Compositions containing glucagon derivatives and long-acting conjugates thereof

Abstract

To provide a pharmaceutical composition for treating or preventing metabolic syndrome containing a glucagon derivative and at least one compound or material having therapeutic activity for metabolic syndrome.SOLUTION: A pharmaceutical composition for treating or preventing metabolic syndrome contains: i) a peptide including the amino acid sequence of the general formula (1), and ii) at least one compound or material having therapeutic activity for metabolic syndrome: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-X29-X30 (General Formula (1), SEQ ID NO: 45).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a glucagon derivative, a long-acting conjugate of a glucagon derivative, and uses thereof. [Background technology]

[0002] In response to recent economic development and changes in eating habits, the incidence of metabolic syndrome-related diseases, including obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, liver disease, etc., is rapidly increasing. Although these diseases may occur separately, they are generally closely related to each other and often occur with multiple symptoms. In particular, according to the World Health Organisation (WHO), over one billion adults worldwide are overweight, of which at least three million are clinically obese, and over 250,000 people die annually in Europe and over 2.5 million people die annually in an overweight-related manner worldwide. Overweight and obesity increase blood pressure and cholesterol levels, and are the cause of the onset or aggravation of various diseases such as heart disease, diabetes, arthritis, etc. Also, overweight and obesity are the main factors increasing the incidence of arteriosclerosis, high blood pressure, hyperlipidemia, heart disease, etc. not only in adults but also in children and adolescents. Thus, obesity is a serious disease that causes various diseases as a global disease, but it is believed that it can be overcome by the self-help efforts of individuals, and it is evaluated that obese patients have weak self-control. However, obesity is surprisingly difficult to treat, because it is a complex disease related to the mechanism of action of appetite regulation and energy metabolism. Therefore, in order to treat obesity, not only the patient's own efforts but also a method of treating the abnormal mechanism of action related to appetite regulation and energy metabolism must be carried out at the same time, and therefore efforts are being made to develop medicines that can treat the abnormal mechanism of action.

[0003] As a result of the aforementioned efforts, anti-obesity drugs such as Rimonabant (Sanofi-Aventis), Sibutramine (Abbott), Contrave (Takeda), and Orlistat (Roche) have been developed, but these drugs have drawbacks such as fatal side effects and insufficient efficacy in treating obesity. For example, Rimonabant has side effects such as central nervous system disorders, Sibutramine and Contrave have cardiovascular side effects, and Orlistat has only been reported to reduce weight by about 4 kg when taken for one year. Therefore, there are currently very few anti-obesity drugs that can be safely prescribed to obese patients. Thus, active research is being conducted to develop new drugs that can solve the problems of conventional obesity treatments, and glucagon derivatives have recently been attracting attention. Glucagon is produced in the pancreas when blood glucose levels begin to drop due to drug treatment, disease, hormone or enzyme deficiency, etc. Glucagon acts to increase blood glucose levels to normal levels by signaling the liver to break down glycogen and release glucose. In addition, glucagon has been reported to have anti-obesity effects by suppressing appetite and activating hormone-sensitive lipase in adipocytes to promote lipolysis in addition to increasing blood glucose. However, glucagon's use as a therapeutic agent has been limited due to its low solubility and precipitation at neutral pH. Therefore, glucagon with improved physical properties can be useful for treating severe hypoglycemia, and for treating non-alcoholic fatty liver disease (NASH) or dyslipidemia by itself, by increasing fat breakdown and beta-oxidation in the liver. Glucagon-like peptide-1 (GLP-1), one of the glucagon derivatives, is being developed as a therapeutic agent to reduce hyperglycemia in diabetic patients. GLP-1 has the functions of promoting insulin synthesis and secretion, inhibiting glucagon secretion, suppressing gastric emptying, promoting glucose utilization, and inhibiting food and drink intake.

[0004] Exendin-4, which is produced from lizard venom and has approximately 50% amino acid homology with GLP-1, is also known to activate the GLP-1 receptor and reduce hyperglycemia in diabetic patients (Non-Patent Document 1). However, it has been reported that obesity treatment drugs containing GLP-1 or exendin-4 have the problem of causing side effects such as vomiting and nausea. Therefore, oxyntomodulin, which can bind to both GLP-1 and glucagon peptide receptors, has been attracting attention as an alternative to GLP-1. Oxyntomodulin is a peptide made from preglucagon, a precursor of glucagon, and shows the effects of GLP-1 in inhibiting food intake and promoting satiety, as well as the lipolysis function of glucagon, raising its potential as an anti-obesity therapeutic agent. However, oxyntomodulin or its derivatives have a serious drawback in that they have a short half-life in vivo, while their low efficacy requires daily administration of excessive amounts of the drug. In addition, when one peptide has both GLP-1 and glucagon activity, the activity ratio is fixed, making it difficult to use dual agonists with various ratios. Therefore, combination therapy that can be used at various activity ratios by adjusting the content of GLP-1 and glucagon may be more effective. However, this requires improving the physical properties of glucagon, which has low solubility and is aggregated at neutral pH and precipitates over time. Therefore, the present inventors have developed a derivative in which the amino acid sequence of glucagon has been partially modified in order to improve the physical properties of glucagon and enhance its therapeutic effect against hypoglycemia and obesity. The inventors have confirmed that the glucagon derivative exhibits improved solubility and high stability at neutral pH due to its pI which is different from that of natural glucagon. The inventors have confirmed through in vitro activity measurement that the developed glucagon derivative activates the glucagon receptor, thereby completing the present invention. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Publication No. 2010-0105877 [Patent Document 2] International Patent Publication No. WO97 / 34631 [Patent Document 3] International Patent Publication No. 96 / 32478 [Non-patent literature]

[0006] [Non-Patent Document 1] J Biol Chem. 1992 Apr 15;267(11):7402-5. [Non-Patent Document 2] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present invention is to provide a pharmaceutical composition for treating or preventing metabolic syndrome, which comprises a glucagon derivative and one or more compounds or substances having therapeutic activity for metabolic syndrome. It is another object of the present invention to provide novel glucagon derivatives. It is another object of the present invention to provide isolated polynucleotides encoding said glucagon derivatives, vectors containing said polynucleotides, and isolated cells containing said polynucleotides or vectors. It is another object of the present invention to provide isolated conjugates of glucagon derivatives linked to biocompatible agents which increase their in vivo half-life. It is another object of the present invention to provide compositions comprising said glucagon derivatives or isolated conjugates. Another object of the present invention is to provide a pharmaceutical composition for treating or preventing hypoglycemia or metabolic syndrome comprising said glucagon derivative or isolated conjugate. Another object of the present invention is to provide a method for preventing or treating hypoglycemia or metabolic syndrome, comprising administering said composition to an individual in need thereof. Another object of the present invention is to provide a use of said glucagon derivative or isolated conjugate, or said composition, in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome. [Means for solving the problem]

[0008] One embodiment of the present invention is a pharmaceutical composition for treating or preventing metabolic syndrome, comprising a glucagon derivative and one or more compounds or substances having therapeutic activity for metabolic syndrome. More specifically, one embodiment of the present invention is a pharmaceutical composition for treating or preventing metabolic syndrome, comprising: i) a peptide comprising an amino acid sequence represented by the following general formula (1); and ii) one or more compounds or substances having therapeutic activity for metabolic syndrome: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (General formula (1), Sequence number 45) In the general formula (1), X1 is histidine, desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan, or tyrosine, or is absent; X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine; X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, alpha-methyl-glutamic acid, cysteine, or is absent; X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or is absent; X18 is alanine, aspartic acid, glutamic acid, arginine, valine, cysteine, or absent; X19 is alanine, arginine, serine, valine, cysteine, or absent; X20 is lysine, histidine, glutamine, aspartic acid, lysine, arginine, alpha-methyl-glutamic acid, cysteine, or is absent; X21 is aspartic acid, glutamic acid, leucine, valine, cysteine, or absent; X23 is isoleucine, valine, arginine, or is absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, leucine, or is absent; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or is absent; X28 is glutamine, lysine, asparagine, arginine, or absent; X29 is lysine, alanine, glycine, or threonine, or is absent; X30 may be cysteine ​​or absent. (However, this does not include the case where the amino acid sequence of the general formula (1) is identical to SEQ ID NO: 1).

[0009] As another specific example, In the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid, lysine or cysteine; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine; X23 is isoleucine, valine, or arginine; X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine; X27 is isoleucine, valine, alanine, methionine, glutamine, or arginine; X28 is glutamine, lysine, asparagine or arginine; X29 is threonine; X30 is characterized as being either cysteine ​​or absent.

[0010] As another specific example, in the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid or lysine; X21 is aspartic acid or glutamic acid; X23 is valine; X24 is valine or glutamine; X27 is isoleucine or methionine; X28 is asparagine or arginine; X29 is threonine; X30 is characterized as being either cysteine ​​or absent.

[0011] As another specific example, in the general formula (1), X1 is tyrosine; X2 is Aib (aminoisobutyric acid); X7 is threonine; X10 is tyrosine; X12 is lysine; X13 is tyrosine; X14 is leucine; X15 is aspartic acid or cysteine; X16 is glutamic acid, serine or cysteine; X17 is lysine or arginine; X18 is arginine; X19 is alanine; X20 is glutamine, cysteine ​​or lysine; X21 is aspartic acid, cysteine, valine, or glutamic acid; X23 is valine; X24 is valine or arginine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; X30 is characterized as being absent.

[0012] As another specific example, the peptide is characterized by being a peptide comprising an amino acid sequence of the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 is cysteine ​​or absent. Among the peptides containing the amino acid sequence of general formula (2), peptides corresponding to SEQ ID NOs: 14, 19, 20, 25, 27, 31, and 33 may be excluded.

