Pegylated interleukin-10

• US 8,697,045 B2
• Filed: 08/26/2011
• Issued: 04/15/2014
• Est. Priority Date: 09/29/2000
• Status: Expired due to Fees

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Inventors: Lee, Seoju, Wylie, David C., Cannon-Carlson, Susan
• Primary Examiner(s): Kwon, Brian-Yong
• Assistant Examiner(s): LEA, CHRISTOPHER RAYMOND
• Application Number:
  US13/218,929
• Publication Number:
  US 20110305665A1
• Time in Patent Office: 963 Days
• Field of Search: None
• 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-pegylated interleukin-10 (mono-PEG-IL-10) comprising one or more polyethylene glycol (PEG) molecules covalently attached via a linker to a single amino acid residue of a single subunit of IL-10,

wherein said amino acid residue is the alpha amino group of the N-terminal amino acid residue or the epsilon amino group of a lysine residue

and wherein the total molecular mass of the one or more PEG molecules and the linker is from 3,000 daltons to 60,000 daltons.

2. The mono-PEG-IL-10 of claim 1, wherein one or two PEG molecules are attached via said linker to said single amino acid residue.

3. The mono-PEG-IL-10 of claim 1, wherein said subunit of said IL-10 has the formula:

(PEG)_b-L-NH—

IL-10

wherein b is 1-9 and L is a C_2-12 alkyl linker moiety covalently attached to a nitrogen (N) of said single amino acid residue.

4. The mono-PEG-IL-10 of claim 3, wherein b is 1 and L is —

CH₂CH₂CH₂—

.

5. The mono-PEG-IL-10 of claim 1, wherein said PEG molecule is covalently attached via said linker to the nitrogen of the alpha amino group of the N-terminal amino acid residue.

6. The mono-PEG-IL-10 of claim 1, wherein said subunit of said IL-10 has the formula:

[X—

O(CH₂CH₂O_n]_b-L-NH—

IL-10,

wherein 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 the 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.

7. The mono-PEG-IL-10 of claim 6, wherein L is —

CH₂CH₂CH₂—

.

8. The mono-PEG-IL-10 according to claim 1, wherein said mono-PEG-IL-10 has greater than 30% of the activity of unconjugated IL-10.

9. A composition of pegylated IL-10 comprising the mono-PEG-IL-10 according to claim 1, wherein the population of mono-PEG-IL-10 is at least 80% of a positional isomer in which the PEG is conjugated to the N-terminal amino acid of one subunit of IL-10.

10. A process for preparing a pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 1, comprising mixing the mono-PEG-IL-10 with a pharmaceutically acceptable carrier.

11. The mono-PEG-IL-10 according to claim 1, wherein the IL-10 is human IL-10.

12. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 11 and a pharmaceutical carrier.

13. The mono-PEG-IL-10 according to claim 11, wherein the PEG molecule has a molecular weight of 12,000 or 20,000 daltons.

14. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 13 and a pharmaceutical carrier.

15. A pharmaceutical composition comprising the mono-PEG-IL-10 according to claim 1 in combination with a pharmaceutically acceptable carrier.

16. A method of treating inflammation in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 15.

17. A process, for preparing the mono-PEG-IL-10 according to claim 1, 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 under conditions in which the linker is covalently attached to one amino acid residue of the IL-10.

18. The process according to claim 17 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 5.6 to 7.8.

19. The process according to claim 17, wherein the ratio of IL-10 to the sodium cyanoborohydride is 1:

5 to 1;

15.

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

21. The process according to claim 17, wherein the reacting step is performed at a pH of 6.3 to 7.5.

22. The process according to claim 17, further comprising a step selected from:

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

or treating the mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HC1 salt.