Synthetic erythropoiesis stimulating proteins

• US 20030191291A1
• Filed: 01/13/2003
• Published: 10/09/2003
• Est. Priority Date: 09/08/2000
• Status: Abandoned Application

• Current Assignee: Gryphon Therapeutics, Inc.
• Original Assignee: Gryphon Therapeutics, Inc.
• Inventors: Hunter, Christine L., Botti, Paolo, Chen, Shiah-yun, Cressman, Sonya, Low, Donald W., Kochendoerfer, Gerd G., Bradburne, James A., Kent, Stephen B.H.
• Application Number:
  US10/332,696
• Publication Number:
  US 20030191291A1
• Time in Patent Office: Days
• US Class Current: 530/397
• 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: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• 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: A61P 31/12 Antivirals
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]

Abstract

Synthetic erythropoiesis stimulating proteins are provided. Also provided are methods for synthesizing the proteins. The invention further relates to derivatives of such synthetic erythropoiesis stimulating proteins that are polymer-modified in a defined manner. Methods and uses for such proteins and derivatized proteins are also provided.

Claims

1. A synthetic erythropoiesis stimulating protein having one or more water-soluble polymers attached thereto.

2. The synthetic erythropoiesis stimulating protein of claim 1 having the in vivo biological activity of increasing production of red blood cells.

3. The synthetic erythropoiesis stimulating protein of claim 1, wherein said protein comprises a polypeptide chain having an amino acid sequence of a ribosomally specified erythropoietin and one or more non-overlapping peptide segments covalently bonded by one or more chemical ligation sites.

4. The synthetic erythropoiesis stimulating protein of claim 3, wherein one or more of said water-soluble polymers is attached to said polypeptide chain at a site corresponding to a glycosylation site of said ribosomally specified erythropoietin.

5. The synthetic erythropoiesis stimulating protein of claim 3, wherein said ribosomally specified erythropoietin is human.

6. The synthetic erythropoiesis stimulating protein of claim 3, wherein said ribosomally specified erythropoietin glycoprotein is non-natural.

7. The synthetic erythropoiesis stimulating protein of claim 6, wherein said non-natural ribosomally specified erythropoietin has one or more non-natural glycosylation sites.

8. The synthetic erythropoiesis stimulating protein of claim 2, wherein said water-soluble polymer is attached exclusively to said polypeptide chain at one or more sites corresponding to a glycosylation site of said ribosomally specified erythropoietin.

9. The synthetic erythropoiesis stimulating protein of claim 1, wherein one or more of said water-soluble polymers comprises a repeat unit comprising a polyalkylene oxide, a polyamide alkylene oxide, or derivatives thereof.

10. The synthetic erythropoiesis stimulating protein of claim 9, wherein said polyalkylene oxide and polyamide alkylene oxide comprise an ethylene oxide repeat unit of the formula —

(CH2-CH2-O)—

.

11. The synthetic erythropoiesis stimulating protein of claim 1, wherein one or more of said water-soluble polymers is branched.

12. The synthetic erythropoiesis stimulating protein of claim 1, wherein one or more of said water-soluble polymers comprises a net charge under physiological conditions that is negative.

13. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein has an isoelectric point between 3 and 7.

14. The synthetic erythropoiesis stimulating protein of claim 1, wherein said water-soluble polymer is mono-disperse.

15. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein is mono-disperse.

16. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 25 kDa.

17. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 40 kDa.

18. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 50 kDa.

19. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 60 kDa.

20. The synthetic erythropoiesis stimulating protein of claim 1, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 70 kDa.

21. The synthetic erythropoiesis stimulating protein of claim 1, wherein said polypeptide chain comprises one or more irregular amino acids.

22. The synthetic erythropoiesis stimulating protein of claim 21, wherein said irregular amino acid comprises a non-natural side chain.

23. The synthetic erythropoiesis stimulating protein of claim 21, wherein said irregular amino acid comprises a pseudo amino acid.

