Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules

• US 5,446,090 A
• Filed: 11/12/1993
• Issued: 08/29/1995
• Est. Priority Date: 11/12/1993
• Status: Expired due to Term

• Current Assignee: Nektar Therapeutics
• Original Assignee: Shearwater Polymers Incorporated (Nektar Therapeutics)
• Inventors: Harris, J. Milton
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US08/151,481
• Time in Patent Office: 655 Days
• Field of Search: 525/535, 525/409, 525/60, 525/50, 525/54.1, 525/906, 525/54.11, 568/32, 568/621, 568/623, 528/374, 435/188, 530/351, 530/404, 514/2, 424/94.3
• US Class Current: 525/54.1
• 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 65/326 containing sulfur
• CPC Class Codes: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/334 containing sulfur
• 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

A poly(ethylene glycol) derivative is disclosed that is activated with a sulfone moiety for selective attachment to thiol moieties on molecules and surfaces. The activated PEG is water soluble, hydrolytically stable for extended periods, and forms hydrolytically stable linkages with thiol moieties. The linkages generally are not reversible in reducing environments. The PEG derivative is useful for modifying the characteristics of substances including modifying biologically active molecules and surfaces for biocompatibility. Methods for synthesizing the active PEG and for preparing conjugates of the active PEG and other substances, including biologically active substances, are also disclosed.

Claims

1. An activated poly(ethylene glycol) derivative having the structure:

space="preserve" listing-type="equation">R--CH.sub.2 CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --Y

wherein n equals 5 to 3,000, Y is --SO₂ --CH═

CH₂, and R is selected from the group consisting of HO--, H₃ CO--, X--CH₂ --CH₂ --SO₂ --, where X is halogen, and CH₂ ═

CH--SO₂ --.

2. The activated poly(ethylene glycol) derivative of claim 1 wherein X is chlorine.

3. The activated poly(ethylene glycol) derivative of claim 1 wherein n equals about 5 to 2200.

4. The activated poly(ethylene glycol), derivative of claim 1 wherein n equals about 34 to 1100.

5. The activated poly(ethylene glycol) derivative of claim 1 wherein n equals about 45 to 110.

6. The activated polymer of claim 1 wherein said activated polymer is stable in aqueous environments of pH about 11 or less.

7. The activated polymer of claim 1 wherein said activated polymer is selective for reaction with thiol moieties at pH conditions of about 9 or less.

8. The activated polymer of claim 1 wherein said activated polymer is stable in reducing environments.

9. The activated polymer of claim 1 wherein said activated polymer is infinitely soluble in water.

10. An activated poly(ethylene glycol) derivative having the structure:

space="preserve" listing-type="equation">CH.sub.3 --(OCH.sub.2 CH.sub.2).sub.n --SO.sub.2 --CH═

CH.sub.2

wherein n equals 5 to 3,000.

11. An activated poly(ethylene glycol) derivative having the structure:

space="preserve" listing-type="equation">CH.sub.2 ═

CH--SO.sub.2 --CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2)hd n--SO.sub.2 --CH═

CH.sub.2

wherein n equals 5 to 3,000.

12. A hydrolytically stable, biologically active conjugate that is the reaction product of 1) at least one biologically active molecule having the structure W-SH, wherein W- is a biologically active moiety and --SH is a reactive thiol moiety, and 2) a poly(ethylene glycol) derivative activated with one or more sulfone moieties and having the structure:

space="preserve" listing-type="equation">R--CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --Y

wherein n equals 5 to 3,000, Y is --SO₂ --CH═

CH₂, and R is selected from the group consisting of HO--, H₃ CO--, X--CH₂ --CH₂ --SO₂ --, wherein X is halogen, and CH₂ ═

CH`SO₂ --, and wherein said reactive thiol moiety is linked to said at least one active sulfone moiety.

13. The conjugate of claim 12 wherein said biologically active molecule is protein and said reactive thiol moiety is contained within a cysteine moiety of said protein.

