System for delivery of protein in insoluble fibrillar or aggregate form

• US 8,632,803 B2
• Filed: 02/18/2009
• Issued: 01/21/2014
• Est. Priority Date: 02/27/2008
• Status: Active Grant

• Current Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Original Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Inventors: Dekel, Yaron, Margalit, Rimona
• Primary Examiner(s): Wax, Robert A
• Assistant Examiner(s): AL-AWADI, DANAH J
• Application Number:
  US12/920,096
• Publication Number:
  US 20110008422A1
• Time in Patent Office: 1,798 Days
• Field of Search: None
• US Class Current: 424/450
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: A61K 9/1617 Organic compounds, e.g. phospholipids, fats
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics

Abstract

A pharmaceutical composition containing a therapeutic protein which is particularly amenable to oral administration by providing stability in the gastrointestinal tract. Insoluble fibrils or aggregates of the therapeutic protein in the pharmaceutical composition are encapsulated in a lipidated glycosaminoglycan particle, coated with a multi-layer of lipids and lipid-glycosaminoglycan conjugates, or present as unencapsulated and uncoated insoluble fibrils or aggregates.

Claims

1. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier, diluent, excipient or auxiliary agent and, as active ingredient, either fibrils or insoluble aggregates of insulin, which fibrils or insoluble aggregates are capable of dissociating and releasing soluble monomers of insulin in therapeutically active form, where said fibrils or insoluble aggregates are (a) encapsulated in a lipidated glycosaminoglycan particle, which comprises the reaction product of at least one glycosaminoglycan with at least one lipid having a primary amino group, (b) coated with a multi-layer of at least two lipids and conjugates of one of said at least two lipids and at least one glycosaminoglycan, or (c) present as unencapsulated and uncoated fibrils or insoluble aggregates.

2. The pharmaceutical composition of claim 1, wherein said fibrils or insoluble aggregates of insulin are encapsulated in said lapidated glycosaminoglycan particle.

3. The pharmaceutical composition of claim 2, wherein one of said lipid(s) is a phosphatidylethanolamine.

4. The pharmaceutical composition of claim 3, wherein said phosphatidylethanolamine is dipalmitoyl phosphatidylethanolamine.

5. The pharmaceutical composition of claim 1, which is adapted for oral administration.

6. The pharmaceutical composition of claim 2, wherein said at least one glycosaminoglycan is hyaluronic acid.

7. The pharmaceutical composition of claim 2, wherein the size of said lipidated glycosaminoglycan particle is in a range of about 10 to 100 microns.

8. The pharmaceutical composition of claim 2, which is adapted for oral administration.

9. A method for treating diabetes, comprising orally administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 8.

10. A method for preparing the active ingredient of the pharmaceutical composition of claim 2, comprising:

reacting at least one glycosaminoglycan with at least one lipid containing a primary amino group to cross-link the carboxylic residue of the glycosaminoglycan with the primary amino group to produce lipidated glycosaminoglycan particles (gagomers);

removing excess glycosaminoglycan, excess lipids and reaction by-products from the produced gagomers;

suspending said gagomers in a salt-free buffer which is capable of being vaporized upon lyophilization;

lyophilizing said gagomers to vaporize the buffer, resulting in a salt-free, buffer-free lyophilized powder of said gagomers; and

rehydrating said salt-free, buffer-free lyophilized powder of said gagomers with an aqueous solution of insulin under conditions suitable for fibrillation or aggregation to encapsulate said therapeutic protein insulin as insoluble fibrils or aggregates in said gagomers.

11. The method of claim 10, wherein said at least one glycosaminoglycan is hyaluronan.

12. The method of claim 10, wherein said at least one of said lipid(s) is a phosphatidylethanolamine.

13. The method of claim 12, wherein said phosphatidylethanolamine is dipalmitoylphosphatidlyethamolamine.

14. The method of claim 10, wherein said salt-free buffer for suspending said gagomers is an ammonium bicarbonate buffer and the insulin is insulin acidified to a pH of about 2 suitable for forming fibrils.

15. The pharmaceutical composition of claim 1, wherein said fibrils or insoluble aggregates of insulin are coated with said multi-layer of at least two lipids and conjugates of one of said at least two lipids said at least one glycosaminoglycan.

16. The pharmaceutical composition of claim 15, which is adapted for oral administration.

17. A method for treating diabetes, comprising orally administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 16.

18. A method for preparing the active ingredient of the pharmaceutical composition of claim 15, comprising:

reacting at least one glycosaminoglycan with liposomes comprising at least two lipids to produce multi-lamellar liposomes having said at least one glycosaminoglycan covalently anchored as a bioadhesive ligand;

removing excess glycosaminoglycan, excess lipids and reaction by-products from the produced multi-lamellar liposomes;

suspending the multi-lamellar liposomes in a salt-free buffer which is capable of being vaporized upon lyophilization;

lyophilizing the multi-lamellar liposomes to vaporize the buffer, resulting in a salt-free, buffer-free lyophilized powder of the multi-lamellar liposomes; and

rehydrating said salt-free, buffer-free lyophilized powder of the multi-lamellar liposomes with an aqueous solution of insulin under conditions suitable for fibrillation or aggregation to coat the insoluble fibrils or aggregates formed under said conditions with a multi-layer of at least two lipids and conjugates of one of said at least two lipids and said at least one glycosaminoglycan.

19. The method of claim 18, wherein said at least one glycosaminoglycan is hyaluronan.

20. The method of claim 18, wherein one of said at least two lipids is a phosphatidylethanolamine.

21. The method of claim 18, wherein the liposomes and said at least one glycosaminoglycan are covalently linked by glutaraldehyde.

22. The method of claim 18, wherein the liposomes and said at least one glycosaminoglycan are cross-linked with 1-ethyl-3(3-dimethylaminoprophyl) carbodiimide.

23. The method of claim 18, wherein said salt-free buffer for suspending said multi-lamellar liposomes is an ammonium bicarbonate buffer and the insulin is insulin acidified to a pH of about 2 suitable for forming fibrils.

24. The pharmaceutical composition of claim 1, wherein said fibrils or insoluble aggregates of insulin are present as unencapsulated and uncoated fibrils or insoluble aggregates.

25. The pharmaceutical composition of claim 24, which is adapted for oral administration.

26. A method for treating diabetes, comprising orally administering to a subject need thereof an effective amount of the pharmaceutical composition of claim 25.

28. The pharmaceutical composition of claim 15, wherein said at least one glycosaminoglycan is hyaluronic acid.

29. The pharmaceutical composition of claim 15, wherein one of said lipid(s) is a phosphatidylethanolamine.

30. The pharmaceutical composition of claim 29, wherein said phosphatidylethanolamine is dipalmitoyl phosphatidylethanolamine.

31. The pharmaceutical composition of claim 24, wherein said at least one glycosaminoglycan is hyaluronic acid.

32. The pharmaceutical composition of claim 24, wherein one of said lipid(s) is a phosphatidylethanolamine.

33. The pharmaceutical composition of claim 32, wherein said phosphatidylethanolamine is dipalmitoyl phosphatidylethanolamine.

27. A scaffold for tissue engineering, comprising insoluble fibrils or aggregates of insulin encapsulated in a lapidated glycosaminoglycan particle, which comprises the reaction product of at least one glycosaminoglycan with a least one lipid having a primary amino group, or coated with a multi-layer of at least two lipids and conjugates of one of said at least two lipids and at least one glycosaminoglycan.