:

1 to about 1;

10.

6. The method of claim 1, wherein the distribution of the inner leaflet component is modulated in hyper-proliferating cells.

7. The method of claim 1, wherein the hyper-proliferating cells are selected from the group consisting of tumor cells and cancer cells.

8. The method of claim 7, wherein the method induces apoptosis in hyper-proliferating cells.

38. The pharmaceutical composition of claim 7, wherein the polypeptide has an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:

1, the amino acid sequence set forth in SEQ ID NO;

1 having one or more conservative substitutions, the amino acid sequence set forth in SEQ ID NO;

2, and the amino acid sequence set forth in SEQ ID NO;

2 having one or more conservative substitutions.

39. The pharmaceutical composition of claim 38, wherein the phosphatidylserine or structural analog thereof is selected from the group consisting of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, and dioleoylphosphatidylserine.

40. The pharmaceutical composition of claim 39, wherein the molar ratio of polypeptide to phospholipid is in the range from about 1:

1 to about 1;

50.

41. The pharmaceutical composition of claim 40, wherein the nanovesicle has a diameter in the range 0.01 to 1 μ

m.

52. A method of treating a cancer in a subject, the method comprising administering to a subject in need of such treatment pharmaceutical composition comprising nanovesicles prepared by the process of claim 38.

9. A method of modulating tumor volume in a subject, the method comprising administering a therapeutically effective amount of a phospholipid nanovesicle incorporating a polypeptide comprising:

a phospholipid selected from the group consisting of phosphatidylserine, phosphatidylethanolamine and structural analogs thereof, an isolated saposin C-related polypeptide, wherein the polypeptide is selected from the group consisting of;

(a) a polypeptide having an amino acid sequence at least 80 percent identical to SEQ ID NO;

2;

(b) a polypeptide having an amino acid sequence as set forth in SEQ ID NO;

2 and having one or more conservative substitutions; and

(c) a polypeptide having an amino acid sequence identical to SEQ ID NO;

2; and

a pharmaceutically acceptable carrier.

2. The method of claim 9, wherein the phosphatidylserine or structural analog thereof is selected from the group consisting of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, and dioleoylphosphatidylserine.

5. The method of claim 2, wherein the polypeptide comprises at least 80 contiguous amino acids SEQ ID NO:

2.

10. The method of claim 9, wherein the phospholipid nanovesicle promotes cell death in hyper-proliferating cells.

11. The method of claim 10, wherein the hyper-proliferating cells are selected from the group consisting of tumor cells and cancer cells.

12. The method of claim 11, wherein the cancer cells are selected from the group consisting of sarcoma, neuroblastoma, breast carcinoma, and squamous cell carcinoma cells.

13. The method of claim 9, wherein the phosphatidylserine or structural analog thereof is selected from the group consisting of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, and dioleoylphosphatidylserine.

14. The method of claim 13, wherein the phosphatidylserine or structural analog thereof is dioleoylphosphatidylserine.

15. The method of claim 9, wherein the subject is a mammal.

16. The method of claim 15, wherein the mammal is a human.

17. The method of claim 9, wherein the tumor volume decreases.

18. The method of claim 9, wherein the molar ratio of polypeptide to phospholipid is in the range from about 1:

1 to about 1;

50.

19. The method of claim 14, wherein the molar ratio of polypeptide to phospholipid is in the range from about 1:

1 to about 1;

10.

20. The method of claim 9, wherein the agent further comprises a pharmaceutically acceptable carrier.

21. A method of treating a cancer in a subject, the method comprising administering a therapeutically effective amount of a phospholipid nanovesicle incorporating a polypeptide comprising:

a phospholipid selected from the group consisting of phosphatidylserine, phosphatidylethanolamine and structural analogs thereof, an isolated saposin C-related polypeptide, wherein the polypeptide is selected from the group consisting of;

(a) a polypeptide having an amino acid sequence at least 80 percent identical to SEQ ID NO;

2;

(b) a polypeptide having an amino acid sequence as set forth in SEQ ID NO;

2 and having one or more conservative substitutions; and

(c) a polypeptide having an amino acid sequence identical to SEQ ID NO;

2; and

a pharmaceutically acceptable carrier.

22. The method of claim 21, wherein the phosphatidylserine or structural analog thereof is selected from the group consisting of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylserine, palmitelaidoyloleoylphosphatidylserine, myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine, palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, and dioleoylphosphatidylserine.

23. The method of claim 22, wherein the phosphatidylserine or structural analog thereof is dioleoylphosphatidylserine.

24. The method of claim 21, wherein the molar ratio of polypeptide phospholipid is in the range from about 1:

1 to about 1;

50.