[0013] As another specific example, the peptide containing the amino acid sequence of the general formula (1) is characterized by having a pI different from the pI of native glucagon, for example, a pI of 6.5 or less, or a pI of 7.0 or more. In another specific example, in the peptide comprising the amino acid sequence of the general formula (1), one or more of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in the general formula (1) are substituted with glutamic acid or lysine, each of which can form a ring. In another specific example, in the peptide comprising the amino acid sequence of the general formula (1), the amino acid pair of X12 and X16 or the amino acid pair of X16 and X20 is substituted with glutamic acid or lysine, respectively, capable of forming a ring. In other specific examples, any one or more of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 form a ring (e.g., a lactam ring).

[0014] As another specific example, the peptide comprising the amino acid sequence of the above general formula (1) is characterized in that the C-terminus thereof is amidated. As another specific example, the peptide containing the amino acid sequence of the general formula (1) is characterized as being a glucagon derivative capable of activating the glucagon receptor. As another specific example, the peptide is characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-44. As another specific example, the peptide is characterized by being selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44. In another embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 12 or 20. In other specific examples, the compound or substance having a therapeutic activity for metabolic syndrome is an insulinotropic peptide, a glucagon like peptide-1 (GLP-1) receptor agonist, a leptin receptor agonist, a dipeptidyl peptidase-IV (DPP-IV) inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2 / 4 receptor agonist, a melanocortin 3 / 4 (MC3 / 4) receptor agonist, a gastric / pancreatic lipase inhibitor, a 5-hydroxytryptamine receptor 2C (5HT2c, G protein-coupled) agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, a peroxisome proliferator-activated receptor α (PPARα), or a combination thereof. The agonist is characterized by being selected from the group consisting of an alpha agonist, a PPAR delta (peroxisome proliferator-activated receptor delta) agonist, a farnesoid X receptor (FXR) agonist, an acetyl-CoA carboxylase inhibitor, peptide YY, CCK (cholecystokinin), xenin, glicentin, obestatin, secretin, nesfatin, insulin, and GIP (glucose-dependent insulinotropic peptide).

[0015] In another specific example, the insulinotropic peptide is selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof. In another embodiment, the insulinotropic peptide is an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is substituted with one selected from the group consisting of des-amino-histidyl, N-dimethyl-histidyl, beta-hydroxyimidazopropionyl, 4-imidazoacetyl and beta-carboxyimidazopropionyl. In another specific example, the insulinotropic peptide is characterized by being selected from the group consisting of native exendin-4, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been removed, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been replaced with a hydroxyl group, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been modified with a dimethyl group, exendin-4 derivatives in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been removed (deleted), exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with serine, and exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with arginine.

[0016] As another specific example, the peptide comprising the amino acid sequence of general formula (1) is in the form of a long-acting conjugate linked to a biocompatible substance that increases its half-life in the body, and the insulinotropic peptide is in the form of a long-acting conjugate linked to a biocompatible substance that increases its half-life in the body. In another specific example, the biocompatible substance is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers. In another specific example, the peptide comprising the amino acid sequence of general formula (1) and the insulinotropic peptide are each linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. In another specific example, the biocompatible substance is an FcRn binding substance, and the peptide comprising the amino acid sequence of general formula (1) and the insulinotropic peptide are each linked to the biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. In another specific example, the FcRn binding substance is a polypeptide comprising an immunoglobulin Fc region. In another embodiment, the immunoglobulin Fc region is characterized as being aglycosylated.

[0017] In another embodiment, the immunoglobulin Fc region comprises (a) the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain; (b) CH1 and CH2 domains; (c) CH1 and CH3 domains; (d) CH2 and CH3 domains; (e) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region; and (f) is characterized in that it is selected from the group consisting of a dimer of each domain of the heavy chain constant region and a light chain constant region. In another embodiment, the polypeptide comprising an immunoglobulin Fc region is in a dimeric form. In another specific example, the immunoglobulin Fc region is characterized in that it is a derivative of native Fc in which a site capable of forming a disulfide bond has been removed, some amino acids at the N-terminus have been removed from native Fc, a methionine residue has been added to the N-terminus of native Fc, a complement binding site has been deleted, or an ADCC (antibody dependent cell mediated cytotoxicity) site has been removed. In another specific example, the immunoglobulin Fc region is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In another specific example, the immunoglobulin Fc region is an IgG4 Fc region. In another specific example, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4. In another specific example, the non-peptide linker is characterized by being linked to a cysteine ​​residue of a peptide containing the amino acid sequence of general formula (1). In another specific example, both ends of the non-peptidic linker are bonded to a biocompatible substance and an amine group or a thiol group of a peptide having the amino acid sequence of general formula (1) or an insulinotropic peptide, respectively. In another embodiment, the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

[0018] Another aspect embodying the present invention is novel glucagon derivatives. In one embodiment, the derivative is an isolated peptide comprising an amino acid sequence of the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 is cysteine ​​or absent. Among the peptides containing the amino acid sequence of general formula (2), peptides corresponding to SEQ ID NOs: 14, 19, 20, 25, 27, 31 and 33 may be excluded.

[0019] In another specific example, the peptide comprising the amino acid sequence of general formula (2) is characterized in that the amino acid pair of X16 and X20 in general formula (2) is replaced with glutamic acid or lysine, each of which can form a ring. As another specific example, the peptide comprising the amino acid sequence of the above general formula (2) is characterized in that the C-terminus thereof is amidated. In another embodiment, the peptide is a glucagon derivative capable of activating the glucagon receptor. As another specific example, the peptide is characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36-44. In another embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:12.

[0020] Further embodiments of the invention are isolated polynucleotides encoding the glucagon derivatives, vectors containing the polynucleotides, and isolated cells containing the polynucleotides or vectors. Another embodiment of the invention is an isolated conjugate of a glucagon derivative linked to a biocompatible agent that increases its half-life in vivo. As one specific example, the biocompatible substance is characterized by being selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers. In another embodiment, the isolated peptide is linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0021] As another specific example, the biocompatible substance is an FcRn-binding substance; The isolated peptide is characterized in that it is linked to a biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. In another specific example, the FcRn binding substance is a polypeptide comprising an immunoglobulin Fc region. Another embodiment of the invention is a composition comprising the glucagon derivative or isolated conjugate. In one embodiment, the composition is a pharmaceutical composition for treating or preventing hypoglycemia or metabolic syndrome. Yet another embodiment of the invention is a method of preventing or treating hypoglycemia or metabolic syndrome comprising administering the composition. Yet another embodiment of the invention is the use of the glucagon derivative or isolated conjugate or the composition in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome. Effect of the Invention

[0022] The glucagon derivative according to the present invention exhibits improved physical properties compared to natural glucagon, and can be usefully used as a therapeutic drug for hypoglycemia by increasing the patient's compliance during use. It can also be usefully used in the prevention or treatment of hypoglycemia and metabolic syndrome including obesity, diabetes, and nonalcoholic steatohepatitis (NASH). [Brief description of the drawings]

[0023] [Figure 1] A long-acting insulinotropic peptide conjugate (named as a long-acting Exendin-4 derivative) and a long-acting glucagon derivative conjugate (named as a long-acting derivative with sequence number 12) were administered alone or in combination to an obese animal model (rats) induced by a high-fat diet; the results of the changes in the rats' body weight every 3 days during 15 days of administration with the dosage adjusted are shown in this figure. [Diagram 2] A long-acting insulinotropic peptide conjugate (named as a long-acting exendin-4 derivative) and a long-acting glucagon derivative conjugate (named as a long-acting derivative with sequence number 12) were administered alone or in combination to a high-fat diet-induced obese animal model (rats); the dosages were adjusted and the mesenteric fat mass was measured after 15 days of administration (*p<0.05, **p<0.01 vs. vehicle by ANOVA test). [Diagram 3]A long-acting insulinotropic peptide conjugate (named as a long-acting Exendin-4 derivative) and a long-acting glucagon derivative conjugate (named as a long-acting derivative with sequence number 12) were administered alone or in combination to a high-fat diet-induced obese animal model (rats); the doses were adjusted and the difference in liver weight was measured after 15 days of administration (***p <0.01, ***p <0.001 vs. vehicle by ANOVA test). [Figure 4] This figure shows the changes in rat body weight after administering a long-acting insulinotropic peptide conjugate (named as a long-acting exendin-4 derivative) and a long-acting glucagon derivative conjugate (named as a long-acting derivative of sequence number 20) alone or in combination to a high-fat diet-induced obesity animal model (mouse) for 22 days at adjusted dosages. [Diagram 5] This figure shows the results of administering a long-acting insulinotropic peptide conjugate (named as a long-acting exendin-4 derivative) and a long-acting glucagon derivative conjugate (named as a long-acting derivative of sequence number 20) alone or in combination to a high-fat diet-induced obesity animal model (mouse) for 22 days at adjusted dosages, and measuring the blood cholesterol content. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Specific details for implementing the present invention are as follows. Meanwhile, each description and embodiment disclosed in the present application can be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in the present application belong to the scope of the present invention. Also, the scope of the present invention is not limited by the specific description described below. Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. Also, the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. Alanine A Arginine R Asparagine N Aspartic acid D Cysteine ​​C Glutamic Acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

[0025] One embodiment of the present invention provides a composition comprising a glucagon derivative and one or more compounds or substances having therapeutic activity for metabolic syndrome. More specifically, the present invention provides a pharmaceutical composition for treating or preventing metabolic syndrome comprising the glucagon derivative and one or more compounds or substances having therapeutic activity for metabolic syndrome. Glucagon derivatives according to the present invention include peptides having one or more differences in their amino acid sequence compared to native glucagon, peptides in which the native glucagon sequence has been altered through modification, and mimetics of native glucagon that can activate the glucagon receptor like native glucagon. Such glucagon derivatives may have an altered pI relative to native glucagon, thereby exhibiting improved physical properties, and may have improved solubility while maintaining the activity of activating the glucagon receptor, but are not limited thereto. The glucagon derivative may also be non-naturally occurring. On the other hand, native glucagon may have the following amino acid sequence: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1)

