Protection of exendin-4 peptides through conjugation
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.
1 - 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) Lys 40 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.
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) Lys 40 or a C-terminal amide thereof.
35 . The conjugate of claim 32 which is according to the formula:
wherein X is S, O, or NH of an amino acid of the blood component;
wherein L is present or absent, and when present is selected from the group consisting of —NH(CH 2 ) 2 NH—, —[C(O)—CH 2 —O—(CH 2 ) 2 —NH] m —, and —[C(O)—CH 2 —O—(CH 2 ) 2 —O—(CH 2 ) 2 —NH] m —;
wherein Z is a peptide comprising exendin-4 or a C-terminal amide thereof; and
wherein m is 1, 2 or 3.
36 . A conjugate according to the formula:
wherein X is S, O, or NH of an amino acid of the blood component;
wherein L is present or absent, and when present is selected from the group consisting of —NH(CH 2 ) 2 NH—, —[C(O)—CH 2 —O—CH 2 ) 2 —NH] m —, and —[C(O)—CH 2 —O—(CH 2 ) 2 —O—(CH 2 ) 2 —NH] m —;
wherein Z is a peptide comprising exendin-4 or a C-terminal amide thereof; and
wherein m is 1, 2 or 3.
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) Lys 40 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)—CH 2 —O—(CH 2 ) 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)—CH 2 —O—(CH 2 ) 2 —O—(CH 2 ) 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.
47 . A conjugate according to the following formula:
wherein X is S of Cysteine 34 of albumin.
48 . The conjugate of claim 47 , wherein said albumin is human serum albumin.
49 . A pharmaceutical composition comprising the modified peptide of any of claims 26 to 31 and a physiologically acceptable medium.
50 . A pharmaceutical composition comprising the conjugate of any of claims 32 to 48 and a physiologically acceptable medium.
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.
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) Lys 40 ; 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.
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) Lys 40 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 .
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 .