Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates

• US 6,562,781 B1
• Filed: 11/30/1995
• Issued: 05/13/2003
• Est. Priority Date: 11/30/1995
• Status: Expired due to Fees

• Current Assignee: Hamilton Civic Hospitals Research Development Inc.
• Original Assignee: Hamilton Civic Hospitals Research Development Inc.
• Inventors: Berry, Leslie, Andrew, Maureen
• Primary Examiner(s): Pak, Michael
• Application Number:
  US08/564,976
• Time in Patent Office: 2,721 Days
• Field of Search: 530/300, 530/330, 530/393, 536/21, 260/123.7, 514/2, 514/21, 435/405
• US Class Current: 514/13.7
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61L 27/20 Polysaccharides
• CPC Class Codes: A61L 33/0029 using an intermediate layer of polymer
• CPC Class Codes: A61L 33/0041 characterised by the choice of an antithrombatic agent other than heparin heparinoïd A61L33/08; polypeptides A61L33/12
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C08L 5/10 Heparin; Derivatives thereof

Abstract

Novel conjugates of glycosaminoglycans, particularly heparin and dermatan sulfate, and amine containing species and therapeutic uses thereof are described. In particular, mild methods of conjugating heparins to proteins, such as antithrombin III and heparin cofactor II, which provide covalent conjugates which retain maximal biological activity are described. Uses of these conjugates to prevent thrombogenesis, in particular in lung airways, such as found in infant and adult respiratory distress syndrome are also described.

Claims

1. A covalent conjugate composition comprising glycosaminoglycans linked by covalent linkages to a species comprising at least one primary amino group, wherein said species is directly covalently linked via said amino group to a terminal aldose residue of said glycosaminoglycans, said covalent linkages comprising an α

-carbonyl amine formed by a substantial amount of subsequent Amadori rearrangement of imines resulting from reaction between said amino group and said terminal aldose residue of said glycosaminoglycans, or a pharmaceutically acceptable salt thereof,

wherein said glycosaminoglycans are heparin (H) and said amino-containing species is antithrombin III (AT).

4. A pharmaceutical preparation, containing a conjugate as in claim 1 as active ingredient in association with a pharmaceutically acceptable carrier.

5. A pharmaceutical preparation as described in claim 4, in the form of an aqueous solution for injection or in the form of an ointment or in the form of an aerosol.

6. The conjugate of claim 1, wherein said covalent linkage comprises a —

CO—

CH₂—

NH—

group formed by Amadori rearrangement of a —

HCOH—

HC═

N—

group resulting from reaction between said amino group and the C1 carbonyl group of said terminal aldose residue.

11. A conjugate of claim 1, wherein the molar ratio of amino-containing species to glycosaminoglycan is less than one.

15. A conjugate composition of claim 1 wherein said linkages comprise an α

-carbonyl amine formed by essentially complete subsequent Amadori rearrangement.

20. An isolated conjugate composition of claim 1.

29. A conjugate composition of claim 1 pharmaceutically suitable for injection in a human.

2. A covalent conjugate composition comprising glycosaminoglycans and molecules comprising at least one amino group, wherein said amino group is directly linked to said glycosaminoglycans by covalent linkages, wherein said conjugate composition is made by the process comprising:

(a) incubating said glycosaminoglycans with said molecules at a pH and for a time sufficient for imine formation between said amino group and a terminal aldose residue of said glycosaminoglycans, and at a time and temperature sufficient for said imines to undergo a substantial amount of subsequent Amadori rearrangement to an α

-carbonyl amine forming said covalent linkages;

(b) isolating said conjugate composition,

or a pharmaceutically acceptable salt thereof,

wherein said glycosaminoglycans are heparin (H) and said amino-containing molecules are antithrombin III (AT).

13. A conjugate of claim 2, wherein the molar ratio of protein to glycosaminoglycan is less than one.

16. A conjugate composition of claim 2 wherein said imine has undergone essentially complete subsequent Amadori rearrangement.

21. An isolated conjugate composition of claim 2.

25. A pharmaceutical composition comprising a conjugate composition of claim 2 and a pharmaceutically acceptable carrier.

30. A conjugate composition of claim 2 pharmaceutically suitable for injection in a human.

34. A conjugate composition of claim 2 wherein said incubating is carried out from about 3 days to two weeks at a temperature of 35°

C. to 45°

C.

