Glucagon-like-peptide-2 (GLP-2) analogues

• US 20070231308A1
• Filed: 05/04/2006
• Published: 10/04/2007
• Est. Priority Date: 05/04/2005
• Status: Active Grant

• Current Assignee: Zealand Pharma A/S
• Original Assignee: Zealand Pharma A/S
• Inventors: Larsen, Bjarne, Petersen, Yvette
• Granted Patent: US 7,563,770 B2
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: A61K 35/54 Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
• CPC Class Codes: A61K 35/74 Bacteria therapeutic use of a bacterial protein A61K38/00
• 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: A61K 38/26 Glucagons
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/19 lyophilised , i.e. freeze-dried, solutions or dispersions lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/12 Antidiarrhoeals
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 19/10 for osteoporosis
• 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 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 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 39/02 Antidotes
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: C07K 14/605 : Glucagons
• CPC Class Codes: C12N 2710/10043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/20043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/22043 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2760/20243 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

GLP-2 analogues are disclosed which comprise one of more substitutions as compared to [hGly2]GLP-2 and which improved biological activity in vivo and/or improved chemical stability, e.g. as assessed in in vitro stability assays. More particularly, preferred GLP-2 analogues disclosed herein comprise substitutions at one or more of positions 8, 16, 24 and/or 28 of the wild-type GLP-2 sequence, optionally in combination with further substitutions at position 2 (as mentioned in the introduction) and one or more of positions 3, 5, 7, 10 and 11, and/or a deletion of one or more of amino acids 31 to 33 and/or the addition of a N-terminal or C-terminal stabilizing peptide sequence. The analogues are particularly useful for the prophylaxis or treatment of stomach and bowel-related disorders and for ameliorating side effects of chemotherapy.

Claims

1. A glucagon-like peptide 2 (GLP-2) analogue represented by general Formula I (SEQ ID NO:

51);

R¹-Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²

wherein;

R¹ is hydrogen, C_1-4 alkyl methyl, acetyl, formyl, benzoyl, or trifluoroacetyl

X2 is Gly, Ala, or Sar

X3 is Glu or Asp

X5 is Ser or Thr

X6 is Phe or Pro

X7 is Ser or Thr

X8 is Asp or Ser

X9 is Glu or Asp

X10 is Met, Leu, Nle, or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Lys, or Ser

X12 is Thr or Lys

X13 is Ile, Glu, or Gln

X14 is Leu, Met, or Nle

X15 is Asp or Glu

X16 is Asn or Ala

X17 is Leu or Glu

X18 is Ala or Aib

X19 is Ala or Thr

X20 is Arg or Lys

X21 is Asp or Ile

X24 is Asn, Ala, or Glu

X28 is Gln, Ala, or Asn

X31 is Pro, Ile, or deleted

X32 is Thr or deleted

X33 is Asp, Asn, or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 amino acid units selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, and Orn; and

wherein the GLP-2 analogue comprises one or more of substitutions selected from the group consisting of X8 is Ser, X16 is Ala, X24 is Ala, and X28 is Ala;

or a pharmaceutically acceptable salt or derivative thereof.

2. The GLP-2 analogue of claim 1 represented by general Formula II (SEQ ID NO:

52);

R¹-Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²

wherein;

R¹ is hydrogen, C_1-4 alkyl, methyl, acetyl, formyl, benzoyl, or trifluoroacetyl

X3 is Glu or Asp

X5 is Ser or Thr

X7 is Ser or Thr

X8 is Asp or Ser

X9 is Glu or Asp

X10 is Met, Leu, Nle, or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Lys, or Ser

X12 is Thr or Lys

X13 is Ile, Glu, or Gln

X14 is Leu, Met, or Nle

X15 is Asp or Glu

X16 is Asn or Ala

X17 is Leu or Glu

X19 is Ala or Thr

X24 is Asn or Ala

X28 is Gln, Ala, or Asn

X31 is Pro, Ile, or deleted

X32 is Thr or deleted

X33 is Asp or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 amino acid units selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, and Orn; and

wherein the GLP-2 analogue comprises one or more of substitutions selected from the group consisting of X8 is Ser, X16 is Ala, X24 is Ala, and X28 is Ala;

or a pharmaceutically acceptable salt or derivative thereof.

