Polymer conjugates of interferon-beta with enhanced biological potency

• US 20040126361A1
• Filed: 12/23/2003
• Published: 07/01/2004
• Est. Priority Date: 12/26/2002
• Status: Active Grant

• Current Assignee: Mountain View Pharmaceuticals, Inc.
• Original Assignee: Mountain View Pharmaceuticals, Inc.
• Inventors: Sherman, Merry R., Saifer, Mark G. P., Williams, L. David, Martinez, Alexa L.
• Granted Patent: US 9,125,880 B2
• Time in Patent Office: Days
• US Class Current: 424/85.6
• CPC Class Codes: A61K 38/215 IFN-beta
• 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 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/565 IFN-beta
• CPC Class Codes: G01N 2333/565 IFN-beta

Abstract

Methods are provided for the synthesis of polymer conjugates of cytokines and receptor-binding antagonists thereof, especially a non-glycosylated interferon-beta, which conjugates retain unusually high biological potency. Preparation of polymer conjugates according to the methods of the present invention diminishes or avoids steric inhibition of receptor-ligand interactions that commonly results from the attachment of polymers to receptor-binding regions of cytokines, as well as to agonistic and antagonistic analogs thereof. The invention also provides conjugates and compositions produced by such methods. The conjugates of the present invention retain a high level of biological potency compared to those produced by traditional polymer coupling methods that are not targeted to avoid receptor-binding domains of cytokines. In assays in vitro, the biological potency of the conjugates of non-glycosylated interferon-beta of the present invention is substantially higher than that of unconjugated interferon-beta and is similar to that of interferon-beta-1a that is glycosylated. The conjugates of the present invention also exhibit an extended half-life in vivo compared to the corresponding unconjugated cytokine. The present invention also provides kits comprising such conjugates and/or compositions, and methods of use of such conjugates and compositions in a variety of diagnostic, prophylactic and therapeutic applications, including treatment of multiple sclerosis.

Claims

1. A method for increasing the biological potency of a non-glycosylated interferon-beta, comprising selectively coupling one or more synthetic water-soluble polymers to the amino-terminal amino acid of said interferon-beta, wherein said amino-terminal amino acid is located remotely from the receptor-binding domain(s) of said interferon-beta.

2. The method of claim 1, wherein said biological potency is measured in a cell-culture assay that responds to interferon-beta.

3. The method of claim 1, wherein said one or more polymers are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylenes), one or more poly(amino acids), one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

4. The method of claim 1, wherein said interferon-beta has the amino acid sequence of interferon-β

-1b specified in SEQ ID NO;

1.

5. The method of claim 1, wherein said interferon-beta has substantially the amino acid sequence in SEQ ID NO:

1.

6. The method of claim 1, wherein said polymer is covalently coupled to the alpha amino group of said amino-terminal amino acid.

7. The method of claim 6, wherein said covalent coupling of said polymer to said alpha amino group is via a secondary amine linkage.

8. The method of claim 1, wherein said polymer is coupled to a chemically reactive side chain group of said amino-terminal amino acid.

9. The method of claim 8, wherein said reactive side chain is an aldehyde group that is introduced by selective oxidative cleavage of an amino-terminal serine residue of an interferon-beta.

10. The method of claim 3, wherein said polymer is a polyalkylene glycol.

11. The method of claim 10, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a mono-methoxypoly(ethylene glycol) and a monohydroxy-poly(ethylene glycol).

12. The method of claim 11, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

13. The method of claim 11, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

14. The method of claim 10, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

15. The method of claim 14, wherein said polyalkylene glycol has a molecular weight of between about 8 kDa and about 14 kDa, inclusive.

16. The method of claim 14, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 30 kDa, inclusive.

17. The method of claim 16, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 22 kDa, inclusive.

18. The method of claim 17, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

19. The method of claim 14, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

20. The method of claim 1, wherein the coupling of said polymer to said interferon-beta at said amino-terminal amino acid mimics the beneficial effects of glycosylation or hyperglycosylation of said interferon-beta.

21. A conjugate produced by the method of claim 1.

22. A pharmaceutical composition comprising one or more of the conjugates of claim 21 and one or more pharmaceutically acceptable excipients or carriers.

45. A pharmaceutical composition comprising the conjugate of any of claims 21, 23 and 24 and a pharmaceutically acceptable carrier or excipient.

46. A kit comprising the conjugate of claim 21.

48. A kit comprising the pharmaceutical composition of claim 22.

49. A kit comprising the pharmaceutical composition of claim 45.

50. A method of preventing, diagnosing, or treating an interferon-beta-responsive physical disorder in an animal suffering from or predisposed to said physical disorder, comprising administering to said animal an effective amount of the conjugate of any one of claims 21, 23 and 25.

