GLUCAGON DERIVATIVES WITH IMPROVED STABILITY

Abstract

The present invention relates to a novel glucagon derivative peptide, and a composition for preventing or treating hypoglycemia containing the novel glucagon derivative peptide as an active ingredient. The glucagon derivative according to the present invention has improved physical properties due to the change in isoelectric point (pI) while being capable of maintaining an activity on glucagon receptors, and thus can improve patient compliance when used as a hypoglycemic agent, and is also suitable for administration in combination with other anti-obesity agents. Accordingly, the glucagon derivative according to the present invention can be effectively used for the prevention and treatment of hypoglycemia and obesity.

Claims

1-15. (canceled)

16. A glucagon derivative, which comprises the amino acid sequence of the following General Formula 1 and has a different isoelectric point (pI) from that of native glucagon: TABLE-US-00007 (General Formula 1) X1-X2-QGTF-X7-SDYS-X12-X13-X14-X15-X16-X17-X18- X19-X20-X21-F-X23-X24-W-L-X27-X28-T wherein, in General Formula 1, X1 is histidine, desamino-histidine, N-dimethyl-histidine, β-hydroxyimidazopropionic acid, 4-imidazoacetic acid, β-carboxyimidazopropionic acid, tryptophan, tyrosine, or deleted; X2 is α-methyl-glutamic acid, aminoisobutyric acid (Aib), D-alanine, glycine, Sar(N-methylglycine), serine, or D-serine; X7 is threonine or valine; X12 is lysine or cysteine; X13 is tyrosine or cysteine; X14 is leucine or cysteine; X15 is aspartic acid, glutamic acid, or cysteine; X16 is glutamic acid, aspartic acid, serine, α-methyl-glutamic acid, or cysteine; X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, valine, or cysteine; X18 is aspartic acid, glutamine, glutamic acid, arginine, or cysteine; X19 is alanine or cysteine; X20 is lysine, glutamic acid, glutamine, aspartic acid, lysine, or cysteine; X21 is aspartic acid, glutamic acid, valine, or cysteine; X23 is valine or arginine; X24 is valine, leucine, glutamine, or arginine; X27 is isoleucine or methionine; and X28 is arginine or asparagine, with the proviso that a glucagon derivative in which the amino acid sequence of General Formula 1 is identical to SEQ ID NO: 1 is excluded.

17. The glucagon derivative of claim 16, wherein, in the amino acid sequence of General Formula 1, X1 is histidine or tryptophan, tyrosine, or deleted; X2 is serine or aminoisobutyric acid (Aib); X7 is threonine or valine; X12 is lysine or cysteine; X13 is tyrosine or cysteine; X14 is leucine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid, aspartic acid, serine, or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, valine, or cysteine; X18 is aspartic acid, glutamic acid, arginine, or cysteine; X19 is alanine or cysteine; X20 is lysine, glutamic acid, glutamine, aspartic acid, lysine, or cysteine; X21 is aspartic acid, glutamic acid, valine, or cysteine; X23 is valine or arginine; X24 is valine, leucine, or glutamine; X27 is isoleucine or methionine; and X28 is arginine or asparagine, with the proviso that a glucagon derivative in which the amino acid sequence of General Formula 1 is identical to SEQ ID NO: 1, it is excluded.

18. The glucagon derivative of claim 16, wherein the glucagon derivative has glucagon receptor-stimulating activity.

19. The glucagon derivative of claim 16, wherein the glucagon derivative comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOS: 2 to 34.

20. A polynucleotide encoding the glucagon derivative according to claim 16.

21. A polynucleotide encoding the glucagon derivative according to claim 17.

22. A polynucleotide encoding the glucagon derivative according to claim 19.

23. A pharmaceutical composition for preventing or treating hypoglycemia comprising the glucagon derivative according to claim 16 as an active ingredient.

24. A pharmaceutical composition for preventing or treating hypoglycemia comprising the glucagon derivative according to claim 17 as an active ingredient.

25. A pharmaceutical composition for preventing or treating hypoglycemia comprising the glucagon derivative according to claim 18 as an active ingredient.

26. A pharmaceutical composition for preventing or treating hypoglycemia comprising the glucagon derivative according to claim 19 as an active ingredient.

27. A pharmaceutical composition for preventing or treating obesity comprising the glucagon derivative according to claim 16 as an active ingredient.