3. The GLP-2 analogue of claim 1 represented by general Formula III (SEQ ID NO:

53);

R¹-Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-Glu-X10-X11-Thr-Ile-Leu-Asp-X16-Leu-Ala-Ala-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²

wherein;

R¹ is hydrogen, C_1-4 alkyl, methyl, acetyl, formyl, benzoyl, or trifluoroacetyl

X3 is Glu or Asp

X5 is Ser or Thr

X7 is Ser or Thr

X8 is Asp or Ser

X10 is Met, Leu, Nle, or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Lys, or Ser

X24 is Asn or Ala

X28 is Gln or Ala

X31 is Pro or deleted

X32 is Thr or deleted

X33 is Asp or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 amino acid units selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, and Orn; and

wherein the GLP-2 analogue comprises one or more of substitutions selected from the group consisting of X8 is Ser, X16 is Ala, X24 is Ala, and X28 is Ala;

or a pharmaceutically acceptable salt or derivative thereof.

4. The GLP-2 analogue of claim 1, having at least 60% amino acid sequence identity to wild-type GLP-2 (1-33) and having the biological activity of causing an increase in intestinal mass in vivo.

5. The GLP-2 analogue of claim 1 comprising a plurality of said substitutions at positions X8, X16, X24, X28;

or comprising one of more of said substitutions in combination with one or more additional substitutions of the wild-type GLP-2 (1-33) sequence at positions selected from the group consisting of X3, X5, X7, X10, and X11.

6. The GLP-2 analogue of claim 5, wherein X10 is Leu, Nle, an oxidatively stable Met-replacement amino acid, Met(O), or Met(O)₂.

7. The GLP-2 analogue of claim 5, wherein X11 is Ala, Ser, or Lys.

8. The GLP-2 analogue of claim 4 comprising at least one group of substitutions selected from the group consisting of:

Ser8, Ala16;

Ser8, Ala24;

Ser8, Ala28;

Ala16, Ala24;

Ala16, Ala28;

Ala24, Ala28;

Ser8, Ala16, Ala24;

Ser8, Ala16, Ala28;

Ser8, Ala24, Ala28;

Ala16, Ala24, Ala28; and

Ser8, Ala16, Ala24, Ala28.

9. The GLP-2 analogue of claim 5, comprising at least one group of substitutions selected from the group consisting of:

Glu3, Leu10, Ala11,24;

Glu3, Thr5, Leu10, Ser11, Ala16,24,28;

Glu3, Thr5, Leu10, Lys11, Ala16,24,28;

Glu3, Thr5, Ser8, Leu10, Lys11, Ala16,24,28;

Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28;

Glu3, Ser8,11, Leu10, Ala16,24,28;

Glu3, Leu10, Ser11, Ala16,24,28;

Glu3, Leu10, Lys11, Ala16,24,28;

Glu3, Thr5, Leu10, Ala11,16,24,28;

Glu3, Thr5, Leu10, Ala11,16,24,28, Ile21;

Glu3, Thr5, Ser8, Leu10, Ala11,16,24,28;

Glu3, Ser8, Leu10, Ala11,16,24,28;

Glu3, Leu10, Ala11,16,24,28;

Thr7, Leu10, Ala11, 24;

Thr7, Leu10, Lys11, Ala24;

Thr7, Leu10, Ser11, Ala24;

Thr7, Leu10, Ser8,11, Ala24;

Thr7, Ser8, Leu10, Ala11,24;

Thr7, Ser8, Leu10, Lys11, Ala24;

Ser8, Leu10, Ala11,24;

Leu10, Ala24;

Leu10, Ala11, Ala24;

Leu10, Ala11,24,28;

Leu10, Ala11,16,24,28;

Leu10, Lys11, Ala24;

Leu10, Ser11, Ala24;

Leu10, Ser8,11, Ala24; and

a deletion at one or more of positions selected from the group consisting of X31-X33.

10. The GLP-2 analogue of claim 1 selected from the group consisting of those disclosed in Table 1 or a pharmaceutically acceptable salt or derivative thereof.