51. A method of preventing, diagnosing, or treating an interferon-beta-responsive physical disorder in an animal suffering from or predisposed to said physical disorder, comprising administering to said animal an effective amount of the pharmaceutical composition of claim 22 or claim 45.

52. The method of claim 50, wherein said animal is a mammal.

53. The method of claim 51, wherein said animal is a mammal.

54. The method of claim 52 or claim 53, wherein said mammal is a human.

55. The method of claim 50, wherein said interferon-beta-responsive physical disorder is selected from the group consisting of a cancer, an infectious disease, a neurodegenerative disorder, an autoimmune disorder and a genetic disorder.

56. The method of claim 55, wherein said cancer is selected from the group consisting of a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a lung cancer, a leukemia, a lymphoma, a colon cancer, a gastrointestinal cancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a bone cancer, a neurological cancer, a head and neck cancer, a skin cancer, a carcinoma, a sarcoma, an adenoma and a myeloma.

57. The method of claim 55, wherein said interferon-beta-responsive infectious disease is selected from the group consisting of a viral hepatitis, a disease caused by a cardiotropic virus and HIV/AIDS.

58. The method of claim 55, wherein said interferon-beta-responsive neurodegenerative disorder is multiple sclerosis.

59. The method of claim 58, wherein said multiple sclerosis is selected from the group consisting of relapsing-remitting, primary progressive and secondary progressive multiple sclerosis.

60. The method of claim 51, wherein said interferon-beta-responsive physical disorder is selected from the group consisting of a cancer, an infectious disease, a neurodegenerative disorder, an autoimmune disorder and a genetic disorder.

61. The method of claim 60, wherein said cancer is selected from the group consisting of a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a lung cancer, a leukemia, a lymphoma, a colon cancer, a gastrointestinal cancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a bone cancer, a neurological cancer, a head and neck cancer, a skin cancer, a carcinoma, a sarcoma, an adenoma and a myeloma.

62. The method of claim 60, wherein said interferon-beta-responsive infectious disease is selected from the group consisting of a viral hepatitis, a disease caused by a cardiotropic virus and HIV/AIDS.

63. The method of claim 60, wherein said interferon-beta-responsive neurodegenerative disorder is multiple sclerosis.

64. The method of claim 63, wherein said multiple sclerosis is selected from the group consisting of relapsing-remitting, primary progressive and secondary progressive multiple sclerosis.

23. A conjugate comprising an interferon-beta covalently coupled at its amino-terminal amino acid to one or more synthetic water-soluble polymers, wherein the biological potency of said interferon-beta is increased compared to the same interferon-beta that has not been so coupled.

25. The conjugate of claim 23 or claim 24, wherein the biological potency of said conjugate is measured in a cell-culture assay that responds to interferon-beta.

26. The conjugate of claim 23 or claim 24, wherein said interferon-beta has the amino acid sequence of interferon-β

-1b specified in SEQ ID NO;

1

27. The conjugate of claim 26, wherein the biological potency of said interferon-β

-1b is increased to approximately the potency of interferon-β

-1a, which has the amino acid sequence specified in SEQ ID NO;

2 and which is glycosylated on asparagine residue 80.

28. The conjugate of claim 25, wherein said interferon-beta responsive cell-culture assay is selected from the group consisting of an antiproliferative assay, an antiviral assay, a signal transduction assay and a gene activation assay.

29. The conjugate of claim 23 or claim 24, wherein said one or more polymers are selected from the group consisting of one or more polyalkylene glycols, one or more polyalkylene oxides, one or more polyvinyl alcohols, one or more polycarboxylates, one or more poly(vinylpyrrolidones), one or more poly(oxyethylene-oxymethylenes), one or more poly(amino acids), one or more polyacryloylmorpholines, one or more copolymers of one or more amides and one or more alkylene oxides, one or more dextrans and one or more hyaluronic acids.

30. The conjugate of claim 23 or claim 24, wherein said polymer is covalently coupled to the alpha amino group of the amino-terminal amino acid of said interferon-beta.

31. The conjugate of claim 30, wherein said covalent coupling of said polymer to said alpha amino group is via a secondary amine linkage.

32. The conjugate of claim 23 or claim 24, wherein said polymer is coupled to a chemically reactive side chain group of said amino-terminal amino acid.