[0026] In the present invention, the term "pi (isoelectric point)" refers to the pH value at which the overall net charge of a molecule such as a polypeptide or peptide is zero (0). For a polypeptide in which various charged functional groups are present, the sum of these charges is zero at the pI. At a pH higher than the pI, the overall net charge of the polypeptide becomes negative, and at a pH value lower than the pI, the overall net charge of the polypeptide becomes positive. The pI can be determined by isoelectric focusing on immobilized pH gradient gels composed of polyacrylamide, starch or agarose, or by estimating the pI from the amino acid sequence, e.g., using the pI / MW tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003). In the present invention, the term "altered pI" means that a part of the amino acid sequence of native glucagon is replaced with negatively and positively charged amino acid residues to have a pI different from that of native glucagon, i.e., a decreased or increased pI. A peptide having such an altered pI can exhibit improved solubility and high stability at neutral pH as a glucagon derivative. More specifically, the glucagon derivative may have a pI value different from that of native glucagon (6.8), more specifically, a pI value less than 6.8, more specifically, 6.7 or less, more specifically, 6.5 or less, or more specifically, a pI value greater than 6.8, 7 or more, more specifically, 7.5 or more, but is not limited thereto, and is within the scope of the present invention as long as it has a pI value different from that of native glucagon. In particular, when the derivative has a pI value different from that of native glucagon and thus shows improved solubility at neutral pH compared to native glucagon, and thus is less prone to aggregation, it is particularly within the scope of the present invention. More specifically, the pI value may be, but is not limited to, 4 to 6.5 and / or 7 to 9.5, more specifically 7.5 to 9.5, and even more specifically 8.0 to 9.3. In this case, since the pI is higher or lower than that of native glucagon, the glucagon can exhibit improved solubility and higher stability at neutral pH compared to native glucagon.

[0027] Specifically, a derivative of native glucagon may be obtained by modifying some amino acids of native glucagon through any one of substitution, addition, deletion and modification, or a combination of these methods. Examples of glucagon derivatives produced by combining these methods include peptides that have one or more amino acid sequences different from those of native glucagon, have deamination at the N-terminal amino acid residue, and have the function of activating the glucagon receptor, but are not limited to these. Native glucagon derivatives applicable to the present invention can be produced by combining several methods for producing derivatives. These modifications for producing derivatives of native glucagon also include modifications using L- or D-amino acids, and / or non-natural amino acids; and / or modifications of the native sequence, such as modifications of side chain functional groups, covalent bonds within the molecule, such as the formation of rings between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. It also includes all forms of glucagon in which one or more amino acids have been added to the amino and / or carboxy termini of native glucagon. The substituted or added amino acids may be any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0028] Glucagon has a pI of about 7 and is insoluble in solutions at physiological pH (pH 4-8) and tends to precipitate at neutral pH. In aqueous solutions below pH 3, glucagon is initially dissolved but precipitates due to gel formation within 1 hour. Gelled glucagon consists mainly of β-sheet fibrils and such precipitated glucagon is not suitable for use as an injection because it will clog blood vessels if the gel is administered into a needle or vein. To retard the precipitation process, acid (pH 2-4) dosage forms are usually used, which maintain glucagon in a relatively aggregate-free state for a short period of time. However, because glucagon fibril formation occurs very rapidly at low pH, such acidic formulations should be injected soon after preparation. In the present invention, the pI of native glucagon has been altered by substitution of negatively and positively charged amino acid residues to develop a glucagon derivative having an extended profile of action. The glucagon derivative of the present invention is characterized by having an altered pI compared to native glucagon, and thus exhibiting improved solubility and / or stability at neutral pH. In one specific embodiment, the glucagon derivative may be a peptide comprising an amino acid sequence of the following general formula (1): X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (General formula (1), Sequence number 45) In the general formula (1), X1 is histidine, desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropIonyl, 4-imidazoacetyl, beta-carboxy imidazopropIonyl, tryptophan, or tyrosine, or is absent; X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine; X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, alpha-methyl-glutamic acid, cysteine, or is absent; X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or is absent; X18 is alanine, aspartic acid, glutamic acid, arginine, valine, cysteine, or absent; X19 is alanine, arginine, serine, valine, cysteine, or absent; X20 is lysine, histidine, glutamine, aspartic acid, lysine, arginine, alpha-methyl-glutamic acid, cysteine, or is absent; X21 is aspartic acid, glutamic acid, leucine, valine, cysteine, or absent; X23 is isoleucine, valine, arginine, or is absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, leucine, or is absent; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or is absent; X28 is glutamine, lysine, asparagine, arginine, or absent; X29 is lysine, alanine, glycine, or threonine, or is absent; X30 may be cysteine ​​or absent. (However, this does not include the case where the amino acid sequence of the general formula (1) is identical to SEQ ID NO: 1). Here, when the amino acid sequence of general formula (1) is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, particularly any of the amino acid sequences of SEQ ID NOs: 13, 15, 36, and 38 to 43, there may be cases where it is excluded from the scope of the peptide containing the amino acid sequence of general formula (1), but this is not limiting.

[0029] More specifically, In the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid, lysine or cysteine; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine; X23 is isoleucine, valine, or arginine; X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine; X27 is isoleucine, valine, alanine, methionine, glutamine, or arginine; X28 is glutamine, lysine, asparagine or arginine; X29 is threonine; X30 may be cysteine ​​or may be absent (excluding the case where the amino acid sequence of general formula (1) is identical to SEQ ID NO: 1). As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 44, and specifically may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 44, but is not limited thereto. In addition, even if the present application describes a "peptide consisting of a specific sequence number," if the peptide has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, it is self-evident that even if it has meaningless sequence additions or naturally occurring mutations or their latent mutations before or after the amino acid sequence of the sequence number, such sequence additions or mutations are not excluded, they fall within the scope of the present application.

[0030] On the other hand, in another aspect, when the amino acid sequence of the general formula (1) is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, particularly any of the amino acid sequences of SEQ ID NOs: 13, 15, 36, and 38 to 43, it may be excluded from the scope of the peptide containing the amino acid sequence of the general formula (1), but is not limited thereto. The above content can be applied to other specific examples or aspects of the present invention, but is not limited thereto. Specifically, in the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid or lysine; X21 is aspartic acid or glutamic acid; X23 is valine; X24 is valine or glutamine; X27 is isoleucine or methionine; X28 is asparagine or arginine; X29 is threonine; X30 may be cysteine ​​or absent (except when the amino acid sequence of general formula (1) is identical to SEQ ID NO: 1). As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, but is not limited thereto.

[0031] Specifically, in the general formula (1), X1 is tyrosine; X2 is Aib (aminoisobutyric acid); X7 is threonine; X10 is tyrosine; X12 is lysine; X13 is tyrosine; X14 is leucine; X15 is aspartic acid or cysteine; X16 is glutamic acid, serine or cysteine; X17 is lysine or arginine; X18 is arginine; X19 is alanine; X20 is glutamine, cysteine ​​or lysine; X21 is aspartic acid, cysteine, valine, or glutamic acid; X23 is valine; X24 is valine or arginine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; X30 may be absent. As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, and specifically may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, but is not limited thereto.

[0032] More specifically, the peptide may be a peptide comprising an amino acid sequence of the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 may be cysteine ​​or absent. Here, the general formula ( 2 ) may be excluded if its amino acid sequence is identical to any one of SEQ ID NOs: 14, 19, 20, 25, 27, 31, and 33. However, the peptides falling within the category of are not limited thereto. For example, the peptide may be one that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, specifically one that is (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, but is not limited thereto. More specifically, the peptide is characterized in that it includes the amino acid sequence of SEQ ID NO: 12 or 20, or is (essentially) composed of the corresponding amino acid sequence, but is not limited thereto.

[0033] In addition, in a peptide comprising the amino acid sequence of general formula (1) or (2), any one or more of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula (1) or (2) may be substituted with glutamic acid or lysine capable of forming a ring, but is not limited thereto. More specifically, the peptide comprising the amino acid sequence of general formula (1) or general formula (2) may have an amino acid pair of X12 and X16 or an amino acid pair of X16 and X20 substituted with glutamic acid or lysine, respectively, capable of forming a ring, but is not limited thereto. More specifically, any one or more of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 may form a ring (e.g., a lactam ring), but is not limited thereto.

[0034] Meanwhile, in order to protect the peptide from in vivo protease cleavage and increase its stability, the amino and carboxy termini of the peptide may be modified or protected with multiple organic groups, for example, the C-terminus may be amidated. Depending on the length, the peptide of the present invention may be synthesized by a method well known in the art, for example, by an automatic peptide synthesizer, or may be produced by genetic engineering techniques. Specifically, the peptides of the present invention may be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the glucagon derivatives of the present invention may be synthesized in a number of ways, including, for example, the following: (a) synthesizing peptides by means of solid-phase or liquid-phase methods, either stepwise or by fragment assembly, and isolating and purifying the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method of obtaining peptide fragments by any combination of (a), (b) and (c), then ligating the fragments to obtain a peptide, and recovering the peptide. As a more specific example, a fusion gene encoding a fusion protein containing a fusion partner and a glucagon derivative can be prepared through genetic engineering, transformed into a host cell, expressed in the form of a fusion protein, and the glucagon derivative can be cleaved and separated from the fusion protein using a protease or a compound to produce the desired glucagon derivative. For this purpose, a DNA sequence encoding an amino acid residue that can be cleaved by a protease such as Factor Xa or enterokinase, or a compound such as CNBr or hydroxylamine can be inserted between the fusion partner and the polynucleotide encoding the glucagon derivative.