14. The conjugate of claim 12 wherein said conjugate has the following structure:

space="preserve" listing-type="equation">R--CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --SO.sub.2 --CH.sub.2 --CH.sub.2 --S-W

wherein n equals 5 to 3,000 and R is selected from the group consisting of HO-- and H₃ CO--.

15. The biologically active conjugate of claim 12 wherein said conjugate has the following structure:

space="preserve" listing-type="equation">W--S--CH.sub.2 --SO.sub.2 --CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --SO.sub.2 --CH.sub.2 --CH.sub.2 --S-W

wherein n equals 5 to 3,000.

16. The biologically active conjugate of claim 12 wherein said conjugate has the following structure:

space="preserve" listing-type="equation">R--CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --SO.sub.2 --CH.sub.2 --CH.sub.2 --S-protein

wherein n equals 5 to 3,000 and R is selected from the group consisting of HO-- and H₃ CO--.

17. The biologically active conjugate of claim 12 wherein said conjugate has the following structure:

space="preserve" listing-type="equation">protein-S--CH.sub.2 --SO.sub.2 --CH.sub.2 --CH.sub.2 --(OCH.sub.2 CH.sub.2).sub.n --SO.sub.2 --CH.sub.2 --CH.sub.2 S-protein

wherein n equals 5 to 3,000.

18. A method for preparing poly(ethylene glycol) vinyl sulfone comprising the steps of:

(a) reacting poly(ethylene glycol) having at least one active hydroxyl moiety with a compound to form either an ester or a halide substituted poly(ethylene glycol);

(b) reacting the ester or a halide substituted poly(ethylene glycol) of step (a) with mercaptoethanol to substitute the mercaptoethanol radical for the ester or halide moiety;

(c) reacting the mercaptoethanol substituted poly(ethylene glycol) of step (b) with an oxidizing agent to oxide sulfur in the mercaptoethanol moiety to sulfone;

(d) reacting the sulfone of step (c) with a compound to convert the hydroxyl of the mercaptoethanol moiety to an ester or halide moiety;

(e) reacting the ethyl sulfone of step (d) with base to form poly(ethylene glycol) vinyl sulfone.

19. The method of claim 18 further comprising the steps of isolating the poly(ethylene glycol) vinyl sulfone.

20. The method of claim 18 wherein the halide of step (d) is chlorine and the product of step (d) is poly(ethylene glycol) chloroethyl sulfone, and wherein the method further comprises the step of isolating the poly(ethylene glycol chloroethyl sulfone by dissolving the chloroethyl sulfone in dichloromethane, precipitating the chloroethyl sulfone in ethyl ether, and recrystallizing the precipitate from ethyl acetate.

21. The method of claim 20 further comprising the steps of dissolving the poly(ethylene gylcol) chloroethyl sulfone crystals in an organic solvent prior to reaction with base to form poly(ethylene glycol) vinyl sulfone, and isolating the poly(ethylene glycol) vinyl sulfone product by filtering to remove the base and evaporating the solvent.

22. A method for preparing a biologically active conjugate of at least one biologically active molecule having the structure W-SH, wherein W-- is a biologically active moiety and --SH is an active thiol moiety, and an activated poly(ethylene glycol) derivative having at least one active sulfone moiety, the activated poly(ethylene glycol) derivative having the structure R--CH₂ CH₂ --(OCH₂ CH₂)_n --Y, wherein n equals 5 to 3,000, Y is --SO₂ --CH═

CH₂, and R is selected from the group consisting of HO--, H₃ CO--, X--CH₂ --CH₂ --SO₂ --, wherein X is halogen, and CH₂ ═

CH--SO₂ --, said method comprising the steps of reacting the biologically active molecule with the activated poly(ethylene glycol) derivative and forming a linkage between the active thiol moiety and the at least one active sulfone moiety of the poly(ethylene glycol).

23. The method of claim 22 wherein W is protein.

24. A method for preparing a biologically active conjugate having the structure

25. A method for preparing a biologically active conjugate having the structure