35. A conjugate composition of claim 34 wherein said incubating is carried out for about 10 days.

36. A conjugate composition of claim 34 wherein said incubating is carried out for about two weeks.

3. A process for preparing a conjugate composition of molecules comprising at least one primary amino group linked to glycosaminoglycans by covalent bonds, said process comprising:

(a) incubating said glycosaminoglycans with said molecules at a pH and for a time sufficient for formation of an imine between said amino group and a terminal aldose residue of said glycosaminoglycans, and at a time and temperature sufficient for said imines to undergo a substantial amount of subsequent Amadori rearrangement to an α

-carbonyl amine forming said covalent bonds; and

(b) isolating said conjugate composition,

wherein said glycosaminoglycans are heparin (H) and said amino-containing molecules are antithrombin III (AT).

17. A process of claim 3 wherein said imine has undergone essentially complete subsequent Amadori rearrangement.

37. A process of claim 3 wherein said incubating is carried out from about 3 days to two weeks at a temperature of 35°

C. to 45°

C.

38. A process of claim 37 wherein said incubating is carried out for about 10 days.

7. A conjugate composition comprising a substantial amount of glycosaminoglycans covalently bonded to an amino-containing species by —

CO—

CH₂—

NH—

, said CO—

CH₂—

portion being derived from said glycosaminoglycan and said NH portion being derived from in amino group of said species,

wherein said glycosaminoglycans are heparin (H) and said amino-containing species is antithrombin III (AT).

12. A conjugate of claim 7, wherein the molar ratio of amino-containing species to glycosaminoglycan is less than one.

18. A conjugate composition of claim 7 wherein said substantial amount is essentially all.

22. An isolated conjugate composition of claim 7.

24. An isolated conjugate composition of claim 18.

26. A pharmaceutical composition comprising a conjugate composition of claim 7 and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising a conjugate composition of claims 18 and a pharmaceutically acceptable carrier.

31. A conjugate composition of claim 7 pharmaceutically suitable for injection in a human.

33. A conjugate composition of claim 18 pharmaceutically suitable for injection in a human.

40. A conjugate composition of claim 7 wherein said conjugate has a longer half-life than heparin.

41. A conjugate composition of claim 7, wherein said conjugate is more effective at inhibiting thrombin than are free ATIII and heparin.

42. A conjugate composition of claim 7, wherein said conjugate inactivates clot-bound thrombin.

43. A conjugate composition of claim 7, wherein in said conjugate the molar ratio of heparin to antithrombin is 1:

1.

44. A conjugate composition of claim 7, wherein the molecular weight of said conjugate is 69 kD-100 kD.

45. A conjugate composition of claim 7, wherein said conjugate possesses >

60% the antithrombin activity of intact unconjugated heparin.

46. A conjugate composition of claim 7, wherein said conjugate possesses >

90% the antithrombin activity of intact unconjugated heparin.

47. A conjugate composition of claim 7, wherein said conjugate possesses >

95% the antithrombin activity of intact unconjugated heparin.

48. A conjugate composition of claim 7, wherein said conjugate possesses >

98% the antithrombin activity of intact unconjugated heparin.

8. A conjugate composition comprising a substantial amount of a complex of the formula:

glycosaminoglycan CO—

CH₂—

NH—

protein,

wherein raid glycosaminoglycan is heparin (H) and said protein is antithrombin III (AT).

9. A pharmaceutical preparation containing a conjugate as in claim 8 as active ingredient in association with a pharmaceutically acceptable carrier.

10. A method of reducing the thrombogenicity of a material comprising coating said material with the conjugate of claim 8.

14. A conjugate of claim 8, wherein the molar ratio of protein to glycosaminoglycan is less than one.

19. A conjugate composition of claim 8 wherein said substantial amount is essentially all.

23. An isolated conjugate composition of claim 8.

27. A pharmaceutical composition comprising a conjugate composition of claim 8 and a pharmaceutically acceptable carrier.

32. A conjugate composition of claim 8 pharmaceutically suitable for injection in a human.

39. A process of claim wherein said incubating is carried out for about two weeks.

49. A covalent conjugate composition comprising glycosaminoglycans linked by covalent linkages to species comprising at least one primary amino group, wherein said species is directly covalently linked via said amino group to a terminal aldose residue of said glycosaminoglycans, said covalent linkages comprising an α

-carbonyl amine formed by a substantial amount of subsequent Amadori rearrangement of imines resulting from reaction between said amino group and said terminal aldose residue of said glycosaminoglycans, or a pharmaceutically acceptable salt thereof,

wherein said glycosaminoglycans are heparin (H) and said amino-containing species is antithrombin III (AT), or said glycosaminoglycans are heparin and said amino-containing species is heparin cofactor II, or said glycosaminoglycans are dermatan sulfate and said amino-containing species is heparin cofactor II.