PEG-urate oxidase conjugates and use thereof

• US 6,576,235 B1
• Filed: 08/06/1999
• Issued: 06/10/2003
• Est. Priority Date: 08/06/1998
• Status: Expired due to Term

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University
• Original Assignee: Duke University, Mountain View Pharmaceuticals, Inc.
• Inventors: Kelly, Susan J., Hershfield, Michael S., Sherman, Merry R., Saifer, Mark G. P., Williams, L. David
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US09/370,084
• Time in Patent Office: 1,404 Days
• Field of Search: 424/94.4, 435/189, 435/191, 435/252.3, 435/320.1, 536/23.2, 530/350
• US Class Current: 424/94.4
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 13/12 of the kidneys
• CPC Class Codes: A61P 19/06 Antigout agents, e.g. antihyperuricemic or uricosuric agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C12N 9/0046 with oxygen as acceptor (1.7.3)
• CPC Class Codes: C12N 9/0048 Uricase (1.7.3.3)
• CPC Class Codes: C12N 9/0093 acting on CH or CH2 groups (1.17)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 107/03003 Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

Abstract

A naturally occurring or recombinant urate oxidase (uricase) covalently coupled to poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), wherein an average of 2 to 10 strands of PEG are conjugated to each uricase subunit and the PEG has an average molecular weight between about 5 kDa and 100 kDa. The resulting PEG-uricase conjugates are substantially non-immunogenic and retain at least 75% of the uricolytic activity of the unmodified enzyme.

Claims

1. A conjugate of uricase that retains at least about 75% of the uricolytic activity of unconjugated uricase and is substantially reduced in immunogenicity, comprising a purified uricase containing no more than about 10% non-tetrameric aggregated uricase, said purified uricase comprising subunits in which each subunit of the uricase is covalently linked to an average of 2 to 10 strands of PEG, wherein each molecule of PEG has a molecular weight between about 5.kDa and 100 kDa.

2. The conjugate of claim 1, wherein the uricase is mammalian uricase.

3. The conjugate of claim 2, wherein the uricase is porcine liver, bovine liver or ovine liver uricase.

4. The conjugate of claim 1, wherein the uricase is recombinant.

5. The conjugate of claim 4, wherein the uricase has the sequence of porcine, bovine, ovine or baboon liver uricase.

6. The conjugate of claim 4, wherein the uricase is chimeric.

7. The conjugate of claim 6, wherein the chimeric uricase contains portions of porcine liver and baboon liver uricase.

8. The conjugate of claim 7, wherein the chimeric uricase is pig-baboon chimeric uricase (PBC uricase).

9. The conjugate of claim 7, wherein the chimeric uricase is porcine uricase in which arginine residue 291 of SEQ ID NO:

1 has been replaced by lysine (R291K) and threonine residue 301 of SEQ ID NO;

1 has been replaced by serine (T301S) (PKS uricase).

10. The conjugate of claim 4, wherein the uricase has the sequence of baboon liver uricase (SEQ ID NO:

2) in which tyrosine 97 has been replaced by histidine.

11. The conjugate of claim 4, wherein the uricase comprises an amino terminus and a carboxy terminus, and wherein the uricase is truncated at one terminus or both termini.

12. The conjugate of claim 1, wherein the uricase is a fungal or microbial uricase.

13. The conjugate of claim 12, wherein the fungal or microbial uricase is isolated from Apergillus flavus, Arthrobacter globiformis or Candida utilis, or is a recombinant enzyme having the sequence of one of those uricases.

14. The conjugate of claim 1, wherein the uricase is an invertebrate uricase.

15. The conjugate of claim 14, wherein the invertebrate uricase is isolated from Drosophila melanogaster or Drosophila pseudoobscura, or is a recombinant enzyme having the sequence of one of those uricases.

16. The conjugate of claim 1, wherein the uricase is a plant uricase.

17. The conjugate of claim 16, wherein the plant uricase is isolated from root nodules of Glycine max or is a recombinant enzyme having the sequence of that uricase.

18. The conjugate of claim 1, wherein the PEG has an average molecular weight between about 10 kDa and 60 kDa.

19. The conjugate of claim 18, wherein the PEG has an average molecular weight between about 20 kDa and 40 kDa.

20. The conjugate of claim 1, wherein the average number of covalently coupled strands of PEG is 3 to 8 strands per uricase subunit.

21. The conjugate of claim 20, wherein the average number of covalently coupled strands of PEG is 4 to 6 strands per uricase subunit.

22. The conjugate of claim 1, wherein the uricase is tetrameric.

23. The conjugate of claim 1, wherein the strands of PEG are covalently coupled to uricase via linkages selected from the group consisting of urethane linkages, secondary amine linkages, and amide linkages.

24. The conjugate of claim 1, wherein the PEG is linear.

25. The conjugate of claim 1, wherein the PEG is branched.

26. A pharmaceutical composition for lowering uric acid levels in a body fluid or tissue, comprising the conjugate of claim 1 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein said composition is stabilized by lyophilization and dissolves promptly upon reconstitution to provide solutions suitable for parenteral administration.

28. The conjugate of claim 11, wherein said uricase is truncated at said amino terminus by deleting at least the first six amino acids from said amino terminus.

29. The conjugate of claim 11, wherein said uricase is truncated at said carboxyl terminus by deleting at least the last three amino acids from said carboxyl terminus.

30. The conjugate of claim 11, wherein said uricase is truncated at said amino terminus and at said carboxyl terminus, by deleting at least the first six amino acids from said amino terminus and by deleting at least the last three amino acids from said carboxyl terminus.

31. The conjugate of claim 11, wherein said uricase is truncated at said amino terminus by deleting the first six amino acids from said amino terminus.

32. The conjugate of claim 11, wherein said uricase is truncated at said carboxyl terminus by deleting the last three amino acids from said carboxyl terminus.

33. The conjugate of claim 11, wherein said uricase is truncated at said amino terminus and at said carboxyl terminus, by deleting the first six amino acids from said amino terminus and by deleting the last three amino acids from said carboxyl terminus.