24. The synthetic erythropoiesis stimulating protein of claim 23, wherein said pseudo amino acid is pseudo glutamate.

25. The synthetic erythropoiesis stimulating protein of claim 22, wherein said non-natural side chain is covalently bonded to a water-soluble polymer.

26. The synthetic erythropoiesis stimulating protein of claim 25, wherein said water-soluble polymer is covalently bonded to said side chain through a bond formed by chemical ligation.

27. The synthetic erythropoiesis stimulating protein of claim 26, wherein said bond formed by chemical ligation is selected from the group consisting of amide, oxime, hydrazone, thaizolidine, oxazolidine and thioester.

28. The synthetic erythropoiesis stimulating protein of claim 3, wherein one or more of said chemical ligation sites comprises an amide bond.

29. The synthetic erythropoiesis stimulating protein of claim 1, wherein said water-soluble polymer is attached to said polypeptide chain through a side chain of an amino acid at a chemical ligation site of said polypeptide chain.

30. A synthetic erythropoiesis stimulating protein according to claim 1 which comprises an amino acid sequence of a synthetic erythropoiesis stimulating protein selected from the group consisting of SEP-1 and SEP-3.

33. A pharmaceutical composition comprising a synthetic erythropoiesis stimulating protein according to claim 1, or pharmaceutically acceptable salts thereof.

34. The pharmaceutical composition according to claim 33 which comprises one or more excipients selected from the group consisting of a buffer, a carrier protein, an amino acid, a detergent, a lipid, a water soluble polymer, and a preservative.

35. The pharmaceutical composition according to claim 33, which comprises one or more additional bioactive agents other than said synthetic erythropoiesis stimulating protein.

36. A method of increasing the hematocrit of a mammal, said method comprising administering to said mammal an effective amount of a synthetic erythropoiesis stimulating protein according to claim 1, whereby the hematocrit in said mammal is increased.

37. A method of increasing production of red blood cells in a mammal, said method comprising administering to said mammal an effective amount of a polymer-modified synthetic erythropoiesis stimulating protein according to claim 1, whereby the production of red blood cells in said mammal is increased.

38. A method of increasing production of hemoglobin in a mammal, said method comprising administering to said mammal an effective amount of a polymer-modified synthetic erythropoiesis stimulating protein according to claim 1, whereby the production of hemoglobin in said mammal is increased.

39. A method of increasing reticulocyte count in a mammal, said method comprising administering to said mammal an effective amount of a synthetic erythropoiesis stimulating protein according to claim 1, whereby the reticulocyte count in said mammal is increased.

31. A synthetic erythropoiesis stimulating protein selected from the group consisting of SEP-0, SEP-1 and SEP-3, and analogs thereof.

32. The synthetic erythropoiesis stimulating protein of claim 31, wherein said analogs are selected from the group consisting of SEP-1-L26, SEP-1-L30, SEP-1-B50, SEP-1-B51, and SEP-1-B52.

40. A method of producing a polypeptide chain comprising a synthetic erythropoiesis stimulating protein, said method comprising chemically ligating peptide segments comprising non-overlapping amino acid sequences of a polypeptide chain of said synthetic erythropoiesis stimulating protein whereby a polypeptide chain comprising said synthetic erythropoiesis stimulating protein is produced.

41. The method of claim 40, which further comprises folding said polypeptide chain whereby a bioactive synthetic erythropoiesis stimulating protein is produced.

42. The method of claim 40, wherein one or more of said peptide segments are partially protected.

43. The method of claim 40, wherein one or more of said peptide segments are unprotected.

44. The method of claim 40, wherein one or more of said peptide segments comprises a water-soluble polymer attached thereto.

45. The method of claim 40, wherein said chemically ligating comprises a chemoselective ligation chemistry selected from native chemical ligation, extended native chemical ligation, pseudo native chemical ligation, oxime forming chemical ligation, hydrazone forming chemical ligation, oxazolidine forming chemical ligation, thaizolidine forming chemical ligation, and thioester forming chemical ligation.