33. The conjugate of claim 32, wherein said reactive side chain is an aldehyde group that is introduced by selective oxidative cleavage of an amino-terminal serine residue of an interferon-beta.

34. The conjugate of claim 23 or claim 24, wherein said water-soluble polymer is a polyalkylene glycol.

35. The conjugate of claim 34, wherein said polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol), a mono-methoxypoly(ethylene glycol) and a monohydroxypoly(ethylene glycol).

36. The conjugate of claim 35, wherein said polyalkylene glycol is a monomethoxypoly(ethylene glycol).

37. The conjugate of claim 35, wherein said polyalkylene glycol is a monohydroxypoly(ethylene glycol).

38. The conjugate of claim 34, wherein said polyalkylene glycol has a molecular weight of between about 1 kDa and about 100 kDa, inclusive.

39. The conjugate of claim 38, wherein said polyalkylene glycol has a molecular weight of between about 8 kDa and about 14 kDa, inclusive

40. The conjugate of claim 38, wherein said polyalkylene glycol has a molecular weight of between about 10 kDa and about 30 kDa, inclusive.

41. The conjugate of claim 40, wherein said polyalkylene glycol has a molecular weight of between about 18 kDa and about 22 kDa, inclusive.

42. The conjugate of claim 41, wherein said polyalkylene glycol has a molecular weight of about 20 kDa.

43. The conjugate of claim 38, wherein said polyalkylene glycol has a molecular weight of about 30 kDa.

44. The conjugate of claim 23 or claim 24, wherein the coupling of said polymer to said interferon-beta at said amino-terminal amino acid mimics the beneficial effects of glycosylation or hyperglycosylation of said interferon-beta.

47. A kit comprising the conjugate of claim 23 or claim 24.

24. A conjugate comprising an interferon-beta covalently coupled at its amino-terminal amino acid to one or more synthetic water-soluble polymers, wherein the biological potency of said conjugate of interferon-beta is increased compared to the same interferon-beta to which one or more of the same synthetic water-soluble polymers has been coupled randomly to solvent-accessible lysine residues.

65. A method for determining the amount of a polymer that is attached to the amino terminus of a protein having an N-terminal serine or threonine residue, in a polymer-protein conjugate synthesized by reductive alkylation, comprising:

a) reacting the conjugate with a sufficient quantity of an oxidizing agent for a sufficient time to cleave the polymer from the serine or threonine residue; and

b) measuring the increase in the portion of unconjugated protein in the preparation.

66. The method of claim 65, wherein said protein is a cytokine.

67. The method of claim 66, wherein said cytokine is an interferon-beta.

68. The method of claim 66, wherein said cytokine is megakaryocyte growth and development factor.

69. The method of claim 65, wherein said oxidizing agent comprises a periodate selected from the group consisting of sodium metaperiodate, potassium metaperiodate, lithium metaperiodate, calcium periodate, barium periodate and periodic acid.

70. The method of claim 65, wherein said measuring is performed by one or more method(s) selected from the group consisting of size-exclusion chromatography, reversed phase chromatography, gel electrophoresis, capillary electrophoresis, ultracentrifugation, ultrafiltration, light scattering and mass spectroscopy.

71. A method for the selective oxidative cleavage of an N-terminal serine or threonine residue of a bioactive protein without oxidizing functionally essential amino acid residues of said bioactive protein, comprising:

a) adjusting the hydrogen ion concentration of a solution of said bioactive protein to a pH between about 7 and about 8;

b) mixing said solution of bioactive protein with about 0.5 to about 5 moles of a periodate per mole of bioactive protein; and

c) incubating said mixture for at least one hour at a temperature of between about 2°

C. and about 40°

C.

72. The method of claim 71, wherein said bioactive protein is an interferon-beta.

73. The method of claim 72, wherein said interferon-beta is interferon-β

-1b having the amino acid sequence specified in SEQ ID NO;

1.

74. A method for increasing the biological potency of a preparation of interferon-beta-1b, comprising removing one or more inhibitory components from said interferon-beta-1b preparation.

75. The method of claim 74, wherein the one or more inhibitory components are, removed from said interferon-beta-1b preparation by one or more chromatographic methods selected from the group consisting of size-exclusion chromatography, reversed phase chromatography, hydrophobic interaction chromatography and affinity chromatography.

76. The method of claim 74, wherein the biological potency of said preparation of interferon-beta-1b is measured in a cell-culture assay that responds to interferon-beta.

77. The method of claim 76, wherein said interferon-beta responsive cell-culture assay is selected from the group consisting of an antiproliferative assay, an antiviral assay, a signal transduction assay and a gene activation assay.