[0035] In a specific embodiment, the peptide of the present invention has been confirmed to have an altered pI compared to native glucagon (see Table 1). Therefore, since the peptide of the present invention has improved solubility and high stability at neutral pH, it can improve patient compliance when used as a therapeutic agent for hypoglycemia, and is suitable for co-administration with other anti-obesity therapeutic agents, making it useful for preventing or treating hypoglycemia and obesity. Thus, the peptides of the present invention may provide an attractive therapeutic option for metabolic syndrome or related disorders. For example, the peptides of the present invention, as the major response regulatory hormone to insulin, may be useful in treating severe hypoglycemia in diabetic patients. The peptides of the present invention may also be used as drugs to prevent weight gain, promote weight loss, reduce overweight, or treat obesity, including morbid obesity (e.g., by regulating appetite, feeding, food intake, caloric intake, and / or energy expenditure), as well as related diseases and conditions, including, but not limited to, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. The peptides of the present invention may also be used to treat metabolic syndromes other than obesity, i.e., glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and related diseases such as stroke and hypoglycemia. However, in these conditions, the effects of the peptides of the present invention may be mediated in whole or in part through the weight-related effects described above, or may be independent therefrom.

[0036] Examples of compounds or substances having therapeutic activity against metabolic syndrome that are included in the combination administration or composition of the present invention include insulinotropic peptides, GLP-1 (glucagon like peptide-1) receptor agonists, leptin receptor agonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, Y5 receptor antagonists, MCH (melanin-concentrating hormone) receptor antagonists, Y2 / 4 receptor agonists, MC3 / 4 (melanocortin 3 / 4) receptor agonists, gastric / pancreatic lipase inhibitors, 5HT2c (5-hydroxytryptamine receptor 2C, G protein-coupled) agonists, β3A receptor agonists, amylin receptor agonists, ghrelin antagonists, ghrelin receptor antagonists, PPARα (peroxisome proliferator-activated receptor agonists), and the like. These include, but are not limited to, alpHa agonists, PPARδ (peroxisome proliferator-activated receptor delta) agonists, FXR (farnesoid X receptor) agonists, acetyl-CoA carboxylase inhibitors, peptide YY, CCK (cholecystokinin), xenin, glicentin, obestatin, secretin, nesfatin, insulin, and GIP (glucose-dependent insulinotropic peptide). In addition, drugs that are effective in treating obesity and drugs that suppress liver inflammation and fibrosis may all be included. Specifically, the insulinotropic peptide may be selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof. More specifically, the insulinotropic peptide may be, but is not limited to, an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is substituted with one selected from the group consisting of des-amino-histidyl, N-dimethyl-histidyl, beta-hydroxyimidazopropionyl, 4-imidazoacetyl and beta-carboxyimidazopropionyl. More specifically, the insulinotropic peptide may be selected from the group consisting of native exendin-4, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been removed, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been replaced with a hydroxyl group, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been modified with a dimethyl group, exendin-4 derivatives in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been removed, exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with serine, and exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with arginine, but are not limited thereto.

[0037] Meanwhile, examples of such insulinotropic peptides or their long-acting conjugates are included in the present application as reference material, but are not limited thereto. In a more specific embodiment, the glucagon derivative, for example, a peptide comprising the amino acid sequence of the general formula (1) or (2) may be in the form of a long-acting conjugate to which a biocompatible substance that increases the half-life in vivo is linked, but is not limited thereto. The biocompatible substance may be mixed with a carrier. The insulinotropic peptide may also be in the form of a long-acting conjugate linked to a biocompatible substance that increases the half-life in the body, but is not limited thereto. In a specific embodiment of the present invention, the conjugate has an increased duration of effect compared to native glucagon or glucagon derivatives not conjugated to a carrier, and these protein conjugates are referred to as "long-acting conjugates" in the present invention. Examples of the biocompatible substance include, but are not limited to, polyethylene glycol, fatty acids, cholesterol, albumin and its fragments, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissues or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and polymeric polymers. For example, one or more amino acid side chains in the peptides of the present invention can be conjugated to such biocompatible substances to increase the solubility and / or half-life in vivo or to increase the bioavailability. Such modifications are known to decrease the clearance of therapeutic proteins and peptides.

[0038] These biocompatible materials are suitably water-soluble (amphiphilic or hydrophilic), non-toxic and pharma- ceutical inert, and examples include, but are not limited to, PEG, homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), or polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid. It is well known to those skilled in the art that such modified glucagon derivatives will exhibit superior therapeutic effects to native glucagon. Therefore, the above-mentioned variants of glucagon derivatives are also included in the scope of the glucagon derivatives of the present invention. In a more specific embodiment, the glucagon derivative, for example, a peptide comprising the amino acid sequence of general formula (1) or (2) and an insulinotropic peptide may be linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof, but is not limited thereto. In a more specific embodiment, the biocompatible substance is an FcRn-binding substance, and the glucagon derivative, for example, a peptide comprising the amino acid sequence of general formula (1) or (2) and an insulinotropic peptide, may be linked to the biocompatible substance via a peptide linker or a non-peptide linker, but is not limited thereto. As a specific example, the FcRn binding agent may be a polypeptide comprising an immunoglobulin Fc region.

[0039] In the present invention, a "non-peptidic linker" includes a biocompatible polymer having two or more repeating units linked together. The repeating units are linked together via any covalent bond other than a peptide bond. The non-peptidic linker may be a component that is part of the long-acting conjugate of the present invention. In the present invention, the non-peptidyl linker may be used in combination with a non-peptidyl polymer. In one specific embodiment, the conjugate may be covalently linked via a non-peptide linker that contains an immunoglobulin Fc region and reactive groups at both ends that can be bound to a protein drug. The non-peptidic linker may be selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) and PLGA (polylactic-glycolic acid); lipid polymers; chitins; hyaluronic acid; polysaccharides, and combinations thereof, but is not limited thereto. In a more specific embodiment, the other peptidic polymer may be polyethylene glycol, but is not limited thereto. In addition, derivatives thereof already known in the art and derivatives that can be easily prepared at the technical level in the art are also included within the scope of the present invention. The non-peptidyl linker that can be used in the present invention can be any polymer that is resistant to in vivo protease. The molecular weight of the non-peptidyl polymer is in the range of 1 to 100 kDa, specifically in the range of 1 to 20 kDa, but is not limited thereto. In addition, the non-peptidyl linker of the present invention that is bound to the polypeptide comprising the immunoglobulin Fc region may be not only one type of polymer, but also a combination of different types of polymers.

[0040] In one specific embodiment, both ends of the non-peptidic linker are capable of binding to an immunoglobulin Fc region and an amine group or a thiol group of the glucagon derivative or insulinotropic peptide, respectively. Specifically, the non-peptidyl polymer may have reactive groups at both ends capable of binding to an immunoglobulin Fc fragment and a glucagon derivative or an insulinotropic peptide, specifically, a reactive group capable of binding to an amine group located at the N-terminus or lysine of an immunoglobulin Fc fragment, or a thiol group of cysteine, but is not limited thereto. Furthermore, the reactive group of the non-peptidyl polymer, which can be bound to the immunoglobulin Fc region and the glucagon derivative or insulinotropic peptide, may be selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto. In the above, the aldehyde group may be, for example, a propionaldehyde group or a butyraldehyde group, but is not limited thereto. The succinimide derivative may be, but is not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate. Furthermore, the final products generated by reductive alkylation with aldehyde bonds are much more stable than those linked with amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0041] In addition, the reactive groups at both ends may be the same or different from each other, for example, one end may have a maleimide group and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group, but is not particularly limited thereto as long as the immunoglobulin Fc region and the glucagon derivative or insulinotropic peptide can be bound to each end of the non-peptide linker. For example, the non-peptidic linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end. When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups can be activated to various reactive groups by known chemical reactions, or the long-acting protein conjugate of the present invention can be produced using commercially available polyethylene glycol having modified reactive groups. In one specific embodiment, the non-peptidyl polymer may be linked to a cysteine ​​residue of the glucagon derivative, more specifically, but not limited to, a -SH group of cysteine. In a specific embodiment, the conjugate is one in which a peptide comprising the amino acid sequence of SEQ ID NO: 12 or 20 is linked to an immunoglobulin Fc region via a non-peptidyl polymer, and the non-peptidyl polymer may be linked to the cysteine ​​residue at position 30 in the amino acid sequence of SEQ ID NO: 12 or the cysteine ​​residue at position 17 in the amino acid sequence of SEQ ID NO: 20, but is not limited thereto. If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of the glucagon derivative through a thioether bond, and the aldehyde group is linked to the -NH 2 The linkage can be, but is not limited to, via a reductive alkylation reaction, which is one example.

[0042] In the present invention, the "immunoglobulin Fc region" refers to a portion of the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) of an immunoglobulin, excluding the heavy and light chain variable regions. The immunoglobulin Fc region may be a component constituting a part of the protein conjugate of the present invention. These immunoglobulin Fc regions may include a hinge portion in the heavy chain constant region, but are not limited thereto. Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes a part or the entire heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding only the heavy and light chain variable regions of the immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of the natural type. It may also be a very long region in which a part of the amino acid sequence corresponding to CH2 and / or CH3 has been deleted. For example, the immunoglobulin Fc region of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains selected from the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region, but is not limited thereto. In one specific example, the immunoglobulin Fc region may be in a dimeric form, and a glucagon derivative or insulinotropic peptide molecule may be covalently linked to one Fc region in the dimeric form, and in this case, the immunoglobulin Fc and the glucagon derivative or insulinotropic peptide may be linked to each other by a non-peptidic polymer. On the other hand, two molecules of the glucagon derivative or insulinotropic peptide may be symmetrically linked to one Fc region in the dimeric form. In this case, the immunoglobulin Fc and the glucagon derivative or insulinotropic peptide may be linked to each other by a non-peptidic linker. However, the present invention is not limited to the above-described examples.

[0043] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof, which means that one or more amino acid residues in the naturally occurring amino acid sequence have a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For example, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding in the case of IgG Fc, may be used as suitable sites for modification. Various types of derivatives are possible, such as those in which sites capable of forming disulfide bonds are removed, those in which several amino acids at the N-terminus of native Fc are removed, or those in which a methionine residue is added to the N-terminus of native Fc. In addition, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing sequence derivatives of these immunoglobulin Fc regions are disclosed in Patent Documents 2 and 3, etc. Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 2). The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / PHe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also be made.