25. The agent of claim 23, wherein the molar ratio of polypeptide to phospholipid is in the range from about 1:

1 to about 1;

10.

26. The method of claim 25, wherein the agent further comprises a pharmaceutically acceptable carrier.

27. The method of claim 26, wherein the agent promotes cell death in hyper-proliferating cells.

28. The method of claim 27, wherein the cell death occurs through apoptosis.

29. The method of claim 27, wherein the hyper-proliferating cells are selected from the group consisting of cancer cells.

30. The method of claim 29, wherein the cancer cells are selected from the group consisting of sarcoma, neuroblastoma, breast carcinoma, and squamous cell carcinoma cells.

31. The method of claim 21, wherein the subject is a mammal.

32. The method of claim 30, wherein the mammal is a human.

33. The method of claim 21, wherein the agent is administered enterally, parenterally, subcutaneously, intravenously, intraperitoneally, or topically.

34. The method of claim 21, wherein multiple doses of the agent are administered to the subject.

35. The method of claim 21, wherein a single dose of the agent is administered to the subject.

36. A process for the manufacture of a pharmaceutical composition comprising the steps of:

(a) combining a composition comprising (i) a phospholipid selected from the group consisting of phosphatidylserine, phosphatidylethanolamine and structural analogs thereof and (ii) an isolated saposin C-related polypeptide, wherein the polypeptide is selected from the group consisting of;

(a) a polypeptide having an amino acid sequence at least 80 percent identical to SEQ ID NO;

2;

(b) a polypeptide having an amino acid sequence as set forth in SEQ ID NO;

2 and having one or more conservative substitutions; and

(c) a polypeptide having an amino acid sequence identical to SEQ ID NO;

2;

in a pharmaceutically acceptable carrier (b) treating the suspension of phospholipid and prosaposin-related polypeptide to form a nanovesicle;

wherein the nanovesicle formed exhibits anti-tumor activity.

37. A pharmaceutical composition comprising nanovesicles prepared by the process of claim 36.

51. A method of treating a cancer in a subject, the method comprising administering to a subject in need of such treatment pharmaceutical composition comprising nanovesicles prepared by the process of claim 37.

42. A process for the manufacture of a pharmaceutical composition comprising the steps of:

(a) combining a composition comprising (i) a dried phospholipid component, wherein the phospholipid component is phosphatidylserine or a structural analog thereof and (ii) a dried and isolated prosaposin-related polypeptide;

wherein the polypeptide has an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO;

1, the amino acid sequence set forth in SEQ ID NO;

1 having one or more conservative substitutions, the amino acid sequence set forth in SEQ ID NO;

2, and the amino acid sequence set forth in SEQ ID NO;

2 having one or more conservative substitutions and wherein the polypeptide retains plasma-membrane affinity;

wherein the molar ratio of polypeptide to phospholipid component in the composition is in the range from 1;

1 to 1;

25;

in a pharmaceutically acceptable carrier;

(b) treating the suspension of phospholipid component and prosaposin-related polypeptide to form a nanovesicle;

wherein the nanovesicle formed has a diameter in the range 10 to 800 nm and exhibits anti-tumor activity.

43. A pharmaceutical composition comprising nanovesicles prepared by the process of claim 42.

44. An anti-tumor composition comprising a nanovesicle prepared by the process of claim 42, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO:

2, wherein the phospholipid component is dioleoylphosphatidylserine and wherein the composition is capable of inducing apoptosis in hyper-proliferating cells.

46. The anti-tumor composition of claim 44, wherein the mass ratio of polypeptide to dioleoylphosphatidylserine is approximately 5:

1.

47. The anti-tumor composition of claim 44, wherein the mass ratio of polypeptide to dioleoylphosphatidylserine is approximately 15:

7.

48. The anti-tumor composition of claim 44, wherein the mass ratio of polypeptide to dioleoylphosphatidylserine is in the range from about 15:

1 to about 3;

10.

49. The anti-tumor composition of claim 44, comprising approximately 10 μ

M polypeptide and approximately 30 μ

M dioleoylphosphatidylserine.

50. The anti-tumor composition of claim 44, comprising approximately 10 μ

M polypeptide and approximately 70 μ

M dioleoylphosphatidylserine.

53. A method of treating a cancer in a subject, the method comprising administering to a subject in need of such treatment pharmaceutical composition comprising nanovesicles prepared by the process of claim 43.

54. A method of treating a cancer in a subject, the method comprising administering to a subject in need of such treatment pharmaceutical composition comprising nanovesicles prepared by the process of claim 44.

45. An anti-tumor agent comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:

2 and dioleoylphosphatidylserin.