11. The GLP-2 analogue of claim 10 selected from the group consisting of:

1834 H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO;

24),

1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆NH₂ (SEQ ID NO;

34),

1847 H-HGEGSFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO;

35),

1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO;

36),

1849 H-HGEGSFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO;

37),

1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO;

43),

1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO;

45), and

1858 H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO;

46).

12. The GLP-2 analogue of claim 1 comprising a plurality of substitutions of the amino acid sequence of wild-type GLP-2 (1-33) at positions selected from the group consisting of X3, X7, X16, X24, X28, X31, X32, and X33.

13. The GLP-2 analogue of claim 12, comprising at least one substitution selected from the group consisting of X3 is Glu, X7 is Ser, X16 is Ala, X24 is Ala, X28 is Ala, X31 is Ile, X32 is Thr, and X33 is Asp, and a deletion of zero, one, two, or three of the amino acid residues in positions X31, X32, and X33 are optionally deleted;

or a pharmaceutically acceptable salt or derivative thereof.

14. The GLP-2 analogue of claim 12 selected from the group consisting of:

1827 H-HGDGSFTDELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 (SEQ ID NO;

17),

1844 H-HGEGTFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

32),

1845 H-HGEGTFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

33),

1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

34),

1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

36),

1849 H-HGEGSFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

37),

1850 H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

38),

1851 H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

39),

1852 H-HGEGTFSSELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 (SEQ ID NO;

40),

1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO;

43),

1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO;

45),

1858 H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO;

46), and

1859 H-HGEGSFSDELKTILDALAARDFIAWLIATKITD-NH2 (SEQ ID NO;

47);

or a pharmaceutically acceptable salt or derivative thereof.

15. The GLP-2 analogue of claim 1, comprising a plurality of substitutions of the amino acid sequence of wild-type GLP-2 (1-33) at positions selected from the group consisting of X3, X8, and X24.

16. The GLP-2 analogue of claim 15 comprising one or more additional substitutions selected from the group consisting of X3 is Asp, X8 is Asp, and X24 is Ala;

and deletion of zero, one, two, or three of the amino acid residues in positions X31, X32 and X33;

or a pharmaceutically acceptable salt or derivative thereof.

17. The GLP-2 analogue of claim 15 selected from the group consisting of:

1830 H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

20),

1831 H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

21),

1835 H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

25),

1836 H-HGDGSFTDELKTILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

26),

1839 H-HGDGSFSDELATILDNLAARDFIAWLIQTKITDKKKKKK-NH2 (SEQ ID NO;

27),

1840 H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 (SEQ ID NO;

28),

1841 H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

29), and

1843 H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH2 (SEQ ID NO;

31);

or a pharmaceutically acceptable salt or derivative thereof.

18. The GLP-2 analogue of claim 1 which possesses a substitution of the amino acid sequence of wild-type GLP-2 (1-33) at one or more of positions selected from the group consisting of X3, X33, X10, X11, X16, and X24.

20. A pharmaceutical composition comprising a GLP-2 analogue of claim 1, or a salt or derivative thereof, in admixture with a carrier.

21. The pharmaceutical composition of claim 20, wherein the GLP-2 analogue is a pharmaceutically acceptable acid addition salt.

22. The pharmaceutical composition of claim 20 or claim 21, which is formulated as a liquid suitable for administration by injection or infusion, or which is formulated to cause slow release of said GLP-2 analogue.

23. A method for treating or preventing a stomach or bowel-related disorder in a patient said method comprising administering a GLP-2 analogue of claim 1 to said patient in need thereof, in an amount sufficient to treat or prevent said stomach or bowel-related disorder.

24. The method of claim 23, wherein the stomach or bowel-related disorder is an ulcer, a digestion disorder, a malabsorption syndrome, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn'"'"'s disease), ulcerative colitis, small intestine damage, or short bowel syndrome.

25. The method of claim 23, wherein the stomach or bowel-related disorder is radiation enteritis, infectious or post-infectious enteritis, or small intestinal damage due to toxic or other chemotherapeutic agents.

26. A method for treating or preventing a side effect of chemotherapy or radiation treatment in a patient, said method comprising administering a GLP-2 analogue of claim 1 to said patient in an amount sufficient to treat or prevent said side effect of chemotherapy or radiation treatment.

