Short-acting heparin-based anticoagulant compounds and methods

1. A synthetic heparin analogue which comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

2. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue has anticoagulant activity.

3. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue has a binding affinity to antithrombin ranging from about 5 Nm to about 30 Nm.

4. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue has anti-Xa activity ranging from about 10 ngml −1 to about 40 ngml −1 IC 50 .

5. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue comprises a structure selected from the group consisting of:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

6. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue consists of the following structure:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

7. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue has a clearance rate about 50% to about 100% faster than that of fondaparinux.

8. The synthetic heparin analogue of claim 1 , wherein the synthetic heparin analogue does not cause heparin-induced thrombocytopenia (HIT).

9. The synthetic heparin analogue of claim 1 , wherein the anticoagulant activity of the synthetic heparin analogue is reversible by andexanet alfa at a rate of 50% or more in the presence of 20 ug/ml or less of andexanet alfa.

10. A method of synthesizing a synthetic heparin analogue of claim 1 , comprising:

providing a saccharide substrate;

elongating the saccharide substrate to a saccharide of a desired or predetermined length; and

performing at least one sulfation reaction using a 3-OST-3 isoform of a 3-O-sulfotransferase (3-OST) enzyme,

whereby a synthetic heparin analogue is synthesized.

11. The method of claim 10 , wherein the saccharide substrate comprises at least one IdoA2S-GlcNS3S disaccharide unit.

12. The method of claim 10 , wherein the saccharide substrate comprises a IdoA2S-GlcNS3 S±6S disaccharide unit, wherein the method further comprises a 6-O-sulfation step using a 6-O-sulfotransferase (6-OST), wherein a 3-O-sulfation by 3-OST-3 occurs prior to the 6-O-sulfation step.

13. The method of claim 10 , wherein the elongation step comprises employing a glycosyl transferase.

14. The method of claim 13 , wherein the glycosyl transferase is selected from the group consisting of N-acetyl glucosaminyl transferase of E. coli K5 (KfiA) and/or heparosan synthase-2 (pmHS2) from Pasteurella multocida.

15. The method of claim 10 , wherein the elongation step comprises employing one or more monosaccharides selected from the group consisting of: glucuronic acid (GlcUA), N-acetylated glucosamine (GlcNAc), and N-trifluoroacetyl glucosamine (GlcNTFA).

16. The method of claim 10 , wherein the method of synthesizing the synthetic heparin analogue has a yield of greater than about 20% to about 50%.

17. A synthetic heparin analogue produced by the method of claim 10 , wherein the synthetic heparin analogue has anticoagulant activity and comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

18. A synthetic heparin analogue produced by the method of claim 10 , wherein the synthetic heparin analogue has a binding affinity to antithrombin ranging from about 5 Nm to about 30 Nm and comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

19. A synthetic heparin analogue produced by the method of claim 10 , wherein the synthetic heparin analogue has anti-Xa activity ranging from about 10 ngml −1 to about 40 ngml −1 IC 50 and comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

20. A synthetic heparin analogue produced by the method of claim 10 , comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit, and wherein the synthetic heparin analogue comprises a structure selected from the group consisting of:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

21. A synthetic heparin analogue produced by the method of claim 10 , comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3S or at least one IdoA2S-GlcNS3S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit, and wherein the synthetic heparin analogue consists of the following structure:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

22. A synthetic heparin analogue produced by the method of claim 10 , comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

23. A synthetic heparin analogue produced by the method of claim 10 , wherein the synthetic heparin analogue has a clearance rate about 50% to about 100% faster than that of fondaparinux and comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

24. A synthetic heparin analogue produced by the method of claim 10 , wherein the synthetic heparin analogue does not cause heparin-induced thrombocytopenia (HIT) and comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

25. A synthetic heparin analogue produced by the method of claim 10 , wherein the anticoagulant activity of the synthetic heparin analogue is reversible by andexanet alfa a rate of 50% or more in the presence of 20 ug/ml or less of andexanet alfa and comprising disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

26. A method of treating a subject in need of anticoagulant therapy, the method comprising:

providing a subject in need of anticoagulant therapy;

administering to the subject a synthetic heparin analogue which comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation and has anticoagulant activity, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

27. The method of claim 26 , further comprising monitoring the subject for heparin-induced thrombocytopenia, and administering to the subject an antidote to reverse the anticoagulant activity of the synthetic heparin analogue if the subject suffers from heparin-induced thrombocytopenia.

28. The method of claim 26 , wherein the antidote to reverse the anticoagulant activity of the synthetic heparin analogue is andexanet alfa.

29. The method of claim 26 , wherein the subject is a human subject.

30. The method of claim 26 , wherein the synthetic heparin analogue has a clearance rate about 50% to about 100% faster than that of fondaparinux.

31. The method of claim 26 , wherein the synthetic heparin analogue comprises an anticoagulant activity of less than about 10% at 4 hours post-administration.

32. The method of claim 26 , wherein the subject has an elevated risk of bleeding.

33. The method of claim 26 , wherein the synthetic heparin analogue comprises a structure selected from the group consisting of:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

34. The method of claim 26 , wherein the synthetic heparin analogue consists of the following structure:

wherein R is selected from the group consisting of —H, alkyl, substituted alkyl, aryl, and substituted aryl.

35. A pharmaceutical composition comprising a synthetic heparin compound of claim 1 .

36. A pharmaceutical composition comprising a synthetic heparin compound produced by the method of claim 10 , wherein the synthetic heparin analogue comprises disaccharide repeating units of a glucuronic acid (GlcA) and iduronic acid (IdoA) residue linked to a glucosamine (GlcN) residue modified by sulfation, comprising:

a 3-O-sulfated oligosaccharide comprising six to eight saccharide units;

at least one disaccharide unit sulfated by a 3-OST-3 enzyme;

at least one IdoA2S-GlcNS3 S or at least one IdoA2S-GlcNS3 S6S disaccharide unit, and is devoid of a GlcA-GlcNS3S6S disaccharide unit; and

at least one GlcNS6S disaccharide unit.

37. The synthetic heparin analogue of claim 5 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.

38. The synthetic heparin analogue of claim 6 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.

39. The method of claim 20 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.

40. The method of claim 21 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.

41. The method of claim 33 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.

42. The method of claim 34 , wherein the alkyl is —CH 3 or —CH 2 CH 3 or the substituted aryl is a p-nitrophenyl group.