Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates

• US 20030124705A1
• Filed: 03/20/2002
• Published: 07/03/2003
• Est. Priority Date: 11/30/1995
• Status: Active Grant

• Current Assignee: Mcmaster University
• Original Assignee: Hamilton Health Sciences Corporation, Mcmaster University
• Inventors: Andrew, Maureen, Berry, Leslie Roy, Chan, Anthony Kam Chuen
• Granted Patent: US 7,045,585 B2
• Time in Patent Office: Days
• US Class Current: 435/226
• 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: C08L 5/10 Heparin; Derivatives thereof
• CPC Class Codes: Y10S 623/92 Method or apparatus for preparing or treating prosthetic

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, and on surfaces in contact with blood are also described.

Claims

1. A covalent conjugate comprising a glycosaminoglycan linked to another species by a covalent linkage wherein the species comprises at least one primary amino group, wherein the species is directly covalently linked via its amino group to a terminal aldose residue of the glycosaminoglycan, or a pharmaceutically acceptable salt thereof.

2. A covalent conjugate as claimed in claim 1, wherein the covalent linkage is an imine (>

C═

N—

) formed between the amino group of the first species and the C1 of the terminal aldose, or the amine reduction product thereof (>

CH—

NH—

).

16. A covalent conjugate as claimed in claim 1 prepared by a process comprising:

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

-carbonyl amine forming the covalent linkage; and

(b) isolating said covalent conjugate;

or a pharmaceutically acceptable salt thereof.

17. A process for preparing a covalent conjugate as claimed in claim 1 comprising (a) incubating the glycosaminoglycan with the species under conditions that allow imine formation between the terminal aldose residue of the glycosaminoglycan and the amino group of the species;

(b) allowing the imine to undergo an Amadori rearrangement to a α

-carbonyl amine; and

(c) isolating the covalent conjugate.

18. A method as claimed in claim 17 wherein in step (b) the imine undergoes a predominant amount of, or essentially complete subsequent Amadori rearrangement.

26. A pharmaceutical composition comprising a covalent conjugate as claimed in claim 1 and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition as claimed in claim 26 wherein the glycosaminoglycan is covalently linked to the amino-group of the species by —

CO—

CH₂—

NH—

, said —

CO—

CH₂-portion being derived from said glycosaminoglycan and said -NH- portion being derived from an amino group of said species.

29. A pharmaceutical composition as claimed in claim 26 wherein the glycosaminoglycan is covalently bonded to the species by —

CO—

CH₂—

NH—

.

30. A pharmaceutical composition as claimed in claim 26 wherein the molar ratio of amino-containing species to glycosaminoglycan is less than one.

31. A pharmaceutical composition as claimed in claim 26 comprising substantially pure covalent conjugates comprising heparin directly covalently linked without an intermediate spacer or linker by a terminal aldose residue to an amino group of antithrombin III.

32. A pharmaceutical composition as claimed in claim 31 wherein the heparin and antithrombin III may be linked by a —

CO—

CH₂—

NH-group formed by Amadori rearrangement of a —

HCOH—

HC═

N-group resulting from reaction between the amino group and a C1 carbonyl group of the terminal aldose residue.

33. A pharmaceutical composition as claimed in claim 31 that has a longer anticoagulant effect when compared to heparin.

34. A pharmaceutical composition as claimed in claim 26 consisting essentially of covalent conjugates comprising heparin directly covalently linked without an intermediate spacer or linker by a terminal aldose residue to an amino group of antithrombin III.

35. A pharmaceutical composition as claimed in claim 26 consisting of covalent conjugates comprising heparin directly covalently linked without an intermediate spacer or linker by a terminal aldose residue to an amino group of antithrombin III.

36. A pharmaceutical composition as claimed in claim 31 that activates antithrombin III to which the covalent conjugate is not conjugated.

37. A pharmaceutical composition as claimed in claim 31 that possesses>

90% of intact unconjugated heparin antithrombin activity.

38. A pharmaceutical composition as claimed in claim 31 wherein the heparin of the covalent conjugate stoichiometrically activates antithrombin III in the covalent conjugate and catalytically activates antithrombin III circulating in a patient.

39. A pharmaceutical composition as claimed in claim 31 that inactivates thrombin and Factor Xa.

42. A method of achieving an anticoagulant effect in a patient comprising administering a pharmaceutical composition as claimed in claim 31.

43. A method for the prophylactic and therapeutic treatment in a patient of an ailment relating to high coagulation activity of blood in a mammal comprising administering to the patient a pharmaceutical composition as claimed in claim 31.