46. The method of claim 40, wherein said polypeptide chain comprises one or more water-soluble polymers attached thereto.

47. The method of claim 40, wherein said polypeptide chain comprises an amino acid sequence of a ribosomally specified erythropoietin.

48. The method of claim 46, wherein one or more of said water-soluble polymers is attached to said polypeptide chain at a site corresponding to a glycosylation site of said ribosomally specified erythropoietin.

49. The method of claim 48, wherein said ribosomally specified erythropoietin glycoprotein is recombinantly produced.

50. The method of claim 48, wherein said ribosomally specified erythropoietin glycoprotein is non-natural.

51. The method of claim 50, wherein said non-natural ribosomally specified erythropoietin glycoprotein has one or more non-natural glycosylation sites.

52. The method of claim 47, wherein said ribosomally specified erythropoietin is human.

53. The method of claim 48, wherein said water-soluble polymer is attached exclusively to said polypeptide chain at a site corresponding to a glycosylation site of said ribosomally specified erythropoietin.

54. The method of claim 46, wherein one or more of said water-soluble polymers comprises a repeat unit comprising a polyalkylene oxide, a polyamide alkylene oxide, or derivatives thereof.

55. The method of claim 54, wherein said polyalkylene oxide and polyamide alkylene oxide comprise an ethylene oxide repeat unit of the formula —

(CH2-CH2-O)—

.

56. The method of claim 46, wherein one or more of said water-soluble polymers is branched.

57. The method of claim 46, wherein one or more of said water-soluble polymers comprises a net charge under physiological conditions that is negative.

58. The method of claim 41, wherein said synthetic erythropoiesis stimulating protein has an isoelectric point between 3 and 7.

59. The method of claim 46, wherein said water-soluble polymer is mono-disperse.

60. The method of claim 40, wherein said synthetic erythropoiesis stimulating protein is mono-disperse.

61. The method of claim 46, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 25 kDa.

62. The method of claim 46, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 40 kDa.

63. The method of claim 46, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 50 kDa.

64. The method of claim 46, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 60 kDa.

65. The method of claim 46, wherein said synthetic erythropoiesis stimulating protein comprises a monomer molecular weight of greater than 70 kDa.

66. The method of claim 40, wherein said polypeptide chain comprises one or more irregular amino acids.

67. The method of claim 66, wherein said irregular amino acid comprises a non-natural side chain.

68. The method of claim 67, wherein said irregular amino acid comprises a pseudo amino acid.

69. The method of claim 40, wherein said polypeptide chain comprises a chemical ligation site having a bond selected from the group consisting of amide, oxime, hydrazone, thaizolidine, oxazolidine and thioester.

70. The method of claim 46, wherein said water-soluble polymer is attached to an amino acid of said polypeptide chain having a non-natural side chain.

71. The method of claim 46, wherein said water-soluble polymer is covalently bonded to said side chain through a bond formed by chemical ligation.

72. The method of claim 71, wherein said bond formed by chemical ligation is selected from the group consisting of amide, oxime, hydrazone, thaizolidine, oxazolidine and thioester.

73. The method of claim 46, wherein said water-soluble polymer is attached to said polypeptide chain through a side chain of an amino acid at a chemical ligation site of said polypeptide chain.

74. The method of claim 41, wherein said bioactive synthetic erythropoiesis stimulating protein has the in vivo biological activity of increasing production of red blood cells.

75. The method of claim 40, wherein said polypeptide chain comprises an amino acid sequence of a synthetic erythropoiesis stimulating protein selected from the group consisting of SEP-1 and SEP-3.

76. The method of claim 40, wherein synthetic erythropoiesis stimulating protein is selected from the group consisting of SEP-0, SEP-1 and SEP-3, and analogs thereof.

77. The method of claim 76, wherein said analogs are selected from the group consisting of SEP-1-L26, SEP-1-L30, SEP-1-B50, SEP-1-B51, and SEP-1-B52.