28. A pharmaceutical composition for preventing or treating obesity comprising the glucagon derivative according to claim 17 as an active ingredient.

29. A pharmaceutical composition for preventing or treating obesity comprising the glucagon derivative according to claim 18 as an active ingredient.

30. A pharmaceutical composition for preventing or treating obesity comprising the glucagon derivative according to claim 19 as an active ingredient.

31. The pharmaceutical composition according to claim 23, further comprising a pharmaceutically acceptable carrier.

32. The pharmaceutical composition according to claim 27, further comprising a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to claim 27, wherein the pharmaceutical composition is administered alone or in combination with a pharmaceutical agent having the effect of preventing or treating obesity.

34. The pharmaceutical composition according to claim 27, further comprising a pharmaceutical agent having the effect of preventing or treating obesity.

35. The pharmaceutical composition according to claim 33, wherein the pharmaceutical agent having the effect of preventing or treating obesity is selected from the group consisting of a GLP-1 receptor agonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3/4 receptor agonist, an MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a β33A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, FGF1, an FGF21 receptor agonist, a cholecystokinin (CCK) receptor agonist, a pancreatic polypeptide (PP) receptor agonist, a dopamine reabsorption inhibitor, and a combination thereof.

36. The pharmaceutical composition according to claim 34, wherein the pharmaceutical agent having the effect of preventing or treating obesity is selected from the group consisting of a GLP-1 receptor agonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3/4 receptor agonist, an MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, FGF1, an FGF21 receptor agonist, a cholecystokinin (CCK) receptor agonist, a pancreatic polypeptide (PP) receptor agonist, a dopamine reabsorption inhibitor, and a combination thereof.

37. The pharmaceutical composition according to claim 28, further comprising a pharmaceutical agent having the effect of preventing or treating obesity.

38. The pharmaceutical composition according to claim 37, wherein the pharmaceutical agent having the effect of preventing or treating obesity is selected from the group consisting of a GLP-1 receptor agonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3/4 receptor agonist, an MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, FGF1, an FGF21 receptor agonist, a cholecystokinin (CCK) receptor agonist, a pancreatic polypeptide (PP) receptor agonist, a dopamine reabsorption inhibitor, and a combination thereof.

39. The pharmaceutical composition according to claim 29, further comprising a pharmaceutical agent having the effect of preventing or treating obesity.

40. The pharmaceutical composition according to claim 39, wherein the pharmaceutical agent having the effect of preventing or treating obesity is selected from the group consisting of a GLP-1 receptor agonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3/4 receptor agonist, an MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, FGF1, an FGF21 receptor agonist, a cholecystokinin (CCK) receptor agonist, a pancreatic polypeptide (PP) receptor agonist, a dopamine reabsorption inhibitor, and a combination thereof.

41. The pharmaceutical composition according to claim 30, further comprising a pharmaceutical agent having the effect of preventing or treating obesity.

42. The pharmaceutical composition according to claim 41, wherein the pharmaceutical agent having the effect of preventing or treating obesity is selected from the group consisting of a GLP-1 receptor agonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3/4 receptor agonist, an MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, FGF1, an FGF21 receptor agonist, a cholecystokinin (CCK) receptor agonist, a pancreatic polypeptide (PP) receptor agonist, a dopamine reabsorption inhibitor, and a combination thereof.

43. A method of preventing or treating hypoglycemia or obesity, comprising administering the glucagon derivative according to claim 16, or a pharmaceutical composition containing the same, to a subject in need thereof.

44. A method of preventing or treating hypoglycemia or obesity, comprising administering the glucagon derivative according to claim 17, or a pharmaceutical composition containing the same, to a subject in need thereof.

45. A method of preventing or treating hypoglycemia or obesity, comprising administering the glucagon derivative according to claim 18, or a pharmaceutical composition containing the same, to a subject in need thereof.

46. A method of preventing or treating hypoglycemia or obesity, comprising administering the glucagon derivative according to claim 19, or a pharmaceutical composition containing the same, to a subject in need thereof.

47. A composition, comprising the glucagon derivative according to claim 16 as an active ingredient.

48. A composition, comprising the glucagon derivative according to claim 17 as an active ingredient.

49. A composition, comprising the glucagon derivative according to claim 19 as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0114] Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only and are not intended to limit the scope Of the present invention.

