Pegylated interleukin-10

• US 20020044921A1
• Filed: 09/28/2001
• Published: 04/18/2002
• Est. Priority Date: 09/29/2000
• Status: Active Grant

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Schering Corporation (Merck & Co., Inc.)
• Inventors: Cannon-Carlson, Susan V., Wylie, David C., Lee, Seoju
• Granted Patent: US 7,052,686 B2
• Time in Patent Office: Days
• US Class Current: 424/85.2
• CPC Class Codes: A61K 38/2066 IL-10
• 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: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07K 14/5428 IL-10
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Interleukin-10 (IL-10) conjugated via a linker to one or more polyethylene glycol (PEG) molecules at a single amino acid residue of the IL-10, and a method for preparing the same, are provided. The method produces a stable mono-pegylated IL-10, which retains IL-10 activity, where pegylation is selective for the N-terminus on one subunit of IL-10 with little or no formation of monomeric IL-10. The method also provides a substantially homogenous population of mono-PEG-IL-10.

Claims

1. A mono-PEG-IL-10.

2. The mono-PEG-IL-10 of claim 1, comprising one or two PEG molecules covalently attached via a linker to one amino acid residue on IL-10, wherein the attachment is at an N-terminal amino acid residue or on a lysine residue.

3. The mono-PEG-IL-10 of claim 2:

(a) which comprises a methoxy PEG;

(b) wherein the IL-10 is human IL-10;

(c) wherein the total molecular mass of all PEG covalently attached to the linker is from 3,000 daltons to 60,000 daltons;

or

(d) wherein the linker is a linear or branched C_1-11 alkyl.

4. The mono-PEG-IL-10 of claim 2, wherein the total molecular mass of all PEG covalently attached to the linker is from 10,000 daltons to 36,000 daltons.

5. The mono-PEG-IL-10 of claim 2, wherein the linker is a linear C₃ alkyl.

6. The mono-PEG-IL-10 of claim 1, wherein a PEG molecule is covalently attached to the alpha amino group of one N-terminus of IL-10 via a linear C₃ alkyl linker.

9. A pharmaceutical composition, comprising a mono-PEG-IL-10 of claim 1 in combination with a pharmaceutically acceptable carrier.

10. A method of treating inflammation in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition of claim 9.

7. A PEG-IL-10 comprising the formula:

[X—

O(CH₂CH₂O)_n]_b—

L—

NH-IL-10,

where X is H or C_1-4 alkyl, n is 20 to 2300, b is 1 to 9 and L is a C_1-11 alkyl linker moiety which is covalently attached to nitrogen (N) of the alpha amino group at the amino terminus of one IL-10 subunit, provided that when b is greater than 1 the total of n does not exceed 2300.

8. A PEG-IL-10 of claim 7, wherein L is —

CH₂CH₂CH₂—

.

11. A process for preparing a mono-PEG-IL-10, comprising the step of:

reacting IL-10 with an activated PEG-aldehyde linker in the presence of a reducing agent to form the mono-PEG-IL-10,

wherein the linker is covalently attached to one amino acid residue of the IL-10.

12. The process of claim 11 wherein:

(a) the reducing agent is sodium cyanoborohydride;

(b) the activated PEG-aldehyde linker is PEG-propionaldehyde;

(c) the PEG is a methoxy-PEG;

(d) the linker is multi-armed;

(e) the ratio of IL-10 to the sodium cyanoborohydride is from about 1;

0.5 to 1;

50;

(f) the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 3,000 daltons to 60,000 daltons;

or

(g) the reacting step is performed at a pH of 5.5 to 7.8.

13. The process of claim 11, wherein the ratio of IL-10 to the sodium cyanoborohydride is 1:

5 to 1;

15.

14. The process of claim 11, wherein the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 10,000 daltons to 36,000 daltons.

15. The process of claim 11, wherein the reacting step is performed at a pH of 6.3 to 7.5.

16. The process of claim 11, further comprising a step selected from:

incubating the mono-PEG-IL-10 product in a buffer at pH 5.0 to 9.0; and

treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.

18. The process of claim 17, wherein the total molecular mass of all PEG comprising the PEG-aldehyde linker is from 10,000 daltons to 36,000 daltons.

19. The process of claim 17, further comprising a step selected from:

incubating the mono-PEG-IL-10 product in a TRIS buffer at pH 7.0 to 8.0; and

treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.

20. A PEG-IL-10 prepared according to a process of claim 11.

17. A process for preparing a mono-PEG-IL-10, comprising the step of:

reacting IL-10 with an activated PEG-propionaldehyde linker in the presence of sodium cyanoborohydride, wherein the molar ratio of IL-10 to sodium cyanoborohydride is from about 1;

5 to about 1;

15, at a pH of about 6.3 to about 7.5 and a temperature of from 18°

C. to 25°

C. form the mono-PEG-IL-10,

wherein the linker is covalently attached to one amino acid residue of the IL-10.