3. A covalent conjugate wherein the glycosaminoglycan is heparin.

4. A covalent conjugate as claimed in claim 3 wherein the species is antithrombin III.

5. A covalent conjugate as claimed in claim 4 wherein the covalent linkage is formed by a substantial amount of subsequent Amadori rearrangement of imines resulting from reaction between the amino group and the terminal aldose residue.

6. A covalent conjugate as claimed in claim 4 characterized by one or more of the following properties:

(a) stable at 4°

C. for at least 60 days;

(b) longer half-life than heparin;

(c) more effective at inhibiting thrombin than free antithrombin III and heparin;

(d) heparin component stoichiometrically activates antithrombin III in the covalent conjugate;

(e) specific anti-factor Xa activity of 20-100 U/mg, preferably 30 to 60 U/mg, more preferably 40 to 50 U/mg heparin;

(f) heparin chains having two pentasaccharides, preferably more than 10%, 20%, 35%, or 50% of the heparin chains having two pentasaccharides;

(g) neutralized with protamine or human platelet factor 4;

(h) inactivates thrombin bound to fibrin;

(i) exosite 2 of heparin is not required for the covalent conjugate to bind to thrombin;

(j) molar ratio of heparin to antithrombin is about 1-2, preferably 1.1;

(k) activates antithrombin III to which the covalent conjugate is not conjugated to act catalytically; and

(l) possesses>

60%, typically>

90, more typically>

95%, and most typically>

98% of intact unconjugated heparin antithrombin activity.

7. A covalent conjugate as claimed in claim 6 which has the characteristics (d), (h), and (k);

(c), (d), (h), and (k);

(c), (d), (f), (h), and (k);

(c), (d), (h), (l), and (k);

(c), (f), (h), and (l);

(c), (f), (h), and (k);

(c), (f), (h), (k), and (l);

(f), (h), and (k);

(a) through (k);

or (a) through (l).

8. A covalent conjugate as claimed in claim 4 which has one or more of the following properties (a) heparin chains with greater than 80 saccharide units;

(b) 1 to 2 catalytic sites per heparin chain;

(c) a catalytic activity about 1-3 fold greater than heparin alone; and

(d) heparin chains with an average molecular weight of 10,000 to 20,000.

41. A method of achieving an anticoagulant effect in a patient comprising administering a covalent conjugate as claimed in claim 4.

44. A method for reducing the thrombogenicity of a material by coating the material with a covalent conjugate as claimed in claim 4.

47. A material for use in a medical or prosthetic device comprising a polymer coated with a covalent conjugate as claimed in claim 4.

56. A medical or prosthetic device having a blood-contacting surface with a covalent conjugate of claim 4 thereon.

57. An implantable medical or prosthetic device with a modified surface comprising a medical device with at least one surface for contacting blood, wherein the contacting surface is modified by treatment with a covalent conjugate of claim 4.

59. A method for coating a medical device or prosthetic device comprising applying covalent conjugates as claimed in claim 4 to the device to form a coating on the device.

61. A method of imparting antithrombotic properties to a surface comprising modifying the surface by treatment with covalent conjugates as claimed in claim 4.

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

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

11. A conjugate composition as claimed in claim 9 wherein the glycosaminoglycan and species are covalently linked by an α

-carbonyl amine formed by a predominant or essentially complete subsequent Amadori rearrangement.

12. A conjugate composition as claimed in claim 9 comprising glycosaminoglycans covalently linked to a protein to a substantial degree by —

CO—

CH₂—

NH—

.

13. A conjugate composition as claimed in claim 9 to a substantial degree of the formula:

glycosaminoglycan—

CO—

CH₂—

NH-protein.

10. A conjugate composition comprising glycosaminoglycans to a substantial degree covalently linked to an amino-group containing species by —

CO—

CH₂—

NH—

said —

CO—

CH₂-portion being derived from said glycosaminoglycans and said —

NH-portion being derived from an amino group of said species.

14. A conjugate composition as claimed in claim 10 wherein the glycosaminoglycan is heparin or a fragment thereof, and the species is antithrombin III.

15. A conjugate composition as claimed in claim 14 wherein the substantial degree is a predominant degree or essentially complete degree.

19. A process for preparing a conjugate composition as claimed in claim 14 comprising:

(a) incubating intact heparin with antithrombin III at a pH and for a time sufficient for formation of an imine between an amino group of the antithrombin III and a terminal aldose residue of heparin, and at a time and temperature sufficient for the imine to undergo a substantial amount of subsequent Amadori rearrangement to an α

-carbonyl amine forming a covalent linkage; and

(b) isolating the conjugate composition.

