PEG-Urate Oxidase Conjugates and Use Thereof

• US 20080031864A1
• Filed: 08/03/2007
• Published: 02/07/2008
• Est. Priority Date: 08/06/1998
• Status: Active Grant

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University
• Original Assignee: MOUNTAIN VIEW PHARMACEUTICALS, INC. AND DUKE UNIVERSITY, MOUNTAIN VIEW PHARMACEUTICALS, INC. AND DUKE UNIVERSITY
• Inventors: Hershfield, Michael, Saifer, Mark, Sherman, Merry, Kelly, Susan, Williams, L.
• Granted Patent: US 7,927,589 B2
• Time in Patent Office: Days
• US Class Current: 424/94.3
• 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

1-41. -41. (canceled)

42. A conjugate of urate oxidase (uricase) that retains uricolytic activity and has a prolonged half-life in vivo compared to an unconjugated uricase, said conjugate comprising a purified uricase conjugated to poly(ethylene glycol) (PEG), wherein greater than 90% of said uricase is in a tetrameric form.

43. The conjugate of claim 42, wherein said PEG has an average molecular weight of about 10 kDa to about 60 kDa.

44. The conjugate of claim 43, wherein said PEG has an average molecular weight of about 10 kDa.

45. The conjugate of claim 43, wherein said PEG has an average molecular weight of about 20 kDa.

46. The conjugate of claim 43, wherein said PEG has an average molecular weight of about 30 kDa.

47. The conjugate of claim 42, wherein the average number of strands of said PEG that are covalently linked to each subunit of said uricase is 2 to 10.

48. The conjugate of claim 42, wherein said uricase is a mammalian uricase.

49. The conjugate of claim 48, wherein said uricase is porcine liver uricase, bovine liver uricase or ovine liver uricase.

50. The conjugate of claim 42, wherein said uricase is recombinant.

51. The conjugate of claim 50, wherein said recombinant uricase has the amino acid sequence of porcine, bovine, ovine or baboon liver uricase.

52. The conjugate of claim 50, wherein said recombinant uricase is chimeric.

53. The conjugate of claim 52, wherein said chimeric uricase contains portions of porcine liver uricase and portions of baboon liver uricase.

54. The conjugate of claim 53, wherein said chimeric uricase is pig-baboon chimeric uricase (PBC uricase).

55. The conjugate of claim 53, wherein said 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).

56. The conjugate of claim 50, wherein said recombinant uricase has the amino acid sequence of baboon liver uricase (SEQ ID NO:

2) in which tyrosine 97 has been replaced by histidine (Y97H).

57. The conjugate of claim 50, wherein said recombinant uricase comprises an amino terminus and a carboxyl terminus, and wherein said uricase is truncated at one terminus or at both termini.

58. The conjugate of claim 47, wherein the average number of covalently linked strands of PEG is 3 to 8 strands per uricase subunit.

59. The conjugate of claim 58, wherein the average number of covalently linked strands of PEG is 4 to 6 strands per uricase subunit.

60. The conjugate of claim 42, wherein the strands of said PEG are covalently linked to said uricase via linkages selected from the group consisting of urethane linkages, secondary amine linkages, and amide linkages.

61. The conjugate of claim 42, wherein said PEG is linear.

62. The conjugate of claim 42, wherein said PEG is branched.

63. The conjugate of claim 42, wherein greater than 95% of said uricase is in a tetrameric form.

64. The conjugate of claim 57, wherein said uricase is truncated at said amino terminus by deleting at least the first six amino acids from said amino terminus.

65. A pharmaceutical composition for lowering uric acid levels in a body fluid or tissue, comprising a purified uricase conjugated to poly(ethylene glycol) (PEG) and a pharmaceutically acceptable carrier, wherein greater than 90% of said uricase is in a tetrameric form.

66. The pharmaceutical composition of claim 65, wherein said composition is stabilized by lyophilization and dissolves promptly upon reconstitution to provide a solution suitable for parenteral administration.

67. A method for isolating a tetrameric form of urate oxidase (uricase) from a solution of uricase comprising tetrameric uricase and uricase aggregates, said method comprising

(a) applying said solution to at least one separation column at a pH between about 9 and about 10.5;

and

(b) recovering from said column one or more fractions that contain isolated tetrameric uricase, wherein greater than 90% of said isolated uricase in said one or more fractions is in a tetrameric form.

68. The method of claim 67, wherein said solution of said uricase is applied to said column at a pH of 10.2.

69. The method of claim 67, wherein the separation of said tetrameric uricase from said solution is based on a property selected from the group consisting of ion exchange and size exclusion.

70. The method of claim 67, further comprising analyzing said fractions to determine at least one property thereof selected from the group consisting of the presence of said tetrameric uricase and the absence of said uricase aggregates.

71. The method of claim 70, wherein said analyzing comprises at least one analytical method selected from the group consisting of chromatography, centrifugation, light scattering and electrophoresis.

72. The method of claim 71, wherein said chromatography is high performance liquid chromatography.