[0044] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have increased structural stability, such as thermal and pH stability, of the Fc region. Furthermore, such an Fc region may be obtained from a natural type isolated in the living body of an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant type obtained from a transformed animal cell or a microorganism, or a derivative thereof. Here, the method of obtaining it from a natural type may be a method of obtaining it by separating the whole immunoglobulin from the living body of a human or animal and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, it is a recombinant immunoglobulin Fc region obtained by obtaining a human-derived Fc region from a microorganism. The immunoglobulin Fc region may also be in a form having a natural sugar chain, an increased sugar chain compared to the natural type, a decreased sugar chain compared to the natural type, or a form in which the sugar chain has been removed. For increasing or decreasing or removing the immunoglobulin Fc sugar chain, a conventional method such as a chemical method, an enzymatic method, or a genetic engineering method using a microorganism may be used. Here, an immunoglobulin Fc region from which the sugar chain has been removed from Fc has a significantly reduced binding ability with complement (C1q) and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. In this respect, it can be said that a form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region from which the sugar chain has been removed or which has been deglycosylated. In the present invention, "deglycosylation" refers to an Fc region from which sugars have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated. On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig pick, etc., and in a more specific embodiment, is of human origin.

[0045] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid of these. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant in human blood, and in a more specific embodiment, it is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In a further specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto. Meanwhile, in the present invention, the term "combination" means that a polypeptide encoding a short-chain immunoglobulin Fc region of the same origin forms a bond with a short-chain polypeptide of a different origin when forming a dimer or multimer. In other words, a dimer or multimer can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments. The compositions of the present invention may be used to prevent or treat hypoglycemia or metabolic syndrome. In the present invention, the term "prevention" means any action that suppresses or delays the onset of hypoglycemia or metabolic syndrome by administering the peptide or composition, and "treatment" means any action that improves or renders advantageous the symptoms of hypoglycemia or metabolic syndrome by administering the peptide or composition. In the present invention, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition is not particularly limited, but may be administered via any common route by which the composition can reach an in vivo target, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, intrarectal administration, etc.

[0046] In the present invention, the term "metabolic syndrome" refers to a condition in which various diseases caused by chronic metabolic disorders occur alone or in combination. In particular, diseases that fall under metabolic syndrome include, but are not limited to, impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke. In the present invention, the term "obesity" is defined as a state in which there is an excess of adipose tissue in the body, with a body mass index (body weight (kg) divided by the square of height (m)) of 25 or more. Obesity is usually induced by an energy imbalance caused by a long-term excess intake of nutrients compared to energy expenditure. Obesity is a metabolic disease that affects the entire body, increases the susceptibility to diabetes and hyperlipidemia, increases the risk of developing sexual dysfunction, arthritis, cardiovascular disease, and is sometimes associated with the development of cancer. The diabetes may exhibit "hypoglycemia" as an acute symptom. In the present invention, the term "hypoglycemia" refers to one of the acute symptoms of diabetes, a state in which blood sugar levels are lower than normal, generally referring to a state in which blood sugar levels are 50 mg / dl or less. Hypoglycemia is generally caused when a person who uses oral hypoglycemic agents or insulin eats less than usual, or when the person is more active or exercises more than usual. Hypoglycemia can also be caused by drinking alcohol or the use of some drugs that lower blood sugar levels, severe physical illnesses, hormone deficiencies such as adrenal cortex hormones and glucagon, insulin-producing pancreatic tumors, autoimmune diseases against insulin, patients who have had gastrectomy, and patients with hereditary carbohydrate metabolic enzyme disorders. Symptoms of hypoglycemia include lethargy, trembling, paleness, cold sweat, dizziness, agitation, anxiety, palpitations, hunger, headache, fatigue, etc. If hypoglycemia continues for a long time, a person may have convulsions or seizures, go into shock, and lose consciousness.

[0047] The pharmaceutical composition of the present invention may further comprise a pharma- ceutically acceptable carrier, excipient or diluent. In the present invention, the term "pharma-ceutically acceptable" means a sufficient amount to exhibit a therapeutic effect and to cause no side effects, and may be easily determined by those skilled in the art according to factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, treatment period, and drugs used in combination or simultaneously. The pharmaceutical composition comprising the peptide of the present invention may further comprise a pharma- ceutically acceptable carrier, which is not particularly limited, and may include, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc., for injections, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc., and for topical administration, bases, excipients, lubricants, preservatives, etc. The composition of the present invention may be prepared in various dosage forms by mixing with the pharma- ceutically acceptable carriers as described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it may be prepared in the form of unit dose ampoules or multiple doses. In addition, it may be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained release preparations, etc. On the other hand, examples of carriers, excipients and diluents suitable for formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. Also, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, preservatives, etc. may be further included.

[0048] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories. The composition may also be formulated into a unit dosage formulation suitable for administration into the body of a patient, preferably in a dosage form useful for administration of peptide pharmaceuticals, by a method commonly used in the pharmaceutical art, and administered orally or via a parenteral route, including dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method commonly used in the art, but is not limited thereto. The peptides may also be mixed with any of a number of pharmaceutical carriers, such as physiological saline or organic solvents, and may include drugs such as carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers, to increase stability and water absorption. The dosage and frequency of administration of the pharmaceutical composition of the present invention will be determined depending on the type of active ingredient, together with several related factors such as the disease to be treated, the administration route, the age, sex, and weight of the patient, and the severity of the disease. The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredients depending on the severity of the disease. Specifically, the preferred total dose of the peptide of the present invention may be about 0.0001 μg to 500 mg per kg of patient body weight per day. However, the effective dose of the peptide is determined taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, diet and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition. Taking these points into consideration, a person having ordinary skill in the art should be able to determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route and administration method as long as it shows the effects of the present invention.

[0049] Since the pharmaceutical composition of the present invention has excellent in vivo durability and potency, it is possible to significantly reduce the number and frequency of administration of the pharmaceutical preparation of the present invention. In particular, since the pharmaceutical composition of the present invention contains a glucagon derivative having an altered pI compared to native glucagon as an active ingredient, it exhibits improved solubility and high stability at neutral pH and is useful for preparing a stable glucagon dosage form for the treatment of hypoglycemia or obesity. Another embodiment of the present invention provides novel glucagon derivatives. The glucagon derivative is as described above. More specifically, the derivative is characterized as being an isolated peptide comprising an amino acid sequence of the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 is cysteine ​​or absent. Here, cases in which the amino acid sequence of the general formula (2) is identical to any one of SEQ ID NOs: 14, 19, 20, 25, 27, 31, and 33 may be excluded. More specifically, the peptide comprising the amino acid sequence of general formula (2) may be one in which the amino acid pair of X16 and X20 in general formula (2) is substituted with glutamic acid or lysine capable of forming a ring, and the amino acid pair of X16 and X20 forms a ring (e.g., a lactam ring), but is not limited thereto. Furthermore, the C-terminus of the peptide containing the amino acid sequence of the general formula (2) may be amidated, but is not limited thereto. The peptide may also be, but is not limited to, a glucagon derivative that can activate the glucagon receptor. More specifically, the peptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36-44, but is not limited thereto.

[0050] Further embodiments of the invention provide isolated polynucleotides encoding the glucagon derivatives, vectors comprising the polynucleotides, and isolated cells comprising the polynucleotides or vectors. The glucagon derivative is as described above. Also included within the scope of the invention are isolated polynucleotides encoding the glucagon derivatives having at least 75% sequence identity with the sequence, specifically at least 85%, more specifically at least 90%, and even more specifically at least 95% sequence identity therewith. The term "homology" is intended to indicate the degree of similarity between wild-type amino acid sequences and wild-type nucleic acid sequences, and homology comparisons can be performed by eye or using readily available comparison programs. Commercially available computer programs can calculate the percentage of homology between two or more sequences. The percentage of homology can be calculated for adjacent sequences. In the present invention, the term "recombinant vector" refers to a DNA construct in which a target peptide, such as a glucagon derivative, is operably linked to a suitable regulatory sequence so as to enable expression of the target peptide, such as a glucagon derivative, in a suitable host. The regulatory sequences include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into an appropriate host cell, a recombinant vector can replicate or function independently of the host genome, or can be integrated into the genome itself.

[0051] The recombinant vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and may be prepared using any vector known in the art. Examples of vectors commonly used include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. The vector usable in the present invention is not particularly limited, and any known expression vector may be used. The recombinant vectors are used to transform host cells to produce the glucagon derivatives of the invention, and such transformed cells, which are also part of the invention, may be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention and for recombinant production of the glucagon derivatives of the invention. In the present invention, the term "transformation" refers to introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it includes both, regardless of whether it is inserted and located within the host cell's chromosome or located outside the chromosome. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a genetic structure that is expressed by itself and contains all necessary elements. The expression cassette may generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of replicating itself. The polynucleotide may also be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. In addition, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the desired peptide of the present invention.

[0052] The host suitable for the present invention is not particularly limited as long as it expresses the polynucleotide of the present invention. Specific examples of the host used in the present invention include Escherichia bacteria such as E. coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria such as Pseudomonas putida, yeast such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (Sf9), and animal cells such as CHO, COS, and BSC. Another embodiment of the present invention provides a separate conjugate of a glucagon derivative linked to a biocompatible agent that increases its half-life in vivo, the conjugate may be a long-acting conjugate. The glucagon derivatives and biocompatible substances, as well as the composition of the conjugates, are all applicable as described above. Specifically, the biocompatible substance may be selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers, but is not limited thereto.

[0053] In addition, the isolated peptide may be linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof, but is not limited thereto. Furthermore, the biocompatible substance may be an FcRn-binding substance, and the separated peptide may be linked to the biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof, but is not limited thereto. The FcRn-binding substance may be, but is not limited to, a polypeptide comprising an immunoglobulin Fc region.

