Thiol-selective water-soluble polymer derivatives

Patent metadata

Field	Value
Publication number	US-9333267-B2
Application number	US-201514694160-A
Country	US
Kind code	B2
Filing date	Apr 23, 2015
Priority date	Jan 6, 2003
Publication date	May 10, 2016
Grant date	May 10, 2016

Abstract

The present invention provides water-soluble, polymer derivatives having a thiol-selective terminus suitable for selective coupling to thiol groups, such as those contained in the cysteine residues of proteins.

First claim

It is claimed:

1. A method for preparing a thiol-selective derivative of a water-soluble polymer comprising the steps of:
(i) providing an electrophilically-activated polymer, POLY-L 0,1 -E, wherein:
POLY is a water-soluble polymer segment,
L is an optional linker,
E is an electrophile, and

(ii) reacting the POLY-L 0,1 -E with a symmetrical disulfide reagent, (NU-Y-S-) 2 , wherein:
NU is a nucleophile, and
Y is alkylene, substituted alkylene, cycloalkylene, substituted cycloalkylene, aryl, or substituted aryl, comprising from about 2 to about 10 carbon atoms,

under conditions effective to promote reaction between E and NU to thereby form POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY, wherein X is a group resulting from the reaction between E and NU.

2. The method of claim 1 , further comprising the step of (iii) reducing the disulfide bond in POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY to form POLY-L 0,1 -X-Y-SH.

3. The method of claim 1 , wherein L 1 is C 1 -C 10 alkylene or C 1 -C 10 substituted alkylene.

4. The method of claim 3 , wherein L 1 is—(CH 2 ) 1-5 —.

5. The method of claim 1 , wherein E is a carboxylic acid or an activated carboxylic acid derivative.

6. The method of claim 1 , wherein E is a carboxylic acid, activated carboxylic acid ester, acid halide, or active anhydride.

7. The method of claim 6 , wherein E is an N-hydroxysuccinimidyl ester.

8. The method of claim 1 , wherein NU is amino, hydroxy, imino, or thiol.

9. The method of claim 1 , wherein NU is —NH 2 .

10. The method of claim 1 , wherein X is an amide, carbamate, carbonate ester, ether, or thioester.

11. The method of claim 1 , wherein POLY is a polyalkylene oxide.

12. The method of claim 11 , wherein POLY is a polyethylene glycol having the structure H 3 CO—(CH 2 CH 2 O) n CH 2 CH 2 — wherein n ranges from 10 to about 4,000.

13. The method of claim 11 , wherein POLY is end-capped.

14. The method of claim 12 , wherein L is absent or —CH 2 —, and E is N-hydroxysuccinimidyl ester or 1-hydroxybenzotriazolyl carbonate.

15. The method of claim 14 , wherein the symmetrical disulfide reagent is cystamine, where NU is primary amino and Y is —(CH 2 ) 2 —.

16. A method for preparing a polymer-protein conjugate comprising the steps of:
(i) providing an electrophilically-activated polymer, POLY-L 0,1 -E, wherein:
POLY is a water-soluble polymer segment,
L is an optional linker, and
E is an electrophile,

(ii) reacting the POLY-L 0,1 -E with a symmetrical disulfide reagent, (NU-Y-S-) 2 , wherein:
NU is a nucleophile, and
Y is selected from the group consisting of C 2 -C 10 alkylene, C 2 -C 10 substituted alkylene, arylene, and substituted arylene,

under conditions effective to promote reaction between E and NU to thereby form POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY wherein X is a group resulting from the reaction between E and NU,
(iii) reducing the disulfide bond in POLY-L 0,1 -X-Y-S-S-Y-X-L 0,1 -POLY to form POLY-L 0,1 -X-Y-SH, and
(iv) reacting POLY-L 0,1 -X-Y-SH with a thiol or protected thiol group of a protein to form a protein conjugate, POLY-L 0,1 -X-Y-S-S-protein.

17. The method of claim 16 , wherein the protein is a therapeutic protein.

Assignees

• Nektar Therapeutics

Inventors

• Kozlowski Antoni
• Gross Iii Remy F
• Mcmanus Samuel P

Classifications

• C08G65/2636

  the other compounds containing sulfur · CPC title

• A61K47/48215 Primary

  Human Necessities · mapped topic

• C07C319/06

  from sulfides, hydropolysulfides or polysulfides · CPC title

• C08G65/329

  with organic compounds · CPC title

• A61K47/48238

  Human Necessities · mapped topic