GLYCOPEGYLATED FACTOR IX

• US 20100330060A1
• Filed: 08/06/2010
• Published: 12/30/2010
• Est. Priority Date: 12/03/2003
• Status: Active Grant

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Novo Nordisk A/S
• Inventors: PANNEERSELVAM, KRISHNASAMY, DEFREES, SHAWN, BAYER, ROBERT J., BOWE, CARYN
• Granted Patent: US 8,632,770 B2
• Time in Patent Office: Days
• US Class Current: 424/94.3
• CPC Class Codes: A61K 38/4846 Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides conjugates between Factor IX and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

Claims

1. A Factor IX peptide conjugate comprising a glycosyl linking group attached to an amino acid residue of said peptide, said glycosyl linking group comprising a modified sialyl residue having the formula:

2. The peptide conjugate according to claim 1, wherein the moiety:

3. The peptide conjugate according to claim 2, wherein said moiety has a formula that is a member selected from:

4. The peptide conjugate according to claim 1, wherein said glycosyl linking group has a formula selected from:

5. The peptide conjugate according to claim 4, wherein said glycosyl linking group has the formula:

6. The peptide conjugate according to claim 5, wherein said glycosyl linking group attached to said amino acid residue has the formula:

7. The peptide conjugate according to claim 6, wherein said amino acid residue is a member selected from serine or threonine.

8. The peptide conjugate according to claim 1, wherein said peptide has the amino acid sequence of SEQ. ID. NO:

1.

9. The peptide according to claim 8, wherein said amino acid residue is a serine at position 61 of SEQ. ID. NO:

1.

10. The peptide conjugate according to claim 1, wherein said peptide comprises at least one glycosyl linking group comprising a substructure having the formula:

11. The peptide conjugate according to claim 10, wherein said at least one glycosyl linking group attached to an amino acid of said peptide has a formula selected from:

12. The peptide conjugate according to claim 11, wherein said amino acid residue is an asparagine residue.

13. The peptide conjugate according to claim 12, wherein said peptide has the amino acid sequence of SEQ ID NO:

1.

14. The peptide conjugate according to claim 13, wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof.

15. A method of preparing a peptide conjugate according to claim 1, said method comprising:

(a) contacting a substrate Factor IX peptide comprising a glycosyl moiety selected from;

16. The method of claim 15, further comprising, prior to step (a):

(b) expressing said substrate Factor IX peptide in a suitable host.

17. The method of claim 16, wherein said host is selected from an insect cell and a mammalian cell.

18. The method of claim 17, wherein said insect cell is a Spodoptera frugiperda cell line.

19. A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal a peptide conjugate according to claim 1 in an amount sufficient to stimulate said blood coagulation.

20. A method of treating hemophilia in a subject, said method comprising administering to said subject a peptide conjugate according to claim 1 in an amount effective to treat said hemophilia.

21. A pharmaceutical formulation comprising the Factor IX peptide conjugate according to claim 1, and a pharmaceutically acceptable carrier.

22. A Factor IX peptide conjugate comprising a glycosyl linking group attached to an amino acid residue of said peptide, said glycosyl linking group comprising a modified sialyl residue having the formula:

23. The peptide conjugate according to claim 22, wherein said modified sialyl residue has the formula:

24. The peptide conjugate according to claim 23, wherein Q is selected from H and CH₃.

25. The peptide conjugate according to claim 22, wherein said peptide comprises at least one glycosyl linking group comprising a substructure having the formula:

26. The peptide conjugate according to claim 25, wherein said at least one glycosyl linking group attached to an amino acid of said peptide has a formula selected from:

27. The peptide conjugate according to claim 22, wherein said amino acid residue is asparagine.

28. The peptide conjugate according to claim 27, wherein said peptide has the amino acid sequence of SEQ ID NO:

1.

29. The peptide conjugate according to claim 28, wherein said glycosyl residue is attached to a member selected from Asn 157, Asn 167 and combinations thereof.

30. The peptide conjugate according to claim 22, wherein said glycosyl linking group comprises the formula:

31. The peptide conjugate according to claim 30, wherein s is 1;

and f is an integer from about 200 to about 300.

32. The peptide conjugate according to claim 22, wherein said amino acid residue is a member selected from serine or threonine.

33. The peptide conjugate according to claim 32, wherein said peptide has the amino acid sequence of SEQ. ID. NO:

1.

34. The peptide conjugate according to claim 33, wherein said amino acid residue is a serine at position 61 of SEQ. ID. NO:

1.

35. A method of preparing the peptide conjugate according to claim 22, said method comprising:

(a) contacting a substrate Factor IX peptide comprising a glycosyl moiety selected from;

36. The method of claim 35, further comprising, prior to step (a):

(b) expressing said substrate Factor IX peptide in a suitable host.

37. The method of claim 36, wherein said host an insect cell.

38. The method of claim 37, wherein said insect cell is a Spodoptera frugiperda cell line.

39. A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal a peptide conjugate according to claim 22 in an amount sufficient to stimulate said blood coagulation.

40. A method of treating hemophilia in a subject, said method comprising administering to said subject a peptide conjugate according to claim 22 in an amount effective to treat said hemophilia.

41. A pharmaceutical formulation comprising the Factor IX peptide conjugate according to claim 22, and a pharmaceutically acceptable carrier.