Glp-1 Fusion Peptides, Their Production and Use

• US 20110130329A1
• Filed: 09/22/2006
• Published: 06/02/2011
• Est. Priority Date: 09/22/2005
• Status: Active Grant

• Current Assignee: Biocompatibles Incorporated (Boston Scientific Corporation)
• Original Assignee: Biocompatibles Incorporated (Boston Scientific Corporation)
• Inventors: Geigle, Peter, Thoenes, Eric, Wallrapp, Christine
• Granted Patent: US 8,431,533 B2
• Time in Patent Office: Days
• US Class Current: 514/5.3
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: C07K 14/605 Glucagons

Abstract

The present invention provides fusion peptides having GLP-1 activity and enhanced stability in vivo, in particular resistancy to dipeptidyl peptidase IV. The fusion peptide comprises as component (I) N-terminally a GLP-1(7-35, 7-36 or 7-37) sequence and as component (II) C-terminally a peptide sequence of at least 9 amino acids or a functional fragment, variant or derivative thereof. Component (II) is preferably a full or partial version of IP2 (intervening peptide 2). A preferred embodiment comprises the sequence GLP-1(7-35, 36 or 37)/IP2/GLP-1(7-35, 36 or 37) or GLP-2. The fusion peptide may be produced in engineered cells or synthetically and may be used for the preparation of a medicament for treating various diseases or disorders, e.g. diabetes type 1 or 2, apoptosis related diseases or neurodegenerative disorders.

Claims

1. A fusion peptide comprising as component (I) N-terminally a sequence according to formula II

Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22-Xaa23-Ala-Xaa25-Xaa26-Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37 (SEQ ID NO:

44),

wherein

Xaa? is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine, homohistidine, N-acetyl-histidine, α

-fluoromethyl-histidine, α

-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaa8 is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl)carboxylic acid;

Xaa16 is Val or Leu;

Xaa18 is Ser, Lys or Arg;

Xaa19 is Tyr or Gln;

Xaa20 is Leu or Met;

Xaa22 is Gly, Glu or Aib;

Xaa23 is Gln, Glu, Lys or Arg;

Xaa25 is Ala or Val;

Xaa26 is Lys, Glu or Arg;

Xaa27 is Glu or Leu;

Xaa30 is Ala, Glu or Arg;

Xaa33 is Val or Lys;

Xaa34 is Lys, Glu, Asn or Arg;

Xaa35 is Gly or Aib;

Xaa36 is Arg, Gly or Lys or amide or absent;

Xaa37 is Gly, Ala, Glu, Pro, Lys, amide or is absent.

or formula III

Xaa?-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Glu-Phe-Ile-Xaa30-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-Xaa37 (SEQ ID NO;

     45),

wherein

Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, 3-hydroxy-histidine, homohistidine, N-acetyl-histidine, α

-fluoromethyl-histidine, α

-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;

Xaa8 is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl)carboxylic acid;

Xaa18 is Ser, Lys or Arg;

Xaa22 is Gly, Glu or Aib;

Xaa23 is Gln, Glu, Lys or Arg;

Xaa26 is Lys, Glu or Arg;

Xaa30 is Ala, Glu or Arg;

Xaa34 is Lys, Glu or Arg;

Xaa35 is Gly or Aib;

Xaa36 is Arg or Lys, amide or is absent;

Xaa37 is Gly, Ala, Glu or Lys, amide or is absent.

and as component (II) C-terminally a peptide sequence of at least 9 amino acids or a functional fragment, variant or derivative thereof.

2. The fusion peptide according to claim 1 comprising as component (I) N-terminally a GLP-1(7-35, 7-36 or 7-37) sequence and as component (II) C-terminally a peptide sequence of at least 9 amino acids or a functional fragment, variant or derivative thereof.

3. The fusion peptide according to claim 1 comprising as component (I) N-terminally a sequence according to formula I

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-X (I) (SEQ ID NO:

43), wherein X is NH₂ or Gly-OH,

and as component (II) C-terminally a peptide sequence of at least 9 amino acids or a functional fragment, variant or derivative thereof.

4. The fusion peptide according to claim 2, wherein component (I) contains a sequence having at least 80% sequence homology with SEQ ID No.:

1.

5. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence forming a β

-turn like structure.

6. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing at least one alanine or proline residue.

7. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing a tetramer with β

turn forming properties or having a proline residue at position 2 of that tetramer.

8. The fusion peptide according to any of preceding claim 1 wherein component (II) is a peptide sequence containing a sequence motif selected from the group consisting of VAIA (SEQ ID NO:

46), IAEE (SEQ ID NO;

     47), PEEV (SEQ ID NO;

     48), AEEV (SEQ ID NO;

     49), EELG (SEQ ID NO;

     50), AAAA (SEQ ID NO;

     51), AAVA (SEQ ID NO;

     52), AALG (SEQ ID NO;

     53), DFPE (SEQ ID NO;

     54), AADX, AXDX, and XADX, wherein X represents any amino acid.

9. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence being linked to the C-terminus of component (I) by its N-terminal sequence motif selected from the group consisting of AA₁ XA, AX, RR₁ RX₁ and XR.

10. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing the sequence motif of SEQ ID No.:

25 (DFPEEVA) or containing a sequence having at least 80% sequence homology with the SEQ ID No.;

25.

11. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing a sequence selected from the group consisting of SEQ ID No.:

22 (RRDFPEEVAI) and SEQ ID No.;

26 (AADFPEEVAI) or containing a sequence having at least 80% sequence homology with SEQ ID No.;

22 or SEQ ID No.;

26.

12. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing a sequence selected from a group consisting of SEQ ID No.:

23 (RRDFPEEVAIVEEL), SEQ ID No. 24 (RRDFPEEVAIAEEL), SEQ ID NO.;

27 (AADFPEEVAIVEEL), and SEQ ID No.;

28 (AADFPEEVAIAEEL), or a sequence having at least 80% sequence homology with any of SEQ ID Nos.;

23, 24, 27, or 28.

13. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence containing a sequence selected from the group consisting of SEQ ID No.:

2 (RRDFPEEVAIVEELG), SEQ ID No.;

3 (RRDFPEEVAIAEELG), SEQ ID NO.;

29 (AADFPEEVAIVEELG), and SEQ ID NO.;

30 (AADFPEEVAIAEELG), or a sequence having at least 80% sequence homology with SEQ ID Nos.;

2, 3, 29 or 30.

14. The fusion peptide according to claim 1, wherein component (II) is a peptide sequence having about 9 to 30, about 9 to 20, or about 9 to 15 amino acids.

15. The fusion peptide according to claim 1, wherein component (I) and component (II) are directly linked or linked via a linker sequence.

16. The fusion peptide according to claim 15, wherein the linker sequence has a length of about 1 to 10 amino acids.

17. The fusion peptide according to claim 1, wherein the fusion peptide contains a sequence selected from the group consisting of

SEQ ID NO.:

8 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIAEELG),

SEQ ID NO.;

12 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELG),

SEQ ID NO.;

31 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGAADFPEEVAIAEELG),

SEQ ID NO.;

32(HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFAEEVAIAEELG),

SEQ ID NO.;

33 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDAAAAVAIAEELG),

SEQ ID NO.;

34 (HAEGTFTSDVSSYLEGQAAKEIAWLVKGRGAADAAAAVAIAAALG),

SEQ ID NO.;

35(HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFP),

SEQ ID NO.;

36 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVA),

SEQ ID NO.;

37 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIAEELGRRHAC),

SEQ ID NO.;

38 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGAADFPEEVAIVEELG),

SEQ ID NO.;

39 (HAEGTFTSDVSSYLEGQAAKE-FIAWLVKGRGRRDFAEEVAIVEELG),

SEQ ID NO.;

40(HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDAAAAVAIVEELG),

SEQ ID NO.;

41 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGAADAAAAVAIVAALG), and

SEQ ID NO.;

42 (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHAC), or a sequence having at least 80% sequence homology with SEQ ID Nos.;

8, 12.

18. The fusion peptide according to claim 1, wherein the fusion peptide contains another component (III) linked to the C-terminus of component (II) and/or to the N-terminus of component (I).

19. The fusion peptide according to claim 18, wherein component (III) comprises at least four amino acid residues, or at least 10 additional amino acid residues, or at least 20 additional amino acid residues, or at least 30 additional amino acid residues.

20. The fusion peptide according to claim 19, wherein component (III) comprises at least 4, at least 10, or at least 20 additional amino acid residues of the N-terminal sequence of GLP-2 as in proglucagon or of GLP-1(7-37).

21. The fusion peptide according to claim 18, wherein component (III) contains the sequence of SEQ ID Nos.:

4 or 5 or a sequence having at least 80% sequence homology with SEQ ID Nos.;

4 or 5.

22. The fusion peptide according to claim 18, wherein the fusion peptide contains a peptide sequence selected from a group consisting of:

SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 10 and SEQ ID No. 11 or a sequence having at, least 80% sequence homology with SEQ ID Nos.;

6, 7, 10, or 11.

23. The fusion peptide according to claim 1, wherein at least one of the amino acids is derivatized by a covalent modification of a side chain of a naturally occurring amino acid, by a modification of the peptide backbone, by modification of the NH₂ or carboxy terminal groups.

24. The fusion peptide according to claim 23, wherein at least one of the amino acids is derivatized by a lipyl or carbohydrate group.

25. The fusion peptide according to claim 23, wherein the N-terminal His residue of GLP-1 (GLP-1(7)) is chemically modified at its NH₂ terminus and/or at its histidyl side chain, in particular by a hydrophobic moiety.

26. The fusion peptide according to claim 1, wherein the fusion peptide further comprises a carrier protein, comprising transferrin or albumin, as component (IV).

27. The fusion peptide according to claim 1, wherein the amino acid sequence of components (I), (II) and/or (III) is reversed and wherein said amino acid sequence(s) is at least partially composed of D amino acid isomers.

28. A method of producing a fusion peptide according to claim 1, by solid state peptide synthesis.

29. A nucleic acid encoding a fusion peptide according to claim 1.

30. A vector comprising a nucleic acid according to claim 29.

31. A host cell comprising exogenously introduced DNA according to claim 30, being capable of expressing said fusion peptide.

32. A method for producing a fusion peptide according to claim 1 in which a micro-organism transformed to include a nucleic acid encoding the fusion peptide is fermented and the protein is recovered.

33. The method of producing a protein according to claim 32 in which animal cells are grown under conditions in which the protein is exported from the cells.

34. A pharmaceutical composition comprising the fusion peptide according to claim 1.

35. A method for the treatment of diabetes mellitus type I or type II, insulin resistance, weight disorders and diseases or conditions associated thereto comprising administering an effective amount of a fusion peptide of claim 1 to a subject in need thereof.

36. A method for the treatment of neurodegenerative disorders and diseases or conditions associated thereto comprising administering an effective amount of a fusion peptide of claim 1 to a subject in need thereof.

37. A method for the treatment of disorders and diseases or conditions associated to apoptosis comprising administering an effective amount of a fusion peptide of claim 1 to a subject in need thereof.