Glucagon antagonists

• US 8,981,047 B2
• Filed: 10/23/2008
• Issued: 03/17/2015
• Est. Priority Date: 10/30/2007
• Status: Expired due to Fees

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Inventors: Dimarchi, Richard D., Yang, Bin
• Primary Examiner(s): Skowronek, Karlheinz R
• Assistant Examiner(s): GARYU, LIANKO G
• Application Number:
  US12/739,342
• Publication Number:
  US 20120122783A1
• Time in Patent Office: 2,336 Days
• Field of Search: None
• US Class Current: 530/308
• 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 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

Glucagon antagonists are provided which comprise amino acid substitutions and/or chemical modifications to glucagon sequence. In one embodiment, the glucagon antagonists comprise a native glucagon peptide that has been modified by the deletion of the first two to five amino acid residues from the N-terminus and (i) an amino acid substitution at position 9 (according to the numbering of native glucagon) or (ii) substitution of the Phe at position 6 (according to the numbering of native glucagon) with phenyl lactic acid (PLA). In another embodiment, the glucagon antagonists comprise the structure A-B-C as described herein, wherein A is PLA, an oxy derivative thereof, or a peptide of 2-6 amino acids in which two consecutive amino acids of the peptide are linked via an ester or ether bond.

Claims

1. A glucagon antagonist comprising the sequence of SEQ ID NO:

42, or an oxy derivative thereof.

2. The glucagon antagonist of claim 1, further comprising a hydrophilic moiety covalently bound to an amino acid residue at position 11, 16 or 19 of SEQ ID NO:

42, or at the N- or C-terminal amino acid of the glucagon antagonist, and pharmaceutically acceptable salts of said glucagon antagonist.

3. A glucagon antagonist comprising the amino acid sequence of native glucagon (SEQ ID NO:

1) modified by (i) deletion of two to five consecutive amino acid residues from the N-terminus of SEQ ID NO;

1, (ii) substitution of the aspartic acid residue at position nine of SEQ ID NO;

1 with homoglutamic acid, β

-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of;

4. The glucagon antagonist of claim 3, wherein the amino acid sequence of native glucagon is further modified by up to three amino acid modifications selected from the group consisting of:

A. substitution of one or two amino acids at positions 10, 20, and 24, according to the amino acid numbering of SEQ ID NO;

1, or the N- or C-terminal amino acid of the glucagon antagonist with an amino acid comprising an acyl or alkyl group;

B. substitution of one or two amino acids at positions 16, 17, 20, 21, and 24, according to the amino acid numbering of SEQ ID NO;

1, or the N- or C-terminal amino acid of the glucagon antagonist with an amino acid selected from the group consisting of;

Cys, Lys, ornithine, homocysteine, and acetyl-phenylalanine (Ac-Phe), wherein the amino acid of the group is covalently bonded to a hydrophilic moiety;

C. addition of an amino acid covalently bonded to a hydrophilic moiety to the N- or C-terminus of the glucagon antagonist;

D. substitution of Asp at position 15, according to the numbering of SEQ ID NO;

1, with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid;

E. substitution of Ser at position 16, according to the numbering of SEQ ID NO;

1, with cysteic acid, glutamic acid, homoglutamic acid, and homocysteic acid;

F. substitution with AIB at one or more of positions 16, 20, 21, and 24 according to the amino acid numbering of SEQ ID NO;

1;

G. deletion of the amino acid at position 29 or the amino acids at positions 28 and 29, according to the numbering of SEQ ID NO;

1;

H. substitution of each or both of the Asn at position 28 and the Thr at position 29, according to the amino acid numbering of SEQ ID NO;

1, with charged amino acids; and

/or addition of one to two charged amino acids at the C-terminus of SEQ ID NO;

1;

I. substitution of the Met at position 27, according to the numbering of SEQ ID NO;

1, with Leu or norleucine;

J. addition of a peptide having the amino acid sequence of any of SEQ ID NOs;

19-21 and 53 to the C-terminus of SEQ ID NO;

1;

wherein Thr at position 29, according to the numbering of SEQ ID NO;

1, is Thr or Gly; and

K. replacement of the C-terminal carboxylate with an amide or ester.

5. A glucagon antagonist comprising the general structure of A-B-C, wherein A is:

(i) phenyl lactic acid (PLA);

or

(ii) an oxy derivative of PLA;

B represents amino acids i to 26 of SEQ ID NO;

1, wherein i is 3, 4, 5, 6, or 7, optionally comprising one or more amino acid modifications selected from the group consisting of;

(iv) Asp at position 9, according to the amino acid numbering of SEQ ID NO;

1, is substituted with a Glu, a sulfonic acid derivative of Cys, homoglutamic acid, β

-homoglutamic acid, or an alkylcarboxylate derivative of cysteine having the structure of;

6. The glucagon antagonist of claim 5, wherein the oxy derivative of PLA is PLA linked to an amino acid, peptide, hydrophilic polymer, acyl group, or alkyl group via an ester bond or ether bond.

