Multiarmed, monofunctional, polymer for coupling to molecules and surfaces

• US 5,932,462 A
• Filed: 05/17/1995
• Issued: 08/03/1999
• Est. Priority Date: 01/10/1995
• Status: Expired due to Term

• Current Assignee: Nektar Therapeutics
• Original Assignee: Shearwater Polymers Incorporated (Nektar Therapeutics)
• Inventors: Harris, J. Milton, Veronese, Francesco Maria, Schiavon, Oddone, Caliceti, Paolo
• Primary Examiner(s): Naff, David M.
• Application Number:
  US08/443,383
• Time in Patent Office: 1,539 Days
• Field of Search: 435/174, 435/177, 435/180, 435/181, 435/188, 424/94.3, 514/2, 525/54.1, 530/402
• US Class Current: 435/188
• 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: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/48 Polymers modified by chemical after-treatment
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y10S 530/815 Carrier is a synthetic polymer
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure ##STR1## wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms poly_a and poly_b, respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings and ester groups in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure ##STR2## where mPEG_a and mPEG_b have the structure CH₃ O--(CH₂ CH₂ O)_n CH₂ CH₂ -- wherein n may be the same or different for mPEG_a and mPEG_b and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000. The mPEG disubstituted lysine can be purified from a reaction mixture by chromatography in water, including gel filtration chromatography and ion exchange chromatography because the carboxyl group is ionizable. Impurities are removed, including unreacted mPEG and mPEG monosubstituted lysine, to provide the polymer in pure form. Ion exchange chromatography permits fractionation of a greater amount of polymer per run.

Claims

1. An essentially pure water soluble polymer having a moiety located along the polymer backbone, which moiety comprises a single ionizable reactive group, wherein said polymer is represented by the structure:

##STR30## wherein C is carbon;

wherein R is a nonreactive moiety;

wherein Z is said moiety comprising a single ionizable reactive group;

wherein poly_a and poly_b are nonpeptidic and nonreactive polymeric arms that may be the game or different;

wherein P and Q are nonreactive linkage fragments that may be the same or different and join polymeric arms poly_a and poly_b, respectively, to C in the absence of aromatic rings and ester groups in said fragments, and wherein said polymer is recovered in essentially pure form by ion exchange chromatography.

2. The polymer of claim 1 wherein said single ionizable reactive group is a carboxyl group.

3. The polymer of claim 1 wherein said recovered polymer is activated for reaction with nucleophilic groups.

4. A conjugate of the polymer of claim 3 and a nucleophilic group.

5. The polymer of claim 1 wherein said nonpeptidic and nonreactive polymeric arms are selected from the group consisting of poly(alkylene oxides), poly(oxyethylated polyols), poly(oxyethylated glucose), and copolymers, terpolymers, and mixtures thereof.

6. The polymer of claim 1 wherein said nonpeptidic and nonreactive polymeric arms are selected from the group consisting of polyethylene glycol), poly(vinyl alcohol), polypropylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), poly(vinylpyrrolidone), and copolymers, terpolymers, and mixtures thereof.

7. The polymer of claim 1 wherein said nonpeptidic and nonreactive polymeric arms are polyethylene glycol arms, each said arm having a molecular weight of from about 50 to 50,000.

8. The polymer of claim 1 wherein said nonpeptidic nonreactive polymeric arms are polyethylene glycol arms and said polymer has a molecular weight of from about 40,000 to 100,000.

9. The polymer of claim 1 wherein said linkage fragments P and Q comprise moieties selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate moieties.

10. The polymer of claim 1 wherein said polymer has a structure selected from the group consisting of:

##STR31##

11. The polymer of claim 3 activated as trifluoroethylsulfonyl, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.

12. The polymer of claim 11 wherein said active ester is N-hydroxylsuccinimidyl ester and wherein said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.

13. The polymer of claim 3 wherein said nucleophilic moieties are selected from the group consisting of amino, thiol, and hydroxyl moieties.

14. The conjugate of claim 4 wherein a biologically active molecule comprise said nucleophilic group.

15. The conjugate of claim 14 wherein said biologically active molecule is selected from the group consisting of enzymes, peptides, polypeptides, nucleotides, polynucleotides, and lipids.

16. The conjugate of claim 4 wherein a solid surface comprises said nucleophilic group.

17. The conjugate of claim 16 wherein said solid surface is a liposome.

18. An essentially pure water soluble polymer represented by the structure:

wherein C is carbon;

wherein Z comprises a single ionizable reactive group;

wherein R comprises -M-poly_d ;

wherein poly_a, poly_b, and poly_d are nonpeptidic and nonreactive polymeric arms that may be the same or different;

wherein P, Q, and M are nonreactive linkage fragments that may be the same or different and join polymeric arms poly_a, poly_b, and poly_d, respectively, to C in the absence of aromatic rings and ester groups in said linkage fragments, and wherein said polymer is recovered in essentially pure form by ion exchange chromatography.

19. An essentially pure water soluble polymer having a molecular weight of from about 40,000 to 100,000 wherein said polymer is represented by the structure:

##STR32## wherein C is carbon;

wherein R is a nonreactive moiety;

wherein Z comprises a single carboxyl moiety, --COOH;

wherein poly_a and poly_b are polyethylene glycol arms that may be of the same or different molecular weight;

wherein P and Q are nonreactive linkage fragments that may be the same or different and join polymeric arms poly_a and poly_b, respectively, to C in the absence of aromatic rings and carbonate ester moieties in said fragments, and wherein said polymer in recovered in essentially sure form by ion exchange chromatography.

