Chemically-modified human growth hormone conjugates

• US 20030171285A1
• Filed: 11/20/2002
• Published: 09/11/2003
• Est. Priority Date: 11/20/2001
• Status: Abandoned Application

• Current Assignee: Pharmacia Corporation (Pfizer Inc.)
• Original Assignee: Pharmacia Corporation (Pfizer Inc.)
• Inventors: Liao, Wei, Finn, Rory F., Siegel, Ned R.
• Application Number:
  US10/300,822
• Publication Number:
  US 20030171285A1
• Time in Patent Office: Days
• US Class Current: 514/12
• 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 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin

Abstract

The present invention provides a chemically modified human Growth Hormone (hGH) prepared by binding a water soluble polymer to the protein. The chemically-modified protein according to the present invention may have a much longer lasting hGH activity than that of the un-modified hGH, enabling reduced dose and scheduling opportunities.

Claims

1. A conjugate comprising at least one water-soluble polymer molecule covalently attached to at least one amino acid residue of a biologically active human growth hormone (hGH) polypeptide or agonist variant thereof.

2. The conjugate of claim 1 wherein said hGH polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

3. The conjugate of claim 1, or 2, wherein said polymer is a poly(ethylene oxide) molecule.

4. The conjugate of claim 3 wherein said poly(ethylene oxide) molecule is a poly(ethylene glycol) molecule.

5. The conjugate of claim 4 wherein the poly(ethylene glycol) is attached at an amino acid residue having a free amino group(s), carboxyl group(s), or sulfhydryl group(s).

6. The conjugate of claim 5 formed using a activated poly(ethylene glycol).

7. The conjugate of claim 6 wherein said activated poly(ethylene glycol) comprises a functional group.

8. The conjugate of claim 7 wherein said attachment is at an amino acid having a free amino group.

9. The conjugate of claim 8 wherein said functional group is selected from the group consisting of:

carbonates, carbonylimidazole, active esters of carboxylic acids, azlactones, cyclic imide thiones, isocyanates or isothiocyanates, imidates, and aldehydes.

10. The conjugate of claim 9 wherein said functional group is a carbonate or carbonylimidazole.

11. The conjugate of claim 10 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

12. The conjugate of claim 11 having the structure

13. The conjugate of claim 12 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

14. The conjugate of claim 9 wherein said functional group is a cyclic imide thione.

15. The conjugate of claim 14 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

16. The conjugate of claim 9 wherein said functional group is a azlactone.

17. The conjugate of claim 16 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

18. The conjugate of claim 9 wherein said functional group is a isocyanate or isothiocyanate.

19. The conjugate of claim 18 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

mPEG-N═

C═

O, and mPEG-N═

C═

S.

20. The conjugate of claim 9 wherein said functional group is an aldehyde or aldehyde hydrate.

21. The conjugate of claim 20 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

22. The conjugate of claim 9 wherein said functional group is an active ester of a carboxylic acid.

23. The conjugate of claim 22 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

24. The conjugate of claim 23 having the structure selected from the group consisting of:

25. The conjugate of claim 24 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

26. The conjugate of claim 9 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

mPEG-O—

SO₂—

CH₂CF₃, and

27. The conjugate of claim 26 having the structure selected from the group consisting of:

PEG-NH—

R, and

28. The conjugate of claim 27 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

29. The conjugate of claim 28 wherein said functional group is an imidate.

30. The conjugate of claim 9 wherein said activated poly(ethylene glycol) is selected from the group consisting of:

31. The conjugate of claim 9 wherein said free amino group is an amino terminal α

-amino group.

32. The conjugate of claim 31 wherein said amino terminal α

-amino group is on a phenylalanine.

33. The conjugate of claim 8 wherein said attachment is at an amino acid having a free carboxyl group.

34. The conjugate of claim 33 wherein said functional group is selected from the group consisting of:

primary amines;

hydrazine; and

hydrazide functional groups.