27. The method of claim 26, wherein the side effect of chemotherapy is diarrhea, abdominal cramping, vomiting, or structural and functional damage of the intestinal epithelium resulting from chemotherapy treatment.

28. A method for treating a neo-natal disorder, obesity, osteoporosis, or a DPP-IV (dipeptidylpetidase-IV) mediated condition in a patient, said method comprising administering a GLP-2 analogue of claim 1 to said patient in an amount sufficient to treat said neo-natal disorder, said obesity, said osteoporosis, or said DPP-IV mediated condition.

29. A method for treating or preventing a condition involving malnutrition in a patient, said method comprising administering a GLP-2 analogue of claim 1 to said patient, in an amount sufficient to treat or prevent said condition involving malnutrition.

30. The method of claim 29, wherein the condition involving malnutrition is cachexia or anorexia.

31. A nucleic acid molecule comprising a nucleic acid sequence encoding a GLP-2 analogue of claim 1.

32. An expression vector comprising the nucleic acid sequence of claim 31, in combination with control sequences to direct the expression of said nucleic acid sequence.

33. A host cell transformed with the expression vector of claim 32.

34. A method of producing the GLP-2 analogue of claim 1 comprising culturing host cells transformed with an expression vector comprising a nucleic acid sequence encoding the GLP-2 analogue under conditions suitable for expressing the GLP-2 analogue and purifying the GLP-2 analogue thus produced.

36. A method for treating or preventing a stomach or bowel-related disorder, treating or preventing a side effect of chemotherapy or radiation treatment, or for treating a neo-natal disorder, obesity, osteoporosis, or a DPP-IV (dipeptidylpeptidase-IV) mediated condition in a patient, said method comprising administering to said patient a nucleic acid molecule of claim 31, an expression vector of claim 32, or a host cell of claim 33, in an amount sufficient for the treatment or prevention of said stomach or bowel-related disorder, for the treatment or prevention of said side effect of chemotherapy or radiation treatment, or for the treatment of said neo-natal disorder, obesity, osteoporosis or a DPP-IV mediated condition.

38. The method of claim 36, wherein the stomach or bowel-related disorder is an ulcer, a digestion disorder, a malabsorption syndrome, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn'"'"'s disease), ulcerative colitis, small intestine damage, or short bowel syndrome.

39. The method of claim 36, wherein the stomach and or bowel-related disorder is radiation enteritis, infectious or post-infectious enteritis, or small intestinal damage due to toxic or other chemotherapeutic agents.

41. The method of claim 36, wherein the side effect of chemotherapy is diarrhea, abdominal cramping, vomiting, or structural or functional damage of the intestinal epithelium resulting from chemotherapy treatment.

43. A therapeutic kit comprising:

(a) a cancer chemotherapy drug; and

(b) a GLP-2 analogue claim 1, a nucleic acid molecule of claim 31, an expression vector of claim 32, or a host cell of claim 33, each optionally in combination with a pharmaceutically acceptable carrier.

44. A pharmaceutical composition comprising (a) a cancer chemotherapy drug and (b) a GLP-2 analogue of claim 1 a nucleic acid molecule of claim 31, an expression vector of claim 32, or a host cell of claim 33, in combination with a pharmaceutically acceptable carrier.

45. A GLP-2 analogue of claim 1, wherein said analogue has enhanced stability relative to Gly2-GLP-2.

46. A GLP-2 analogue of claim 1 having an observed purity of at least 70% relative to the initial purity following a treatment in a solution selected from the group consisting of 0.1 M HCl for 12 days, 0.5% H₂O₂ for 3 days, and 0.1 M NH₄HCO₃ for 6 days.

47. A GLP-2 analogue of claim 1 having an observed purity of at least 60% relative to initial purity following treatment in a solution of HCl 0.1 M for 12 days.

48. A GLP-2 analogue of claim 1 having an observed purity of at least 70% relative to initial purity following treatment in a solution of NH₄HCO₃ 0.1 M for 6 days.

19. (canceled)

35. (canceled)

37. (canceled)

40. (canceled)

42. (canceled)