7. The glucagon antagonist of claim 5, wherein the amino acid is Xaa₃, or wherein the peptide comprises Xaa₂-Xaa₃ or Xaa₁-Xaa₂-Xaa₃, wherein Xaa₃ is Gln or Glu, Xaa₂ is selected from a group consisting of:

Ser, D-serine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB); and

Xaa₁ is selected from a group consisting of;

His, D-histidine, alpha, alpha-dimethyl imidiazole acetic acid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine and homo-histidine.

8. The glucagon antagonist of claim 5, wherein the peptide of (iii) comprises amino acids j to 6 of SEQ ID NO:

1, wherein j is 1, 2, 3, 4, or 5.

9. The glucagon antagonist of claim 5, wherein B represents amino acids 7 to 26 of SEQ ID NO:

1.

10. The glucagon antagonist of claim 5, comprising

a hydrophilic moiety covalently bound to an amino acid residue at position 16, 21, or 24, according to the numbering of SEQ ID NO:

1, or the N- or C-terminal residue of the glucagon antagonist,

an amino acid covalently attached to an acyl group or alkyl group via an ester, ether, thioether, amide, or alkyl amine linkage, wherein the amino acid is at position 10, 20, or 24, according to the amino acid numbering of SEQ ID NO;

1, or is the N- or C-terminal residue of the glucagon antagonist, or

a combination thereof.

11. The glucagon antagonist of claim 5, comprising the amino acid sequence selected from the group consisting of:

SEQ ID NOs;

62, 64-67, 71, [PLA6, D9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     72), [PLA6, D9]Glucagon(aa6-29)-COOH (SEQ ID NO;

     73), [PLA6, E9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     74), [PLA6, hCys(SO3)9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     75), [PLA6, D9, D28] Glucagon(aa6-29)-NH2 (SEQ ID NO;

     76), [PLA6, E9]Glucagon (aa6-29+CEX)-NH2 (SEQ ID NO;

     77), [Ac-PLA⁶, E⁹]Glucagon(aa6-29)-NH₂ (SEQ ID NO;

     78), [PLA⁵ E⁹]Glucagon(aa5-29)-NH₂ (SEQ ID NO;

     79), [PLA⁴, E⁹]Glucagon(aa-4-29)-NH₂ (SEQ ID NO;

     80), [PLA6, C8(20 kDaPEG), E9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     81), [PLA6, E9, C11(20 kDaPEG)]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     82), [PLA6, E9, C24(20 kDaPEG)]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     83), [PLA6, C8(1.2 kDaPEG), E9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     84), [PLA6, C8(5 kDaPEG), E9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     85), [PLA6, C8(20 kDaPEG), E9]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     86), [PLA6, E9, C24(1.2 kDaPEG)]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     87), and [PLA6, E9, C24(20 kDaPEG)]Glucagon(aa6-29)-NH2 (SEQ ID NO;

     88).

12. The glucagon antagonist of claim 5, comprising a Glu at position 9, according to the amino acid numbering of SEQ ID NO:

1.

13. The glucagon antagonist of claim 5, wherein C is X-Y-Z, wherein X is Met, Leu, or Nle;

Y is Asn or a charged amino acid;

Z is Thr, Gly, Cys, Lys, ornithine (Orn), homocysteine, acetyl phenylalanine (Ac-Phe), or a charged amino acid.

14. The glucagon antagonist of claim 13, wherein X is Met, Y is Asn, and Z is Thr.

15. The glucagon antagonist of claim 13, wherein X is Met, Y is a charged amino acid, and Z is Thr.

16. The glucagon antagonist of claim 15, wherein Y is Asp.

20. A dimer or multimer comprising two or more glucagon antagonists of claim 5.

21. A conjugate comprising a glucagon antagonist of claim 5 and a heterologous peptide or polypeptide.

22. A pharmaceutical composition comprising a glucagon antagonist of claim 5 and a pharmaceutically acceptable carrier.

23. A method comprising administering to a patient an effective amount of a pharmaceutical composition of claim 22.

17. A peptide comprising the amino acid sequence of SEQ ID NO:

72.

18. A peptide comprising the amino acid sequence of SEQ ID NO:

73.

19. A peptide comprising the amino acid sequence of SEQ ID NO:

76.