[0054] Another embodiment of the invention is a composition comprising the glucagon derivative or isolated conjugate. The glucagon derivative or isolated conjugate is as described above. Specifically, the composition may be a pharmaceutical composition for treating or preventing hypoglycemia or metabolic syndrome, but is not limited thereto. The pharmaceutical composition is as described above. The composition may also be a composition containing a peptide comprising an amino acid sequence of the general formula (2) described below. Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 is cysteine ​​or absent. Although not particularly limited thereto, the general formula ( 2 ) may be excluded if its amino acid sequence is the same as SEQ ID NOs: 14, 19, 20, 25, 27, 31, and 33.

[0055] Another embodiment of the invention provides a method of preventing or treating hypoglycemia or metabolic syndrome comprising administering to an individual the composition described above. The composition, hypoglycemia, metabolic syndrome, prevention and treatment are as described above. In the present invention, the individual is an individual suspected of hypoglycemia or metabolic syndrome, and the individual suspected of hypoglycemia or metabolic syndrome refers to mammals including mice, including humans, and livestock, which have or may develop the relevant disease, but includes, without limitation, individuals that can be treated with the glucagon derivative of the present invention or a composition containing the same. In addition, by administering a pharmaceutical composition containing the glucagon derivative of the present invention to an individual suspected of hypoglycemia or obesity, the individual can be effectively treated. Hypoglycemia and obesity are as described above. The method of the present invention may include administering a pharmaceutical composition comprising the peptide in a pharma- ceutical effective amount. The appropriate total daily dosage may be determined by the treating physician within the scope of sound medical judgment, and may be administered in one or several divided doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will desirably vary depending on a variety of factors, including the type and extent of the response to be achieved, the specific composition, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration, and the excretion rate of the composition, the duration of treatment, drugs used in conjunction with or concurrently with the specific composition, including, in some cases, whether other formulations are used, and similar factors well known in the pharmaceutical arts. Yet another aspect embodying the invention provides the use of the glucagon derivative or isolated conjugate or the composition in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome. The glucagon derivatives, the isolated conjugates, the compositions, hypoglycemia, and metabolic syndrome are as described above. The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. EXAMPLES

[0056] Example 1: Generation of cell lines that exhibit cAMP response to glucagon PCR was performed using a portion corresponding to the ORF of the cDNA of the human glucagon receptor gene (OriGene Technologies, Inc., USA) as a template and forward and reverse primers of SEQ ID NOs: 47 and 48, which contain an EcoRI cleavage site and an XhoI cleavage site, respectively. In this case, the PCR reaction was repeated 30 times, with denaturation at 95° C. for 60 seconds, annealing at 55° C. for 60 seconds, and elongation at 68° C. for 30 seconds. The amplified PCR product was electrophoresed on a 1.0% agarose gel, and a 450 bp band was eluted and obtained. Forward primer (SEQ ID NO: 47): 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' Reverse primer (SEQ ID NO: 48): 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3' The PCR product was cloned into a known animal cell expression vector, x0GC / dhfr, to prepare a recombinant vector, x0GC / GCGR. The recombinant vector x0GC / GCGR was transformed into CHO DG44 cells cultured in 10% FBS-containing DMEM / F12 medium using Lipofectamine, and selectively cultured in a selection medium containing 1 mg / mL G418 and 10 nM methotrexate. From this, monoclonal cell lines were selected by limiting dilution, and finally, cell lines showing excellent concentration-dependent cAMP response to glucagon were selected.

[0057] Example 2: Synthesis of glucagon derivatives In order to develop glucagon derivatives with improved physical properties, the amino acid sequence of natural glucagon of SEQ ID NO: 1 was replaced with negatively and positively charged amino acid residues to synthesize the glucagon derivatives shown in Table 1 below, in which the relative in vitro activities described herein were measured by the method described in Example 4 below.

[0058] Table 1. Amino acid sequences of native glucagon and glucagon derivatives TIFF0007626393000001.tif202166 TIFF0007626393000002.tif208161 In the sequences shown in Table 1, an amino acid represented by X is an unnatural amino acid, aminoisobutyric acid (Aib), an underline in an amino acid symbol indicates that the amino acid forms a ring, and "-" indicates that there is no amino acid residue at the corresponding position.

[0059] Example 3: pI measurement of glucagon derivatives To confirm the improved physical properties of the glucagon derivative synthesized in Example 2, the pI was estimated from the amino acid sequence using the pI / Mw tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003). As shown in Table 1, native glucagon of SEQ ID NO: 1 has a pI of 6.8, whereas some glucagon derivatives according to the present invention have a pI in the range of about 4 to 6. These glucagon derivatives have a lower or higher pI than native glucagon, and therefore can exhibit improved solubility and higher stability at neutral pH, etc., compared to native glucagon. The glucagon derivative according to the present invention can improve patient compliance when used as a therapeutic agent for hypoglycemia, and is suitable for combined administration with other anti-obesity therapeutic agents or anti-diabetic agents. It can be useful as a therapeutic agent for metabolic syndrome including hypoglycemia, obesity, diabetes, nonalcoholic steatohepatitis (NASH), dyslipidemia, and coronary heart disease.

[0060] Example 4: Measurement of cAMP activity of glucagon derivatives The activity of the glucagon derivative synthesized in Example 2 was measured in the cell line having the human glucagon receptor prepared in Example 1. Specifically, the transformed cell line was subcultured three or four times a week, and then cultured in a 384-well plate at 6x10 cells per well. 3The subcultured cell lines were dispensed and cultured for 24 hours. The native glucagon was suspended at 200 nM and the glucagon derivatives were suspended at 1600 nM in HBSS (Hank's Balanced Salt Solution) buffer containing 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 0.1% BSA (bovine serum albumin), and 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and then serially diluted 10 times by 4. This was applied to a cAMP assay kit (LANCE cAMP 384 kit, PerkinElmer), added to the cells, and the fluorescence value was measured. After the measurement, the highest fluorescence value was selected as 100%, and the EC 50 The values ​​were calculated and compared with those of natural glucagon, and the results are shown in Table 1 above.

[0061] Example 5: Preparation of conjugates containing glucagon derivatives and immunoglobulin Fc (SEQ ID NOs: 12, 20 - immunoglobulin Fc region conjugates) In order to PEGylate the cysteine ​​residue of the glucagon derivative (SEQ ID NO: 12, 20) with 10 kDa PEG having a maleimide group and an aldehyde group at both ends, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the glucagon derivative and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1-5 and a protein concentration of 3-10 mg / ml at low temperature for 1-3 hours. The reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative mono-PEGylated to cysteine. Next, the purified monoPEGylated glucagon derivative and immunoglobulin Fc were reacted at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg / ml at 4-8°C for 12-18 hours. The reaction solution was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was added with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-20% isopropanol. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing the glucagon derivative and immunoglobulin Fc. After preparation, the purity was determined to be 95% or more by reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography. Here, the conjugate in which the glucagon derivative of SEQ ID NO: 12 and immunoglobulin Fc are linked via PEG is named "a conjugate comprising the glucagon derivative of SEQ ID NO: 12 and immunoglobulin Fc" or "SEQ ID NO: 12, long-acting derivative", and these terms are used interchangeably in this application. Herein, the conjugate in which the glucagon derivative of SEQ ID NO: 20 and immunoglobulin Fc are linked via PEG is named "a conjugate comprising the glucagon derivative of SEQ ID NO: 20 and immunoglobulin Fc" or "SEQ ID NO: 20, long-acting derivative", and these terms are used interchangeably in this application.

[0062] Example 6: Preparation of a conjugate containing an exendin-4 derivative and immunoglobulin Fc Imidazoacetyl Exendin-4 (CA Exendin-4, AP, USA), in which the alpha carbon of the N-terminal histidine has been deleted, was reacted with 3.4 kDa PEG having propionaldehyde groups at both ends, i.e., 3.4K PropionALD(2)PEG, to Lys of CA Exendin-4. Coupling was then performed using the rearmost isomer peak (Lys27 positional isomer), which reacted more than the other of the two Lys isomer peaks and was clearly distinguishable from the N-terminal isomer. The peptide and immunoglobulin Fc molar ratio was 1:8, and the total protein concentration was 60 mg / ml, and the reaction was carried out at 4°C for 20 hours. The reaction solution was 100 mM KP pH 6.0, and 20 mM SCB, a reducing agent, was added. The coupling reaction solution was purified through two purification columns. First, SOURCE Q (XK-16mL, Amersham Biosciences) was used to remove a large amount of immunoglobulin Fc that was not involved in the coupling reaction. When a salt gradient was applied using 20 mM Tris (pH 7.5) and 1 M NaCl, immunoglobulin Fc with relatively weak binding strength was eluted first, followed by exendin-4 immunoglobulin Fc. Although a certain amount of immunoglobulin Fc was removed through the primary purification, it was not completely separated because the difference in binding strength between immunoglobulin Fc and exendin-4 immunoglobulin Fc in the ion exchange column was not large. Therefore, secondary purification was carried out by utilizing the difference in hydrophobicity between the two substances. After binding the primary purified sample to SOURCE ISO (HR16ml, Amersham Biosciences) using 20mM Tris (pH7.5) and 1.5M ammonium sulfate, the sample was eluted by gradually decreasing the concentration of ammonium sulfate. Immunoglobulin Fc with weak binding to the HIC column was eluted first, and the exendin-4 immunoglobulin Fc sample with strong binding was eluted later. The large difference in hydrophobicity between these samples made it much easier to separate them than with the ion exchange column. Column: SOURCE Q (XK 16ml, Amersham Biosciences) Flow rate: 2.0ml / min Gradient: A0 -> 25% B in 70 min (A: 20 mM Tris pH 7.5, B: A + 1 M NaCl) Column: SOURCE ISO (HR 16ml, Amersham Biosciences) Flow rate: 7.0ml / min Gradient: B 100 -> 0% 60 min B (A: 20 mM Tris pH 7.5, B: A + 1.5 M ammonium sulfate) The conjugate prepared as above, in which the Exendin-4 derivative and the immunoglobulin Fc region are linked via PEG, is named "long-acting Exendin-4 derivative", which is used interchangeably with the long-acting Exendin derivative.