20. A process as claimed in claim 19 wherein the conjugate composition comprises a substantial amount of heparin directly covalently linked to antithrombin III wherein more than 10%, 20%, 35%, or 50% of the heparin chains have two pentasaccharides.

21. A process as claimed in claim 19 wherein in step (a), the incubation is carried out from about 3 days to two weeks.

22. A process as claimed in claim 19 wherein in step (a), the incubation is carried out for 5 days, 10 days, or 2 weeks.

23. A process as claimed in claim 19 wherein in step (a), the incubation is carried out at a temperature of 35°

C. to 45°

C.

24. A process as claimed in claim 19 wherein in step (a), the incubation is carried out at a temperature of 40°

C.

25. A process as claimed in claim 19 wherein the substantial amount is a predominant amount, or essentially complete amount.

27. A pharmaceutical composition comprising a conjugate composition as claimed in claim 14 and a pharmaceutically acceptable carrier.

45. A method for reducing the thrombogenicity of a material by coating the material with a conjugate composition as claimed in claim 14.

46. A material for use in a medical or prosthetic device comprising a polymer coated with a conjugate composition as claimed in claim 14.

48. A material of claim 46 pharmaceutically suitable for insertion into a human.

40. A substantially pure composition comprising covalent conjugates comprising glycosaminoglycans and molecules comprising at least one amino group, wherein the glycosaminoglycan is heparin, the species is antithrombin III and the covalent conjugate is antithrombin-heparin (ATH), and wherein the amino group is directly linked without an intermediate spacer or linker to the glycosaminoglycan by a covalent linkage, wherein the covalent conjugate is made by a process comprising:

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

-carbonyl amine to form a covalent linkage between the glycosarninoglycan and molecule; and

(b) isolating the conjugate conjugate.

49. A material for use in a medical or prosthetic device comprising a polymer in contact with a covalent conjugate, the covalent conjugate comprising glycosaminoglycans linked to a species by covalent linkages, wherein the species comprises at least one primary amino group, and wherein the species is directly covalently linked via the amino group to a terminal aldose residue of the glycosaminoglycans, the covalent linkages comprising an α

-carbonyl amine formed by a substantial amount of subsequent Amadori rearrangement of an imine resulting from reaction between the amino group and the terminal aldose residue of the glycosaminoglycans.

50. A material for use in a medical or prosthetic device comprising a polymer in contact with a covalent conjugate composition, the covalent conjugate composition comprising glycosaminoglycans linked to a species by covalent linkages, wherein the glycosaminoglycans are heparins, the species is antithrombin III, and the covalent conjugate composition comprises antithrombin III-heparin (ATH), the ATH being covalently attached to the polymer, and wherein the species is directly covalently linked via the amino group to a terminal aldose residue of the glycosaminoglycans to a substantial degree by an α

-carbonyl linkage.

51. A material as claimed in claim 50 wherein the substantial degree is a predominant degree or essentially complete degree.

52. A material of claim 50, wherein the polymer is a synthetic polymer selected from the group consisting of poly 2-hydroxyethyl methacrylate, poly acrylamide, poly ether polyurethane urea (PEUU), polyethylene, polypropylene, polytetrafluoroethylene, poly(vinylchloride), polydimethylsiloxane, an ethylene-acrylic acid copolymer, Dacron, polyester-polyurethane, polyurethane, polycarbonate-polyurethane, polyamide (Nylon) and polystyrene.

53. A material as claimed in claim 50 wherein the ATH is covalently attached to the polymer.

54. A prosthetic or medical device comprising a material of claim 50.

55. The device of claim 54 which is endovascular tubing, an arterial or central venous line, a cardiac catheter, a dialysis catheter, a cardiopulmonary bypass circuit, a dialysis circuit, a pacemaker lead, an arterial or venous catheter, a thrombectomy catheter, a suture, a blood filter, an intravenous line, a mechanical valve, a stent, or an artificial kidney, lung, heart, or liver.

58. An implantable medical or prosthetic device with at least one contacting surface for contacting blood comprising a layer on the contacting surface comprising the product of incubating heparin with antithrombin II under conditions which allow imine formation between the terminal aldose residue of the heparin and an amine of the antithrombin III and allowing Amadori rearrangement to an α

-carbonyl amine.

60. A method of forming a coating on a surface of a medical or prosthetic device comprising applying a product on the surface that is prepared by (a) incubating heparin with antithrombin III under conditions which allow imine formation between the terminal aldose residue of the heparin and an amine of the antithrombin III and (b) allowing Amadori rearrangement to an α

-carbonyl amine.