Peptides for heparin and low molecular weight heparin anticoagulation reversal

• US 5,614,494 A
• Filed: 09/08/1994
• Issued: 03/25/1997
• Est. Priority Date: 11/12/1993
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Stanley, James C., Wakefield, Thomas W., Andrews, Philip C.
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): Prickril, Benet
• Application Number:
  US08/303,025
• Time in Patent Office: 929 Days
• Field of Search: 530/300, 530/324-326, 514/2, 514/12, 514/13
• US Class Current: 514/13.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68

Abstract

Less toxic agents for reversal of heparin or low molecular weight heparin anticoagulation which are synthetic protamine-like polycationic peptides having a total cationic charge which is less than that of n-protamine. In preferred embodiments, arginine residues of n-protamine are replaced with lysine residues for ease of manufacture. Selective positively charged arginine residues have been replaced with an uncharged amino acid residue or its analog, such as glycine or glutamine, in order to reduce the total cationic charge on the polycationic peptide to the range of about [+14] to [+18], preferably [+16] to [+18]. In specific embodiments, there are sequences of 29 and 32 amino acid residues wherein 4 to 5 clusters of 2 to 4 positively charged amino acids are separated by 2 to 6 neutral amino acids. The C-terminus and the N-terminus can be modified to mitigate against in vivo degradation by carboxypeptidases and aminopeptidases. Another modification, specifically use of α-helix forming amino acids, such as glutamic acid, further promotes anticoagulation reversal.

Claims

1. A method of making an agent for reversing at least partially the anticoagulation effects of heparin and/or low molecular weight heparin comprising the steps of:

preparing a synthetic peptide comprising a sequence of 20-40 uncharged and charged amino acids having a total cationic charge of less than [+21], but not lower than [+14], as determined by the number of positively charged amino acids in the sequence, the uncharged amino acids in the sequence being selected from the group consisting of glycine, glutamine, serine, threonine, asparagine, proline, valine, and isoleucine, and having the ability to at least partially reverse the effects of heparin and/or low molecular weight heparin anticoagulation; and

combining the synthetic peptide with a delivery vehicle.

2. The method of claim 1 wherein the total cationic charge is in the range of [+14] to [+18].

3. The method of claim 1 wherein the total cationic charge is in the range of [+16] to [+18].

4. The method of claim 1 wherein the step of preparing includes modifying the C-terminus and/or N-terminus of the sequence of amino acids to render the synthetic peptide resistant to in vivo degradation.

5. The method of claim 4 wherein the step of modifying the C-terminus of the sequence of amino acids comprises the step of amidating.

6. The method of claim 4 wherein the step of modifying the N-terminus of the sequence of amino acids comprises the step of acetylating.

7. The method of claim 1 wherein the step of preparing includes distributing the positively charged amino acids evenly along the peptide sequence.

8. The method of claim 1 wherein the step of preparing includes distributing the positively charged amino acids randomly along the peptide sequence.

9. The method of claim 1 wherein the step of preparing includes grouping the positively charged amino acids in clusters which are separated by uncharged amino acids.

10. The method of claim 9 wherein the step of grouping comprises grouping 29-32 amino acids into 4 to 5 clusters of 2 to 4 positively charged amino acids separated by 2 to 6 uncharged amino acids so that the total charge on the peptide is in the range of [+14] to [+18].

11. The method of claim 10 wherein the charged and uncharged amino acids are selected to facilitate α

-helix formation.

12. A method of at least partially reversing the anticoagulation effects of heparin and/or low molecular weight heparin comprising administering to a living being an anticoagulation-reversing effective amount of a synthetic peptide which is an analog of n-protamine wherein the positive charge on the amino acid sequence of n-protamine is reduced by replacement of selective positively charged arginine residues with an uncharged amino acid residue so that the total cationic charge less than [+21], but not lower than [+14], the uncharged amino acids being selected from the group consisting of glycine, glutamine, serine, threonine, asparagine, proline, valine, and isoleucine.

13. The method of claim 12 wherein the total cationic charge is in the range of [+14] to [+18].

14. The method of claim 13 wherein, in the total cationic charge is in the range of [+16] to [+18].

15. A method of at least partially reverting the anticoagulation effects of heparin and/or low molecular weight heparin comprising the steps of:

preparing a synthetic peptide which is an analog of n-protamine wherein the positive charge on the amino acid sequence of n-protamine is reduced by replacement of selective positively charged arginine residues with an uncharged amino acid residue so that the total cationic charge is less than [+21], but not lower than [+14];

combining the synthetic peptide with a delivery vehicle to form an anticoagulation-reversal agent; and

administering to a living being an anticoagulation-reversing effective amount of the anticoagulation-reversal agent.

16. The method of claim 15 wherein there is further provided the step of sterilizing the synthetic peptide and/or the anticoagulation agent.

17. The method of claim 15 wherein the step of administering comprising administering the anticoagulation agent slowly over a predetermined period of time.

18. The method of claim 17 wherein the period of time is about 3 minutes.

19. The method of claim 15 wherein the total cationic charge is in the range of [+14] to [+18].

20. The method of claim 15 wherein the total cationic charge is in the range of [+16] to [+18].

21. The method of claim 15 wherein the step of preparing includes modifying the C-terminus and/or N-terminus of the sequence of amino acids to render the synthetic peptide resistant to in vivo degradation.

22. The method of claim 21 wherein the step of modifying the C-terminus of the sequence of amino acids comprises the step of amidating.

23. The method of claim 21 wherein the step of modifying the N-terminus of the sequence of amino acids comprises the step of acetylating.

24. The method of claim 15 wherein the step of preparing includes distributing the positively charged amino adds evenly along the peptide sequence.

25. The method of claim 15 wherein the step of preparing includes distributing the positively charged amino adds randomly along the peptide sequence.

26. The method of claim 15 wherein the step of preparing includes grouping the positively charged amino acids in clusters which are separated by uncharged amino acids.

27. The method of claim 26 wherein the step of grouping comprises grouping 29-32 amino adds into 4 to 5 clusters of 2 to 4 positively charged amino acids separated by 2 to 6 uncharged amino acids so that the total charge on the peptide is in the range of [+14] to [+18].

28. The method of claim 27 wherein the charged and uncharged amino acids are selected to facilitate α

-helix formation.

29. The method of claim 15 wherein the synthetic peptide is acetyl-PAK₂ (AK₂ A₂ K₂)₃ AK₂ -amide (SEQ ID NO:

12).

30. The method of claim 15 wherein the synthetic peptide is acetyl-E(AK₂ A₂ K₂) ₄ -amide (SEQ ID NO:

13).

31. The method of claim 15 wherein the synthetic peptide is PK(K₂ A₂ K₂ A) ₃ K₂ AK₃ (SEQ ID NO:

14).

32. The method of claim 15 wherein the synthetic peptide is acetyl-PA(K₂ A₂ K₂ A)₄ K₂ -amide (SEQ ID NO:

15).

33. The method of claim 15 wherein the synthetic peptide is acetyl-EA₂ (K₂ A₂ K₂ A)₄ K₂ -amide (SEQ ID NO:

16).