Protection of exendin-4 peptides through conjugation

• US 20090093408A1
• Filed: 08/01/2008
• Published: 04/09/2009
• Est. Priority Date: 05/17/1999
• Status: Abandoned Application

• Current Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Original Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Inventors: Bridon, Dominique P., St. Pierre, Serge, Leblanc, Anouk, L'Archeveque, Benoit, Holmes, Darren L.
• Application Number:
  US12/221,554
• Publication Number:
  US 20090093408A1
• Time in Patent Office: Days
• US Class Current: 514/1.1
• CPC Class Codes: A61K 38/38 Albumins
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: C07K 14/57563 Vasoactive intestinal peptide [VIP]; Related peptides

Abstract

A method for protecting a peptide from peptidase activity in vivo, the peptide being composed of between 2 and 50 amino acids and having a C-terminus and an N-terminus and a C-terminus amino acid and an N-terminus amino acid is described. In the first step of the method, the peptide is modified by attaching a react group to the C-terminus amino acid, to the N-terminus amino acid, or to an amino acid located between the N-terminus and the C-terminus, such that the modified peptide is capable of forming a covalent bond in vivo with a reactive functionality on a blood component. In the next step, a covalent bond is formed between the reactive group and a reactive functionality on a blood component to form a peptide-blood component conjugate, thereby protecting said peptide from peptidase activity. The final step of the method involves the analyzing of the stability of the peptide-blood component conjugate to assess the protection of the peptide from peptidase activity.

Claims

1-25. -25. (canceled)

26. A modified peptide comprising:

a peptide; and

a maleimide or a maleimido-containing reactive group coupled, optionally via a linker, to the peptide,

the modified peptide being capable of covalently binding in vivo or in vitro by reaction of the reactive group, with a functionality on a blood component;

wherein the peptide comprises exendin-4 or a C-terminal amide thereof.

27. The modified peptide of claim 26, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) or a C-terminal amide thereof.

28. The modified peptide of claim 26, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) Lys⁴⁰ or a C-terminal amide thereof.

29. The modified peptide of claim 28 which is according to the following formula:

30. The modified peptide of any one of claims 26-29, wherein the blood component is albumin.

31. The modified peptide of claim 26, wherein the functionality on the blood component is an amino group, an hydroxyl group, or a thiol group.

49. A pharmaceutical composition comprising the modified peptide of any of claims 26 to 31 and a physiologically acceptable medium.

55. A method of preparing a conjugate comprising:

contacting a modified peptide of claim 26 with a blood component under conditions suitable for formation of a conjugate between said modified peptide and said blood component.

56. The method of claim 55, wherein the amino acid sequence of the modified peptide is that of exendin-4(1-39) or a C-terminal amide thereof.

57. The method of claim 55, wherein the amino acid sequence of the modified peptide is that of exendin-4(1-39) Lys⁴⁰ or a C-terminal amide thereof.

58. The method of claim 55, wherein the reactive group of the modified peptide is a maleimido-containing group.

59. The method of claim 55, wherein the blood component is albumin.

60. The method of claim 59 wherein said modified peptide is conjugated to Cys-34 of human serum albumin.

61. The method of claim 55, wherein the contacting occurs in vitro.

62. The method of claim 55, wherein the modified peptide is according to the following formula:

63. A method for the treatment of brittle diabetes or iatrogenic hypoglycemia in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the modified peptide of claim 26.

32. A conjugate formed by conjugating

(a) a peptide coupled, optionally via a linker, to a maleimide or a maleimido-containing reactive group, with

(b) a blood component, by covalent linking of the reactive group with a functionality on the blood component,

wherein the peptide comprises exendin-4 or a C-terminal amide thereof.

33. The conjugate of claim 32, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) or a C-terminal amide thereof.

34. The conjugate of claim 32, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) Lys⁴⁰ or a C-terminal amide thereof.

35. The conjugate of claim 32 which is according to the formula:

50. A pharmaceutical composition comprising the conjugate of any of claims 32 to 48 and a physiologically acceptable medium.

36. A conjugate according to the formula:

37. The conjugate of claim 36, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) or a C-terminal amide thereof.

38. The conjugate of claim 36, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) Lys⁴⁰ or a C-terminal amide thereof.

39. The conjugate of claim 36, wherein L is present.

40. The conjugate of claim 39, wherein Z is covalently attached to L through the NH of an amino acid of Z.

41. The conjugate of claim 39, wherein L is a poly ethoxy amino acid.

42. The conjugate of claim 41, wherein the poly ethoxy amino acid is according to the formula:

—

[C(O)—

CH₂—

O—

(CH₂)₂—

NH]_m—

wherein m is 1, 2 or 3.

43. The conjugate of claim 41, wherein the poly ethoxy amino acid is according to the formula:

—

[C(O)—

CH₂—

O—

(CH₂)₂—

O—

(CH₂)₂—

NH]_m—

wherein m is 1, 2 or 3.

44. The conjugate of claim 36, wherein X is S.

45. The conjugate of claim 36, wherein the blood component is albumin.

46. The conjugate of claim 44, wherein the S is of Cys-34 of human serum albumin.

64. A method for the treatment of brittle diabetes or iatrogenic hypoglycemia in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate of claim 36.

47. A conjugate according to the following formula:

48. The conjugate of claim 47, wherein said albumin is human serum albumin.

51. A method of synthesizing a modified exendin-4(1-39) peptide comprising:

(a) synthesizing a peptide from the carboxy terminal amino acid, wherein the amino acid sequence of the peptide is that of exendin-4(1-39); and

(b) adding a reactive group, optionally via a linking group, to the carboxy terminal amino acid.

53. The method of claim 51 or 52, wherein the reactive group is maleimide or a maleimido-containing group.

54. The method of claim 51 or 52, wherein the reactive group is succinimide or a succinimido-containing group.

52. A method of synthesizing a modified exendin-4(1-39) peptide comprising:

(a) synthesizing a peptide from the carboxy terminal amino acid, wherein the amino acid sequence of the peptide is that of exendin-4(1-39) Lys⁴⁰; and

(b) adding a reactive group, optionally via a linking group, to the carboxy terminal amino acid.