Compositions containing glucagon and dual GLP-1 and GIP receptor agonists and therapeutic uses thereof

Abstract

The present invention relates to compositions comprising a glucagon derivative and a dual agonist of the GLP-1 receptor and GIP receptor, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising a compound or substance having glucagon activity and a dual agonist of the GLP-1 receptor and GIP receptor, and to therapeutic uses thereof. [Background technology]

[0002] Recently, due to economic development and changes in eating habits, there has been a rapid increase in the incidence of metabolic syndrome-related diseases, including obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, liver disease, etc. Although these diseases may occur individually, in most cases they occur together with multiple symptoms, which are closely related to each other.

[0003] Overweight and obesity increase blood pressure and cholesterol levels, causing or worsening various diseases such as heart disease, diabetes, arthritis, etc. In addition, overweight and obesity are major factors that increase the incidence of arteriosclerosis, high blood pressure, hyperlipidemia, and heart disease not only in adults but also in children and adolescents.

[0004] Obesity is a complex disease involving the mechanisms of appetite regulation and energy metabolism. Therefore, methods for treating abnormal mechanisms related to appetite regulation and energy metabolism must be developed simultaneously. Therefore, efforts to develop drugs that treat these abnormal mechanisms are ongoing. As a result of these efforts, anti-obesity drugs such as rimonabant (Sanofi-Aventis), sibutramine (Abbott), Contrave (Takeda), and orlistat (Roche) have been developed. However, these drugs have drawbacks, such as fatal side effects and ineffectiveness in treating obesity. For example, rimonabant has been reported to cause central nervous system disorders, sibutramine and Contrave have cardiovascular side effects, and orlistat has only been reported to reduce weight by approximately 4 kg after one year of administration.

[0005] Meanwhile, metabolic syndrome, including obesity, increases the likelihood of liver diseases. Examples include metabolic liver disease, fatty liver, non-alcoholic fatty liver disease, steatohepatitis, and hepatic fibrosis. These liver diseases are on the rise as the obese and diabetic populations increase, with the annual incidence rate reaching approximately 16% in Korea. Because liver disease has no noticeable symptoms in the early stages and is only discovered when the disease is well advanced, it is one of the leading causes of death not only in Korea but globally, making drug development highly necessary.

[0006] Glucagon is produced in the pancreas when blood glucose levels drop due to drug treatment, disease, or hormone or enzyme deficiency. Glucagon signals the liver to break down glycogen and release glucose, thereby raising blood glucose levels to normal. However, glucagon's use as a therapeutic agent has been limited due to its low solubility and precipitation at neutral pH.

[0007] Glucagon-like peptide-1 (GLP-1), a glucagon derivative, is a hormone secreted from the small intestine in response to food intake. It promotes insulin secretion in the pancreas in a blood glucose-dependent manner, suppresses glucagon secretion, and helps lower blood glucose levels. It also acts as a satiety factor, slowing gastrointestinal digestion and slowing the gastrointestinal transit time of food digestion, thereby reducing food intake.

[0008] GIP, one of the gastrointestinal hormones that is secreted in response to food intake, along with GLP-1, is a hormone consisting of 42 amino acids secreted from K cells in the small intestine. It promotes insulin secretion in the pancreas depending on blood glucose concentration, and helps lower blood glucose levels. It has been reported to have effects such as increasing GLP-1 activity, anti-inflammatory effects, and improving lipid metabolism.

[0009] Therefore, active research is underway to develop GLP-1 as a treatment for diabetes and obesity, taking advantage of its blood glucose control and weight loss effects. However, GLP-1 alone only reduces HbA1c by 0.5% to 1.8%, and is considered appropriate for patients with HbA1c levels of 9% or less (Ther Adv Endocrinol Metab. 2015 Feb; 6(1): 3-18). In other words, there is a limit to the blood glucose regulation that can be expected from the administration of GLP-1 alone.

[0010] Therefore, research has been conducted into dual agonists that simultaneously activate GLP-1 and GIP receptors, and examples of such dual agonists are described in International Publications WO2013164483, WO2016111971, WO2014192284, etc.

[0011] The glucagon and GLP-1 receptor and GIP receptor dual agonists are generally known to have opposing effects and have been used as therapeutic agents for different diseases. That is, because glucagon and GLP-1 receptor and GIP receptor dual agonists have opposing roles in the body, no drug therapy has been reported in which they are administered together. Meanwhile, when various therapeutic agents related to metabolic syndrome are administered to patients, there are risks of side effects such as weight gain, overdose, and hypoglycemia. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication WO2013 / 164483 [Patent Document 2] International Publication WO2016 / 111971 [Patent Document 3] International Publication WO2014 / 192284 [Patent Document 4] International Patent Publication No. WO97 / 34631 [Patent Document 5] International Patent Publication No. 96 / 32478 [Non-patent literature]

[0013] [Non-Patent Document 1] Ther Adv Endocrinol Metab. 2015 Feb; 6(1): 3-18 [Non-patent document 2] H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979 [Non-patent document 3] GJ Webb et al., J. Autoimmunity, 2015 Nov;64:42-52 Summary of the Invention [Problem to be solved by the invention]

[0014] One object of the present invention relates to a pharmaceutical composition for preventing or treating metabolic syndrome, which comprises a substance active against the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor.

