N-maleimidyl polymer derivatives

• US 6,602,498 B2
• Filed: 02/21/2001
• Issued: 08/05/2003
• Est. Priority Date: 02/22/2000
• Status: Expired due to Term

• Current Assignee: Nektar Therapeutics
• Original Assignee: Shearwater Corp. (Nektar Therapeutics)
• Inventors: Shen, Xiaoming
• Primary Examiner(s): Page, Thurman K.
• Assistant Examiner(s): Fubara, Blessing
• Application Number:
  US09/790,250
• Publication Number:
  US 20010044526A1
• Time in Patent Office: 895 Days
• Field of Search: 424/78.08, 424/78.31, 424/78.32, 424/78.33
• US Class Current: 424/78.08
• CPC Class Codes: A61K 31/74 Synthetic polymeric materials
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• 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: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: C08G 65/00 Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
• CPC Class Codes: C08G 65/33337 cyclic
• CPC Class Codes: C08G 65/33396 having oxygen in addition to nitrogen
• CPC Class Codes: C08G 65/48 Polymers modified by chemical after-treatment

Abstract

The invention is directed to multi-functional N-maleimidyl polymer derivatives comprising a water soluble and non-peptidic polymer backbone having a terminal carbon, such as a poly(alkylene glycol), the terminal carbon of the polymer backbone being directly bonded to the nitrogen atom of a N-maleimidyl moiety without a linking group therebetween. The invention also provides two methods of preparing such linkerless N-maleimidyl polymer derivatives.

Claims

1. A multi-functional N-maleimidyl polymer derivative comprising a water soluble and non-peptidic polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a first terminus bonded to a first functional group and a second terminus having a terminal carbon, wherein said terminal carbon of said second terminus is directly bonded to a N-maleimidyl moiety having the structure:

wherein L is the point of bonding to the terminal carbon of the second terminus of the polymer backbone, said first functional group being different from said N-maleimidyl moiety bonded to the terminal carbon of said second terminus.

2. The polymer of claim 1, wherein said water soluble and non-peptidic polymer backbone has from about 2 to about 300 termini.

3. The polymer of claim 1, wherein said water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(alkylene glycol), copolymers thereof, terpolymers thereof, and mixtures thereof.

4. The polymer of claim 1, wherein said water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), and copolymers of ethylene glycol and propylene glycol.

5. The polymer of claim 1, having the structure:

wherein;

POLY is a linear water soluble and non-peptidic polymer having a terminal carbon, the terminal carbon being directly bonded to the nitrogen atom of the N-maleimidyl moiety; and

X is said first functional group.

6. The polymer of claim 5, wherein POLY is selected from the group consisting of poly(alkylene glycol), copolymers thereof, terpolymers thereof, and mixtures thereof.

7. The polymer of claim 5, wherein POLY is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), and copolymers of ethylene glycol and propylene glycol.

8. The polymer of claim 5, wherein X is selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

9. The polymer of claim 1, wherein the water soluble and non-peptidic polymer backbone has the structure:

10. The polymer of claim 9, wherein X is selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

11. The polymer of claim 9, wherein X is selected from the group consisting of —

OH, —

NH₂, —

CO₂H, —

CHO, —

CH(OC₂H₅)₂, N-hydroxysuccinimidyl esters, 1-benzotriazolyl esters, N-hydroxysuccinimidyl carbonates, 1-benzotriazolyl carbonates, and tresylate.

12. The polymer of claim 9, wherein n is about 20 to about 2,000.

13. The polymer of claim 1, wherein the polymer backbone has an average molecular weight from about 6,000 Da to about 80,000 Da.

14. The polymer of claim 1, wherein the polymer backbone is poly(ethylene glycol) having an average molecular weight from about 6,000 Da to about 80,000 Da.

15. The polymer of claim 1, wherein the polymer backbone is selected from the group consisting of linear poly(ethylene glycol), branched poly(ethylene glycol), forked poly(ethylene glycol), pendant poly(ethylene glycol), and poly(ethylene glycol) having degradable linkages therein.

16. The polymer of claim 5, wherein POLY is poly(ethylene glycol) having an average molecular weight from about 6,000 Da to about 80,000 Da.

17. The polymer of claim 5, wherein X is selected from the group consisting of —

OH, —

NH₂, —

CO₂H, —

CHO, —

CH(OC₂H₅)₂, N-hydroxysuccinimidyl esters, 1-benzotriazolyl esters, N-hydroxysuccinimidyl carbonates, 1-benzothiazolyl carbonates, and tresylate.

18. A method of preparing a multi-functional N-maleimidyl polymer derivative, comprising:

providing a water-soluble and non-peptidic polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a first terminus bonded to a first functional group and a second terminus bonded to an amine group;

reacting the polymer backbone with maleic anhydride to form an open ring intermediate; and

heating the open ring intermediate in the presence of acetic anhydride and a salt of acetic acid to form a multi-functional N-maleimidyl polymer derivative product, wherein a terminal carbon at the second terminus of the polymer backbone of the N-maleimidyl polymer derivative product is bonded to the nitrogen atom of an N-maleimidyl moiety and the first functional group is different from the N-maleimidyl moiety.