[0063] Experimental Example 1: Effect of weight loss in high-fat diet-induced obese rats High-fat diet-induced obese rats, widely used as an obesity animal model, were used for this study. The rats weighed approximately 600 g before dosing. The rats were housed individually and allowed free access to water during the study period. The lights were turned off from 6AM to 6PM. The test groups fed with a high fat diet were: Group 1: vehicle (injected once every 3 days) control group; Group 2: 3.3 nmol / kg of the sustained release exendin derivative of Example 6 (injected once every 3 days); Group 3: 1.6 nmol / kg of the sustained release derivative of SEQ ID NO: 12 (injected once every 3 days); Group 4: 3.3 nmol / kg of the sustained release derivative of SEQ ID NO: 12 (injected once every 3 days), group 5: 6.6 nmol / kg of the long-acting derivative of sequence number 12 (injected once every 3 days), group 6: 3.3 nmol / kg of the long-acting exendin derivative of Example 6 + 1.6 nmol / kg of the long-acting derivative of sequence number 12 (injected once every 3 days), group 7: 3.3 nmol / kg of the long-acting exendin derivative of Example 6 + 3.3 nmol / kg of the long-acting derivative of sequence number 12 (injected once every 3 days), group 8: 3.3 nmol / kg of the long-acting exendin derivative of Example 6 + 6.6 nmol / kg of the long-acting derivative of sequence number 12 (injected once every 3 days), group 9: paired with group 4 and fed, group 10: paired with group 7 and fed; The experiment was completed on the 15th day, and the weight change of the rats corresponding to each group was measured every 3 days during the experiment. After the experiment was completed, the mesenteric fat amount and liver weight were measured through autopsy. Statistical analysis was performed using one-way ANOVA to compare the vehicle group (control group) and the test groups. As a result of measuring the change in body weight, as shown in Figure 1, the groups administered the sustained-release Exendin derivative and the sustained-release derivative of sequence number 12 alone showed a weight loss of -8% and -7% to -22% compared to before the start of administration, respectively, whereas when the sustained-release Exendin derivative and the sustained-release derivative of sequence number 12 were administered in combination, the weight loss effect was confirmed to be further increased to -22% to -35%. In addition, when comparing the weight loss effect between the group administered only the long-acting derivative of sequence number 12 and the paired feeding group administered a combination of a long-acting exendin derivative and a long-acting derivative of sequence number 12, the weight loss effect differed by approximately -11% and -17%, respectively, indicating that the weight loss effect was due to factors other than food intake when glucagon derivatives were administered alone or in combination. That is, it was confirmed that the long-acting glucagon derivative of the present invention plays another role in weight loss in addition to reducing appetite. In addition, the results of measuring the amount of mesenteric fat and liver weight showed that when the sustained-release exendin derivative and the sustained-release derivative of SEQ ID NO: 12 were administered in combination, the amount of fat in the body was significantly reduced, and the liver weight was also reduced, compared to the vehicle administration group, as shown in Figures 2 and 3. Meanwhile, the increase or decrease in liver weight is generally due to the increase or decrease in fat in the liver, and the effect of reducing liver weight is the effect of reducing liver fat, and the reduction in liver fat may be measured as a method for measuring the therapeutic effect of metabolic syndromes such as obesity, diabetes, and non-alcoholic steatohepatitis.