35. The conjugate of claim 34 wherein said functional group is selected from the group consisting of:

36. The conjugate of claim 8 wherein said attachment is at an amino acid having a free sulfhydryl group.

37. The conjugate of claim 36 wherein said functional group is selected from the group consisting of:

thiols;

maleimides;

vinyl sulfones; and

phenyl glyoxals.

38. The conjugate of claim 37 wherein said functional group is selected from the group consisting of:

39. The conjugate of claim 38 having the structure selected from the group consisting of:

40. The conjugate of claim 39 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

41. The conjugate of claim 8 wherein said poly(ethylene glycol) has a molecular weight of between about 0.5 kDa and about 100 kDa.

42. The conjugate of claim 41 wherein said poly(ethylene glycol) has a molecular weight of between about 5 kDa and about 40 kDa.

43. The conjugate of claim 8 wherein said poly(ethylene glycol) is a branched polymer.

44. The conjugate of claim 43 wherein said branched polymer is selected from the group consisting of:

60. The conjugate of claim 8 wherein said poly(ethylene glycol) is a bifunctional polymer.

61. The conjugate of claim 8 wherein said poly(ethylene glycol) is a prodrug.

62. A composition comprising the hGH of claim 1 and at least one pharmaceutically acceptable carrier.

63. A method of treating a patient having a growth or development disorder or comprising administering to said patient a therapeutically effective amount of the hGH conjugate of claim 1.

64. The method of claim 63 wherein said growth or development disorder is Growth Hormone Deficiency (GHD).

65. The method of claim,63 wherein said growth or development disorder is Turner'"'"'s syndrome.

66. The method of claim 63 wherein said growth or development disorder is Chronic Renal Insufficiency.

67. The method of claim 63 wherein said growth or development disorder is small for gestational age (SGA).

75. A method of treating a patient having a growth or development disorder or comprising administering to said patient a therapeutically effective amount of the hGH conjugate of claim 1.

76. The method of claim 75 wherein said growth or development disorder is Growth Hormone Deficiency (GHD).

77. The method of claim 75 wherein said growth or development disorder is Turner'"'"'s syndrome.

78. The method of claim 75 wherein said growth or development disorder is Chronic Renal Insufficiency.

79. The method of claim 75 wherein said growth or development disorder is small for gestational age (SGA).

45. A human growth hormone-PEG conjugate having the structure

46. The conjugate of claim 45 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

47. The conjugate of claim 46 wherein at least 80% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

48. The conjugate of claim 46 wherein at least 90% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

49. The conjugate of claim 47 or 48 wherein each mPEG has a molecular weight of about 20 kDa.

68. A composition comprising the hGH of claim 48 and at least one pharmaceutically acceptable carrier.

69. A method of treating a patient having a growth or development disorder or comprising administering to said patient a therapeutically effective amount of the hGH conjugate of claim 48.

70. The method of claim 69 wherein said growth or development disorder is Growth Hormone Deficiency (GHD).

71. The method of claim 69 wherein said growth or development disorder is Turner'"'"'s syndrome.

72. The method of claim 69 wherein said growth or development disorder is Chronic Renal Insufficiency.

73. The method of claim 69 wherein said growth or development disorder is small for gestational age (SGA).

50. A human growth hormone-PEG conjugate having the structure

51. The conjugate of claim 50 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

52. The conjugate of claim 51 wherein at least 80% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

53. The conjugate of claim 51 wherein at least 90% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

54. The conjugate of claim 52 or 53 wherein each mPEG has a molecular weight of about 20 kDa.

74. A composition comprising the hGH of claim 50 or 55 and at least one pharmaceutically acceptable carrier.

55. A human growth hormone-PEG conjugate having the structure

mPEG-OCH₂CH₂CH₂NH—

R

wherein R is a human growth hormone polypeptide.

56. The conjugate of claim 55 wherein said human growth hormone polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

57. The conjugate of claim 56 wherein at least 80% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

58. The conjugate of claim 56 wherein at least 90% of said polyethylene glycol is conjugated to the amino-terminal phenylalanine.

59. The conjugate of claim 57 or 58 wherein each mPEG has a molecular weight of about 20 kDa.