19. The method of claim 18, wherein the water soluble and non-peptidic polymer backbone has from about 2 to about 300 termini.

20. The method of claim 18, wherein the water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(alkylene glycol), copolymers thereof, terpolymers thereof, and mixtures thereof.

21. The method of claim 18, wherein the water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), and copolymers of ethylene glycol and propylene glycol.

22. The method of claim 18, wherein said reacting step comprises reacting maleic anhydride with X-PEG-N₂, wherein PEG is poly(ethylene glycol) and X is the first functional group, to form an open ring intermediate having the following structure:

23. The method of claim 22, wherein X is selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

24. The method of claim 22, wherein X is selected from the group consisting of hydroxyl, protected hydroxyl, acetal, alkenyl, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, dithiopyridine, and vinylpyridine.

25. The method of claim 18, further comprising purifying the open ring intermediate by precipitation prior to said heating step.

26. The method of claim 18, wherein said heating step comprises heating the open ring intermediate at a temperature of about 50°

C. to about 140°

C. for about 0.2 hours to about 5 hours.

27. The method of claim 18, further comprising purifying the multi-functional N-maleimidyl polymer derivative product.

28. The method of claim 27, wherein said purifying step comprises:

passing the N-maleimidyl polymer derivative product through an ion exchange column;

collecting an eluent containing the N-maleimidyl polymer derivative product from the column; and

precipitating the N-maleimidyl polymer derivative product by contacting the product with a solvent.

29. The method of claim 28, wherein the solvent is selected from the group consisting of ethyl ether, isopropanol, and mixtures thereof.

30. The method of claim 18, further comprising the step of reacting the N-maleimidyl polymer derivative product with a biologically active agent to form a biologically active polymer conjugate.

31. The method of claim 30, wherein the biologically active agent is selected from the group consisting of peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles and micelles.

32. A method of preparing a N-maleimidyl polymer derivative, comprising

providing a water-soluble and non-peptidic polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a terminal amine group;

and

reacting the polymer backbone with an N-alkoxycarbonylmaleimide to form a N-maleimidyl polymer derivative product.

33. The method of claim 32, wherein the water-soluble and non-peptidic polymer backbone has the formula X-PEG-NH₂, wherein PEG is poly(ethylene glycol) and X is a capping group or a functional group.

34. The method of claim 33, wherein X is selected from the group consisting of alkoxy, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine, and vinylpyridine.

35. The method of claim 33, wherein X is selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

36. The method of claim 32, further comprising purifying the N-maleimidyl polymer derivative product.

37. The method of claim 32, wherein the water soluble and non-peptidic polymer backbone has from about 2 to about 300 termini.

38. The method of claim 32, wherein the water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(alkylene glycol), copolymers thereof, terpolymers thereof, and mixtures thereof.

39. The method of claim 32, wherein the water soluble and non-peptidic polymer backbone is selected from the group consisting of poly(ethylene glycol), poly(propylene glycol), and copolymers of ethylene glycol and propylene glycol.

40. The method of claim 32, further comprising the step of reacting the N-maleimidyl polymer derivative product with a biologically active agent to form a biologically active polymer derivative.

41. The method of claim 40, wherein the biologically active agent is selected from the group consisting of peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles and micelles.

42. The method of claim 32, wherein the N-alkoxycarbonylmaleimide has the structure:

43. A multi-functional branched N-maleimidyl polymer derivative comprising a central branched core moiety and a plurality of water soluble and non-peptidic polymer backbones attached to said central branched core moiety, each polymer backbone comprising a terminal carbon and having an average molecular weight from about 800 Da to about 100,000 Da, wherein said terminal carbon of each polymer backbone is directly bonded to a N-maleimidyl moiety having the structure:

wherein L is the point of bonding to the terminal carbon of each polymer backbone.

44. The polymer of claim 43, wherein each polymer backbone is poly(ethylene glycol) having an average molecular weight from about 6,000 Da to about 80,000 Da.

45. The polymer of claim 43, wherein the central branched core moiety is a polyol or an amino acid.

46. The polymer of claim 43, wherein the central branched core moiety is selected from the group consisting of glycerol, glycerol oligomers, pentaerythritol, sorbitol, and lysine.

47. The polymer of claim 43, having the structure:

wherein;

POLY is a water-soluble and non-peptidic polymer backbone having a terminal carbon, the terminal carbon being directly bonded to the nitrogen atom of the N-maleimidyl moiety;

R is a central core molecule; and

q is an integer from 2 to about 300.

48. The polymer of claim 47, wherein R is selected from the group consisting of glycerol, glycerol oligomers, pentaerythritol, sorbitol, and lysine.

49. The polymer of claim 47, wherein POLY is poly(ethylene glycol) having an average molecular weight from about 6,000 Da to about 80,000 Da.