[0064] Experimental Example 2: Effect of weight loss in high-fat diet-induced obese mice High-fat diet-induced obese mice, widely used as an obesity animal model, were used for this study. The mice weighed approximately 55 g before administration. The mice were housed in groups of seven and had free access to water during the study period. The lights were turned off from 6 PM to 6 AM. The test groups fed with a high fat diet were as follows: Group 1: vehicle (injected once every 2 days) - control group; Group 2: 4.3 nmol / kg of the sustained exendin derivative of Example 6 (injected once every 2 days); Group 3: 4.4 nmol / kg of the sustained derivative of SEQ ID NO:20 (injected once every 2 days); Group 4: 8.8 nmol / kg of the sustained derivative of SEQ ID NO:20 (injected once every 2 days); Group 5: 4.3 nmol / kg of the sustained exendin derivative of Example 6 + 4.4 nmol / kg of the sustained derivative of SEQ ID NO:20 (injected once every 2 days); Group 6: 2.1 nmol / kg of the sustained exendin derivative of Example 6 + 6.6 nmol / kg of the sustained derivative of SEQ ID NO:20 (injected once every 2 days); Group 7: 0.8 nmol / kg of the sustained exendin derivative of Example 6 + 8.0 nmol / kg of the sustained derivative of SEQ ID NO:20 (injected once every 2 days); The experiment was completed on the 22nd day, and the weight change of the mice in each group was measured every 2 days during the experiment. After the experiment was completed, the mice were autopsied and the liver weight was measured. As a result of measuring the change in body weight, as shown in FIG. 4, it was confirmed that the long-acting derivative of SEQ ID NO: 20 (administered at 8.8 nmol / kg once every two days) showed a weight loss of -25% and -29% compared to before the start of administration in each single group, respectively, and the effect was greater than that when administered in combination with a long-acting exendin derivative. It was confirmed that the effect of weight loss was -50% or more and further increased when the long-acting exendin derivative of SEQ ID NO: 20 was administered in combination at a ratio of 1:1, 1:3, and 1:10. In addition, the effect of weight loss did not change significantly depending on the ratio of the long-acting derivative of SEQ ID NO: 20 and the long-acting exendin derivative, but the decrease in appetite was greater with a higher ratio of the long-acting exendin derivative, so it was confirmed that the long-acting glucagon derivative of the present invention plays another role in weight loss in addition to decreasing appetite. In addition, the total cholesterol level in the blood was measured, and as shown in FIG. 5, the groups of the long-acting exendin derivative (4.4 nmol / kg, administered once every two days) and the long-acting derivative of sequence number 20 (8.8 nmol / kg, administered once every two days), which showed similar weight loss effects, showed cholesterol reductions of -35% and -71%, respectively. From this, it was confirmed that the long-acting glucagon derivative of the present invention plays a different role in reducing blood cholesterol in addition to the weight loss effect. For statistical processing, one-way ANOVA was used to compare the vehicle group (control group) and the test group. From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description. Next, preferred embodiments of the present invention will be described. 1. A pharmaceutical composition for treating or preventing metabolic syndrome, comprising: i) a peptide comprising an amino acid sequence represented by the following general formula (1); and ii) one or more compounds or substances having therapeutic activity against metabolic syndrome: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (General formula (1), Sequence number 45) In the general formula (1), X1 is histidine, desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan or tyrosine, or is absent; X2 is alpha-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine; X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, alpha-methyl-glutamic acid, cysteine, or absent; X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or is absent; X18 is alanine, aspartic acid, glutamic acid, arginine, valine, cysteine, or absent; X19 is alanine, arginine, serine, valine, cysteine, or absent; X20 is lysine, histidine, glutamine, aspartic acid, lysine, arginine, alpha-methyl-glutamic acid, cysteine, or is absent; X21 is aspartic acid, glutamic acid, leucine, valine, cysteine, or absent; X23 is isoleucine, valine, arginine, or absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, leucine, or absent; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or is absent; X28 is glutamine, lysine, asparagine, arginine, or absent; X29 is lysine, alanine, glycine, or threonine, or is absent; X30 may be cysteine ​​or absent. (However, this does not include cases where the amino acid sequence of the general formula (1) is identical to SEQ ID NO: 1.) 2. In the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid, lysine or cysteine; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine; X23 is isoleucine, valine, or arginine; X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine; X27 is isoleucine, valine, alanine, methionine, glutamine, or arginine; X28 is glutamine, lysine, asparagine or arginine; X29 is threonine; 2. The pharmaceutical composition of claim 1, wherein X30 is cysteine ​​or absent. 3. In the general formula (1), X1 is histidine, tryptophan, tyrosine, or absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; 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, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid or lysine; X21 is aspartic acid or glutamic acid; X23 is valine; X24 is valine or glutamine; X27 is isoleucine or methionine; X28 is asparagine or arginine; X29 is threonine; 2. The pharmaceutical composition of claim 1, wherein X30 is cysteine ​​or absent. 4. In the general formula (1), X1 is tyrosine; X2 is Aib (aminoisobutyric acid); X7 is threonine; X10 is tyrosine; X12 is lysine; X13 is tyrosine; X14 is leucine; X15 is aspartic acid or cysteine; X16 is glutamic acid, serine or cysteine; X17 is lysine or arginine; X18 is arginine; X19 is alanine; X20 is glutamine, cysteine ​​or lysine; X21 is aspartic acid, cysteine, valine, or glutamic acid; X23 is valine; X24 is valine or arginine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; The pharmaceutical composition described in claim 1, wherein X30 is absent. 5. A pharmaceutical composition, wherein the peptide is a peptide comprising an amino acid sequence represented by the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; 2. The pharmaceutical composition of claim 1, wherein X30 is cysteine ​​or absent. 6. The pharmaceutical composition described in 1 above, wherein the peptide comprising the amino acid sequence of general formula (1) has a pI different from the pI of native glucagon, which is 6.8. 7. The pharmaceutical composition according to 1 above, wherein in the peptide comprising the amino acid sequence of general formula (1), one or more of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula (1) are substituted with glutamic acid or lysine capable of forming a ring, respectively. 8. The pharmaceutical composition according to claim 7, wherein in the peptide comprising the amino acid sequence of the general formula (1), the amino acid pair of X12 and X16 or the amino acid pair of X16 and X20 is substituted with glutamic acid or lysine, respectively, capable of forming a ring. 9. The pharmaceutical composition described in 1 above, wherein the C-terminus of the peptide comprising the amino acid sequence of general formula (1) is amidated. 10. The pharmaceutical composition according to claim 1, wherein the peptide comprising the amino acid sequence of the general formula (1) is a glucagon derivative capable of activating the glucagon receptor. 11. The pharmaceutical composition described in 1 above, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 44. 12. The pharmaceutical composition described in 5 above, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44. 13. The pharmaceutical composition described in 1 above, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 12 or 20. 14. The compound or substance having a therapeutic activity for metabolic syndrome is an insulinotropic peptide, a glucagon like peptide-1 (GLP-1) receptor agonist, a leptin receptor agonist, a dipeptidyl peptidase-IV (DPP-IV) inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y2 / 4 receptor agonist, a melanocortin 3 / 4 (MC3 / 4) receptor agonist, a gastric / pancreatic lipase inhibitor, a 5-hydroxytryptamine receptor 2C (5HT2c, G protein-coupled) agonist, a β3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, a ghrelin receptor antagonist, a peroxisome proliferator-activated receptor α (PPARα) antagonist, a serotonin receptor ... alpha agonists, PPAR delta (peroxisome proliferator-activated receptor delta) agonists, farnesoid X receptor (FXR) agonists, acetyl-CoA carboxylase inhibitors, peptide YY, CCK (cholecystokinin), xenin, glicentin, obestatin, secretin, nesfatin, insulin, and glucose-dependent insulinotropic peptide (GIP). 15. The pharmaceutical composition according to claim 14, wherein the insulinotropic peptide is selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants and combinations thereof. 16. The pharmaceutical composition according to claim 15, wherein the insulinotropic peptide is an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is replaced with one selected from the group consisting of des-amino-histidyl, N-dimethyl-histidyl, beta-hydroxyimidazopropionyl, 4-imidazoacetyl and beta-carboxyimidazopropionyl. 17. The pharmaceutical composition according to claim 15, wherein the insulinotropic peptide is selected from the group consisting of native exendin-4, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been removed, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been replaced with a hydroxyl group, exendin-4 derivatives in which the N-terminal amine group of exendin-4 has been modified with a dimethyl group, exendin-4 derivatives in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been removed, exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with serine, and exendin-4 derivatives in which the 12th amino acid (lysine) of exendin-4 has been replaced with arginine. 18. The peptide comprising the amino acid sequence of the general formula (1) is in the form of a long-acting conjugate linked to a biocompatible substance that increases the half-life in vivo; 15. The pharmaceutical composition according to claim 14, wherein the insulinotropic peptide is in the form of a long-acting conjugate linked to a biocompatible substance that increases its half-life in vivo. 19. The pharmaceutical composition described in 18 above, wherein the biocompatible substance is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers. 20. The pharmaceutical composition described in 18 above, wherein the peptide comprising the amino acid sequence of general formula (1) and the insulinotropic peptide are each linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. 21. The biocompatible substance is an FcRn-binding substance; 19. The pharmaceutical composition according to claim 18, wherein the peptide comprising the amino acid sequence of the general formula (1) and the insulinotropic peptide are linked to a biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. 22. The pharmaceutical composition according to claim 21, wherein the FcRn-binding substance is a polypeptide comprising an immunoglobulin Fc region. 23. The pharmaceutical composition described in claim 22, wherein the immunoglobulin Fc region is non-glycosylated. 24. The immunoglobulin Fc region, (a) the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain; (b) CH1 and CH2 domains; (c) CH1 and CH3 domains; (d) CH2 and CH3 domains; (e) CH1 domain, CH2 domain, CH3 domain, and CH4 domain with an immunoglobulin hinge region or a portion of said hinge region; and (f) The pharmaceutical composition described in claim 22, wherein the pharmaceutical composition is selected from the group consisting of a dimer of each domain of the heavy chain constant region and a dimer of the light chain constant region. 25. The pharmaceutical composition described in claim 22, wherein the polypeptide comprising the immunoglobulin Fc region is in a dimeric form. 26. The pharmaceutical composition described in claim 22, wherein the immunoglobulin Fc region is a derivative of a native Fc in which sites capable of forming disulfide bonds have been removed, some amino acids at the N-terminus have been removed from the native Fc, a methionine residue has been added to the N-terminus of the native Fc, the complement binding site has been deleted, or the ADCC (antibody dependent cell mediated cytotoxicity) site has been removed. 27. The pharmaceutical composition described in claim 22, wherein the immunoglobulin Fc region is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. 28. The pharmaceutical composition described in claim 27, wherein the immunoglobulin Fc region is an IgG4 Fc region. 29. The pharmaceutical composition described in claim 22, wherein the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4. 30. The pharmaceutical composition according to claim 21, wherein the non-peptide linker is linked to a cysteine ​​residue of a peptide comprising the amino acid sequence of general formula (1). 31. The pharmaceutical composition described in claim 21, wherein both ends of the non-peptidic linker are linked to a biocompatible substance and an amine group or a thiol group of a peptide comprising the amino acid sequence of general formula (1) or an insulinotropic peptide, respectively. 32. The pharmaceutical composition described in 1 above, wherein the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke. 33. An isolated peptide comprising an amino acid sequence represented by the following general formula (2): Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula (2), SEQ ID NO: 46) In the general formula (2), X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is valine or glutamine; X30 is cysteine ​​or absent. (However, this does not include cases where the amino acid sequence of the general formula (1) is identical to any one of SEQ ID NOs: 14, 19, 20, 25, 27, 31, and 33.) 34. The peptide according to claim 33, comprising the amino acid sequence of the general formula (2), wherein the amino acid pair of X12 and X16 in the general formula (2) is substituted with glutamic acid or lysine capable of forming a ring. 35. The peptide according to claim 33, wherein the C-terminus of the peptide is amidated. 36. The peptide according to claim 33, wherein the peptide is a glucagon derivative capable of activating the glucagon receptor. 37. The peptide according to claim 33, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44. 38. An isolated polynucleotide encoding the isolated peptide according to any one of claims 33 to 37. 39. A vector comprising the isolated polynucleotide described in 38 above. 40. An isolated conjugate comprising the isolated peptide according to claim 33 linked to a biocompatible substance that increases the in vivo half-life. 41. The isolated conjugate according to claim 40, wherein the biocompatible substance is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissues or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers. 42. The isolated conjugate according to claim 40, characterized in that the isolated peptide is linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. 43. The biocompatible substance is an FcRn-binding substance; 41. The isolated conjugate according to claim 40, wherein the peptide comprising the amino acid sequence of general formula (1) and the insulinotropic peptide are each linked to a biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof. 44. The isolated conjugate according to claim 43, wherein the FcRn-binding substance is a polypeptide comprising an immunoglobulin Fc region. 45. A composition comprising an isolated peptide according to claim 33 or an isolated conjugate according to claim 40. 46. ​​The composition according to claim 45, which is a pharmaceutical composition for treating or preventing hypoglycemia or metabolic syndrome. 47. A method for treating metabolic syndrome, comprising administering the composition described in 1 above to an individual in need thereof. 48. A method for treating hypoglycemia or metabolic syndrome, comprising administering to an individual in need thereof an isolated peptide described in 33 above, an isolated conjugate described in 40 above, or a composition described in 46 above.

Claims

1. A pharmaceutical composition for treating or preventing metabolic syndrome, comprising: i) a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38 and 44; and ii) one or more compounds or substances having therapeutic activity against metabolic syndrome, wherein the compounds or substances having therapeutic activity against metabolic syndrome are selected from the group consisting of dipeptidyl peptidase-IV (DPP-IV) inhibitors, amylin, peptide YY, cholesterol (CCK), xenin, glicentin, obestatin, secretin, nesfatin, insulin and glucose-dependent insulinotropic peptide (GIP).

2. 2. The pharmaceutical composition of claim 1, wherein the peptide has an isoelectric point (pi) different from 6.8, which is the pI of native glucagon.

3. The pharmaceutical composition of claim 1 , wherein in the peptide, the amino acids at positions 16 and 20 form a ring.

4. The pharmaceutical composition of claim 1 , wherein the peptide is a glucagon derivative capable of activating the glucagon receptor.

5. The pharmaceutical composition of claim 1 , wherein the peptide is in the form of a long-acting conjugate linked to a biocompatible substance that increases its half-life in vivo.

6. 6. The pharmaceutical composition according to claim 5, wherein the biocompatible substance is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, saccharides, and high molecular weight polymers.

7. The pharmaceutical composition according to claim 5, wherein the peptide is linked to a biocompatible substance via a linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

8. the biocompatible substance is an FcRn-binding substance; 6. The pharmaceutical composition of claim 5, wherein the peptide is linked to a biocompatible material via a peptide linker or a non-peptide linker selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) or PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof.

9. The pharmaceutical composition of claim 8 , wherein the FcRn binding agent is a polypeptide comprising an immunoglobulin Fc region.

10. The pharmaceutical composition of claim 9, wherein the immunoglobulin Fc region is aglycosylated.

11. The immunoglobulin Fc region is (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain with an immunoglobulin hinge region or a portion of said hinge region; and (f) a dimer of each of the heavy chain constant region and light chain constant region domains.

12. The pharmaceutical composition of claim 9 , wherein the polypeptide comprising an immunoglobulin Fc region is in a dimeric form.

13. The pharmaceutical composition of claim 9, wherein the immunoglobulin Fc region is a derivative of a native Fc in which a site capable of forming a disulfide bond has been removed, some amino acids at the N-terminus have been removed from the native Fc, a methionine residue has been added to the N-terminus of the native Fc, a complement binding site has been deleted, or an ADCC (antibody dependent cell mediated cytotoxicity) site has been removed.

14. 10. The pharmaceutical composition of claim 9, wherein the immunoglobulin Fc region is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

15. The pharmaceutical composition of claim 14 , wherein the immunoglobulin Fc region is an IgG4 Fc region.

16. The pharmaceutical composition of claim 9, wherein the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4.

17. The pharmaceutical composition of claim 8 , wherein the non-peptidic linker is linked to a cysteine ​​residue of the peptide.

18. The pharmaceutical composition according to claim 8 , wherein both ends of the non-peptidic linker are linked to a biocompatible substance and an amine group or a thiol group of a peptide, respectively.

19. 2. The pharmaceutical composition of claim 1, wherein the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

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