Example 1

Production of a Cell Line Having a cAMP Response to Glucagon

[0115] PCR was performed using a region corresponding to an open reading frame (ORF) in cDNA (OriGene Technologies, Inc., USA) of human glucagon receptor gene as a template, and the following forward and reverse primers of SEQ ID NOS: 35 and 36, including each of the HindIII and EcoRI restriction sites.

[0116] In particular, PCR was performed for a total of 30 cycles using the following conditions: 95° C. denaturation for 60 seconds, annealing at 55° C. for 60 seconds, and extension at 68° C. for 30 seconds. The thus-obtained PCR product was electrophoresed in a 1.0% agarose gel, and a band with a size of 450 bp was obtained therefrom by elution.

TABLE-US-00004 Forward primer: (SEQ ID NO: 35) 5′-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3′ Reverse primer: (SEQ ID NO: 36) 5′-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3′

[0117] The PCR product was cloned into a known animal cell expression vector, x0GC/dhfr, to prepare a recombinant vector x0GC/GCGR. CHO DG44 cell line cultured in DMEM/F12 (10% FBS) medium was transfected with the recombinant vector x0GC/GCGR using Lipofectamine, and selectively cultured in a selection medium containing 1 mg/mL. G418 and 10 nM Methotraxate. Single clone cell lines were selected therefrom by a limit dilution method, and among them, a cell line showing excellent cAMP response to glucagon in a concentration-dependent manner was finally selected therefrom.

Example 2

Synthesis of Glucagon Derivatives

[0118] It is widely known in the art that the solubility, activity, and stability of a protein in a solution can vary according to pI (Shaw, K. L. et al., Protein Science 10, pp 1206-1215, 2001). In order to prepare glucagon derivatives with improved physical properties, the amino acid sequence of native glucagon represented by SEQ ID NO: 1 was substituted with amino acid residues having positive and negative charges, and thereby glucagon derivatives were synthesized as shown in Table 1 below.

TABLE-US-00005 TABLE 11 SEQ ID NO Amino Acid Sequence Ring Formation SEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT — SEQ ID NO: 2 HSQGTFTSDYSKYLDCDRAQDFVQWLMNT — SEQ ID NO: 3 HSQGTFTSDYSKYLDCERAQDFVQWLMNT — SEQ ID NO: 4 HSQGTFTSDYSKYLDSCDAQDFVQWLMNT — SEQ ID NO: 5 HSQGTFTSDYSKYLDSCEAQDFVQWLMNT — SEQ ID NO: 6 HSQGTFTSDYSKYLDSCEADDFVQWLMNT — SEQ ID NO: 7 YSQGTFTSDYSKYLDSCEADDFVQWLMNT — SEQ ID NO: 8 YXQGTFTSDYSKYLDSCDAQDFVQWLINT — SEQ ID NO: 9 YXQGTFTSDYSKYLDSCDAQDFVVWLINT — SEQ ID NO: 10 YXQGTFTSDYSKYLDSCDADDFVVWLINT — SEQ ID NO: 11 YXQGTFTSDYSKYLDEKCAKEFVQWLMNT — SEQ ID NO: 12 YXQGTFTSDYSKYLDSRRAQDFVQWLMNT — SEQ ID NO: 13 YXQGTFTSDYSCYLDEKRAKEFVQWLMNT — SEQ ID NO: 14 YXQGTFTSDYSKYLDCKRAKEFVQWLMNT — SEQ ID NO: 15 YXQGTFTSDYSKYLCEKRAQDFVQWLMNT — SEQ ID NO: 16 YXQGTFTSDYSKYLDCRRAQVFVQWLMRT — SEQ ID NO: 17 YXQGTFTSDYSKYLDCVRAQDFVQWLMRT — SEQ ID NO: 18 YXQGTFTSDYSKYLDSRRACDFRLWLMNT — SEQ ID NO: 19 YXQGTFTSDYSKYLCEKRAKEFVQWLMNT ring formed SEQ ID NO: 20 YXQGTFTSDYSKYLDECRAKEFVQWLMNT ring formed SEQ ID NO: 21 YXQGTFTSDYSKYLDEKCAKEFVQWLMNT ring formed SEQ ID NO: 22 YXQGTFTSDYSKYLDEKRCKEFVQWLMNT ring formed SEQ ID NO: 23 YXQGTFTSDYSKYCDEKRAKEFVQWLMNT ring formed SEQ ID NO: 24 YXQGTFTSDYSKCLDEKRAKEFVQWLMNT ring formed SEQ ID NO: 25 YXQGTFTSDYSKYLDEKRAKCFVQWLMNT ring formed SEQ ID NO: 26 WXQGTFTSDYSKYLDECRAKDRVQWLMNT ring formed SEQ ID NO: 27 YXQGTFVSDYSKYLDECRAKDRVQWLMNT ring formed SEQ ID NO: 28 WXQGTFVSDYSKYLDECRAKDFVQWLMNT ring formed SEQ ID NO: 29 YXQFTFTSDYSKCLDERRAKDFVQWLMNT ring formed SEQ ID NO: 30 WXQGTFTSDYSKCLDERRAKDFVQWLMNT ring formed SEQ ID NO: 31 YXQGTFTSDYSKYLDCKRAKEFVQWLMNT ring formed SEQ ID NO: 32 -SQGTFTSDYSKYLDECRAKEFVQWLMNT ring formed SEQ ID NO: 33 WXQGTFTSDYSKYCDERRAKEFVQWLMNT ring formed SEQ ID NO: 34 YXQGTFTSDYSKYCDERRAKEFVQWLMNT ring formed