[0015] Another object of the present invention is to provide a method for preventing or treating metabolic syndrome, comprising administering to an individual in need thereof a substance having activity at the glucagon receptor and a GLP-1 receptor and GIP receptor dual agonist.

[0016] Another object of the present invention is to provide the use of a substance having activity against the glucagon receptor and a dual agonist of the GLP-1 receptor and GIP receptor for the prevention or treatment of metabolic syndrome. [Means for solving the problem]

[0017] One embodiment of the present invention is the therapeutic use of a combination of an agent having activity at the glucagon receptor and a dual GLP-1 and GIP receptor agonist.

[0018] In one embodiment, the present invention relates to a composition comprising an agent having activity at the glucagon receptor and a dual GLP-1 receptor and GIP receptor agonist.

[0019] In the above embodiment, the present invention relates to a pharmaceutical composition for preventing or treating metabolic syndrome, comprising a substance having activity on the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor.

[0020] In the above specific example(s), the substance having activity against the glucagon receptor is characterized in that it is a peptide comprising the amino acid sequence of the following general formula 1:

[0021] 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 46)

[0022] In the above formula, X1 is tyrosine (Y), X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine; X7 is threonine (T), valine (V), or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X13 is tyrosine (Y) or cysteine ​​(C); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C); X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methyl-glutamic acid, or cysteine ​​(C), or is absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), alpha-methyl-glutamic acid, or cysteine ​​(C), or is absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methyl-glutamic acid, or leucine (L), or is absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X29 is threonine (T), X30 is cysteine ​​(C) or absent. (However, this does not include cases where the amino acid sequence of the above general formula 1 is identical to SEQ ID NO: 1 or SEQ ID NO: 12).

[0023] In the preceding specific example(s), The peptide is in the form of a long-acting conjugate, and the long-acting conjugate is characterized by being represented by the following chemical formula (1):

[0024] XLF···(1)

[0025] In this case, X is a peptide containing the amino acid sequence of the above general formula 1, L is a linker comprising an ethylene glycol repeat unit; F is an immunoglobulin Fc region, - represents a covalent bond between X and L, and between L and F.

[0026] In the above specific examples (a), in the above general formula 1, X2 is Aib (aminoisobutyric acid), X7 is threonine (T), valine (V), or cysteine ​​(C); X10 is tyrosine (Y), X12 is lysine (K), X13 is tyrosine (Y), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), X16 is glutamic acid (E) or serine (S), X17 is lysine (K), arginine (R), or cysteine ​​(C); X18 is arginine (R), X19 is alanine (A) or cysteine ​​(C), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X23 is valine (V), X24 is glutamine (Q), X27 is methionine (M), X28 is asparagine (N), X29 is threonine (T), X30 is characterized by being either cysteine ​​(C) or absent.

[0027] In the above specific example(s), the peptide is characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-45.

[0028] In the above specific example(s), the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, and 13 to 25, 27, 29, 31, 33, and 35 to 45.

[0029] In the above embodiment(s), the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44.

[0030] In the above specific example(s), the amino acids in at least one 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 characterized in that each amino acid forms a ring.

[0031] In the above specific example(s), the C-terminus of the peptide is amidated.

[0032] In the above specific example(s), the GLP-1 receptor and GIP receptor dual agonist is characterized by being a substance having activity against the GLP-1 (Glucagon-like peptide-1) receptor and the GIP (Glucose-dependent insulinotropic polypeptide) receptor.

[0033] In the above embodiment(s), the GLP-1 receptor and GIP receptor dual agonist is one or more selected from the group consisting of tirzepatide, NN9709, and SAR-438335.

[0034] In the above specific example (s), the chemical formula weight of the ethylene glycol repeating unit moiety in L is characterized by being in the range of 1 to 100 kDa.

[0035] In the above specific example(s), the metabolic syndrome is characterized in that it is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

[0036] In the above specific example(s), the liver disease is characterized as being at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, hepatic fibrosis, cirrhosis, liver failure, and liver cancer.

[0037] In the above specific example(s), the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

[0038] In the above embodiment(s), the liver disease is characterized as being caused by or associated with non-alcoholic steatohepatitis.

[0039] In the above embodiment(s), the composition is characterized by one or more of the following properties:

[0040] (a) Reduction in body weight and fat mass; (b) improvement in blood lipid levels; (c) improved insulin sensitivity; (d) decreased UCP-1 and PGC-1α gene expression; (e) reduction in NAS (NAFLD activity score); and (f) Decreased collagen expression in liver tissue.