20. An essentially pure monomethoxy poly(ethylene glycol) disubstituted lysine represented by the following structure:

##STR33## wherein mPEG_a and mPEG_b are monomethoxypoly (ethylene glycols) having the structure CH--(CH₂ CH₂ O)_n CH₂ CH₂ --, wherein n equals from 1 to about one thousand one hundred fifty (1,150), wherein n may be the same or different for mPEG_a and mPEG_b, and wherein said disubstituted lysine is recovered in essentially pure form by ion exchange chromatography.

21. The monomethoxy polyethylene glycol) disubstituted lysine of claim 20 activated for reaction with nucleophilic moieties.

22. A conjugate of the activated monomethoxy poly(ethylene glycol) disubstituted lysine of claim 21 and a nucleophilic moiety.

23. The monomethoxy poly(ethylene glycol) disubstituted lysine of claim 21 activated as the succinimidyl ester.

24. A conjugate of a biologically active molecule and the monomethoxy poly(ethylene glycol) disubstituted lysine of claim 23 activated as the succinimidyl ester.

25. A method of synthesizing a water soluble, branched polymer having a single ionizable reactive group, which ionizable reactive group is located along the polymer backbone, said method comprising the steps of:

a) reacting two or more nonpeptidic and monofunctional polymers of the structure poly-W, wherein W is an active moiety located at the terminus of the polymer that provides the monofunctionality for the polymer, with a linger molecule having two or more active sites with which W is reactive and forming linkages therewith to create a branched polymer, wherein said linker molecule and said linkages do not comprise aromatic rings and ester groups and wherein the linker molecule further comprises a single reactive group with which active moiety --W is not reactive and that provides the single ionizable reactive group on the branched polymer backbone; and

b) recovering the branched polymer in essentially pure form by ion exchange chromatography.

26. The method of claim 25 wherein said single ionizable reactive group is carboxyl.

27. The method of claim 25 wherein the linkages between the linker molecule and monofunctional polymers poly-W are formed in a single step.

28. The method of claim 25 wherein a first linkage formed between the linker molecule and the monofunctional polymer poly-W in a first step, and wherein a second linkage of the linker molecule and poly-W is formed in a second step.

29. The method of claim 28 wherein the first step occurs in aqueous buffer and the second step occurs in a nonaqueous medium.

30. The method of claim 25 wherein the active moiety W is an electrophilic moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.

31. The method of claim 30 wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.

32. The method of claim 25 wherein the active moiety W is a nucleophilic moiety selected from the group consisting of amino, thiol, and hydroxyl moieties.

33. The method of claim 32 wherein the active sites on the linker moiety are electrophilic moieties selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.

34. The method of claim 33 wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.

35. The method of claim 25 wherein the linkages are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.

36. The method of claim 25 further comprising the step of activating the single ionizable reactive group on said polymer backbone for reaction with nucleophilic moieties.

37. The method of claim 36 wherein the group activated for reaction with nucleophilic moieties is an electrophilic moiety that is reactive with nucleophilic moieties selected from the group consisting of amino, thiol, and hydroxyl moieties.

38. The method of claim 37 wherein the electrophilic moiety is selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.

39. A method for forming monofunctional monomethoxy-poly(ethylene glycol) disubstituted lysine comprising carrying out steps to perform the following rejection:

##STR34## and recovering the disubstituted lysine in essentially pure form by ion exchange chromatography.

40. The method of claim 39 wherein the reaction takes place in water at a pH of about 8.0.

41. The method of claim 40 further comprising carrying out steps to perform the following reactions:

##STR35##

42. The method of claim 41 wherein reactions a) and b) take place in methylene chloride.

43. The method of claim 39 further comprising the steps of activating the carboxyl moiety of the recovered disubstituted lysine for reaction with nucleophilic moieties and then reacting the activated carboxyl moiety with a nucleophilic moiety to join the disubstituted lysine to the nucleophilic moiety.

48. A method of preparing monomethoxy poly(ethylene glycol)-disubstituted lysine activated as the succinimidyl ester, comprising carrying out steps to react the recovered disubstituted lysine produced by the method of claim 39 or claim 44 an follows:

##STR36##

44. A method for forming a monofunctional monomethoxy-poly(ethylene glycol) disubstituted lysine comprising carrying out steps to perform the following reactions:

and recovering the disubstituted lysine in essentially pure form by ion exchange chromatography.

45. The method of claim 44 further comprising the steps of activating the carboxyl moiety of the recovered disubstituted lysine for reaction with nucleophilic moieties and then reacting the activated carboxyl moiety with a nucleophilic moiety to join the disubstituted lysine to the nucleophilic moiety.

46. The method of claim 44 wherein reaction (a) takes place in aqueous buffer.

47. The method of claim 44 wherein reaction (b) takes place in methylene chloride.

49. A polymeric derivative having a structure selected from the group consisting of:

wherein poly_a and poly_b are nonpeptidic and nonreactive polymeric arms that may be the same or different; and

wherein Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.