[0119] Regarding the SEQ ID NOS: 8 to 31 and 33 to 34 shown in Table 1, the amino acid represented by X represents a non-native amino acid. aminoisobutyric acid (Aib); “-” in the amino acid sequence of SEQ ID NO: 32 means that no amino acid residue is present on the corresponding position; and the two bold and underlined amino acid residues represent formation of a ring between the two amino acid residues.

Example 3

Measurement of pI of Glucagon Derivatives

[0120] In order to measure the improved physical properties of glucagon derivatives synthesized in Example 2, pI values were calculated based on the amino acid sequences using the pI/Mw tool (http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003) in the ExPASy server.

TABLE-US-00006 TABLE 2 Glucagon Derivatives pI SEQ ID NO: 1 6.8 SEQ ID NO: 2 4.56 SEQ ID NO: 3 4.66 SEQ ID NO: 4 4.13 SEQ ID NO: 5 4.22 SEQ ID NO: 6 4.03 SEQ ID NO: 7 3.71 SEQ ID NO: 8 3.77 SEQ ID NO: 9 3.77 SEQ ID NO: 10 3.66 SEQ ID NO: 11 4.78 SEQ ID NO: 12 6.04 SEQ ID NO: 13 4.78 SEQ ID NO: 14 8.12 SEQ ID NO: 15 6.11 SEQ ID NO: 16 9.11 SEQ ID NO: 17 6.03 SEQ ID NO: 18 8.15 SEQ ID NO: 19 8.12 SEQ ID NO: 20 4.78 SEQ ID NO: 21 4.78 SEQ ID NO: 22 6.20 SEQ ID NO: 23 6.20 SEQ ID NO: 24 6.21 SEQ ID NO: 25 8.12 SEQ ID NO: 26 4.68 SEQ ID NO: 27 4.68 SEQ ID NO: 28 4.68 SEQ ID NO: 29 6.15 SEQ ID NO: 30 4.44 SEQ ID NO: 31 8.12 SEQ ID NO: 32 4.78 SEQ ID NO: 33 6.21 SEQ ID NO: 34 6.21

[0121] As shown in Table 2 above, while the native glucagon of SEQ ID NO: 1 had a pI of 6.8, the glucagon derivatives according to the present invention showed pI values in the range of from about 4 to about 9, thus showing improved physical properties. Since the glucagon derivatives according to the present invention have pI values different from that of native glucagon, they can exhibit improved solubility and higher stability according to the pH conditions of a given solution.

[0122] Accordingly, when the glucagon derivatives according to the present invention are used as a therapeutic agent for treating hypoglycemia, they can improve patient compliance, and are also suitable for administration in combination with other anti-obesity agents, e.g., a GLP-1 receptor antagonist, a glucose-dependent insulinotropic peptide (GIP) receptor antagonist, etc., and thus can be effectively used as a therapeutic agent for treating hypoglycemia and obesity.

[0123] Those of ordinary skill in the art will recognize that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within the scope of the present invention.