[0041] Another embodiment of the present invention is a pharmaceutical kit for preventing or treating metabolic syndrome, comprising a substance active against the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor; or a composition containing the same.

[0042] Another embodiment of the present invention is a method for preventing or treating metabolic syndrome, comprising administering to an individual in need thereof a substance having activity at the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor; or a composition comprising same.

[0043] Another embodiment of the present invention is the use of a substance having activity at the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor; or a composition containing the same, for the prevention or treatment of metabolic syndrome.

[0044] Another embodiment of the present invention is the use of a substance having activity at the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor; or a composition containing same, in the manufacture of a medicament for the prevention or treatment of metabolic syndrome. [Effects of the Invention]

[0045] The combined administration of a substance active against the glucagon receptor and a dual agonist for the GLP-1 receptor and GIP receptor according to the present invention can be useful for the prevention or treatment of metabolic syndromes, including obesity, diabetes, and nonalcoholic steatohepatitis (NASH), and associated liver diseases. [Brief explanation of the drawings]

[0046] [Figure 1]The results show changes in body weight due to the combined administration of a long-acting glucagon derivative and tirzepatide, a dual agonist of GLP-1 receptors and GIP receptors. [Figure 2] (A) and (B) are results confirming the reduction in fat weight and blood lipids by the combined administration of a long-acting glucagon derivative and tirzepatide, a dual agonist of GLP-1 receptors and GIP receptors. [Figure 3] (A) and (B): Results confirming the increase in insulin sensitivity and UCP-1 and PGC-1α gene expression following coadministration of a long-acting glucagon derivative and tirzepatide, a dual agonist for GLP-1 and GIP receptors. [Figure 4] These results confirm the change in NAS (NAFLD activity score) following combined administration of a long-acting glucagon derivative and tirzepatide, a dual agonist of GLP-1 receptors and GIP receptors. [Figure 5] The results confirmed that the combined administration of a long-acting glucagon derivative and tirzepatide, a dual agonist of GLP-1 and GIP receptors, reduced collagen expression in liver tissue. DETAILED DESCRIPTION OF THE INVENTION

[0047] Specific details for implementing the present invention are as follows. Meanwhile, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific description below.

[0048] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein and such equivalents are intended to be encompassed by the present invention.

[0049] 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 in this specification by abbreviation are written according to the IUPAC-IUB nomenclature system.

[0050] 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

[0051] One embodiment of the present invention provides the therapeutic use of a combination of a substance having activity at the glucagon receptor and a dual agonist of the GLP-1 receptor and GIP receptor.

[0052] Specifically, one aspect of the present invention provides a composition comprising a substance active against the glucagon receptor and a dual agonist of the GLP-1 receptor and the GIP receptor. In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating metabolic syndrome.

[0053] The pharmaceutical composition of the present invention comprising (i) a substance having activity against the glucagon receptor; and (ii) a GLP-1 receptor and GIP receptor dual agonist, a) (i) a substance with activity at the glucagon receptor; and (ii) a GLP-1 receptor and GIP receptor dual agonist are administered in a single mixture; or b) (i) a substance having activity against the glucagon receptor; and (ii) a form in which the GLP-1 receptor and GIP receptor dual agonist can be administered in a separated form, but is not limited to these.

[0054] For example, the substance having activity at the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist may be formulated into a single formulation or may be formulated separately. When the substance having activity at the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist are in separate forms, the substance having activity at the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist may be formulated into separate formulations and administered simultaneously, separately, sequentially, or in the reverse order.

[0055] In the present invention, "co-administration" does not simply mean simultaneous administration, but should be understood as an administration form in which a substance active on the glucagon receptor and a dual agonist for GLP-1 and GIP receptors act together in an individual to perform the same or higher level of their inherent functions. Therefore, when the term "co-administration" is used in this application, it should be understood to refer to simultaneous, separate, sequential, or reverse-order administration of a substance active on the glucagon receptor and a dual agonist for GLP-1 and GIP receptors. When the administration is sequential, reverse, or separate, the order of administration is not particularly limited; simply, the interval between administrations of the two components should not eliminate the beneficial effects of the combination.

[0056] The substance active against the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist of the present invention, or a composition containing them, may be provided in the form of a kit, but is not limited thereto. The "kit" of the present invention may include a composition according to the present invention for co-administering a substance active against the glucagon receptor and a GLP-1 receptor and GIP receptor dual agonist. Specifically, the kit of the present invention may include a substance active against the glucagon receptor and a GLP-1 receptor and GIP receptor dual agonist formulated into a single formulation, or separate formulations of a substance active against the glucagon receptor and a GLP-1 receptor and GIP receptor dual agonist, and may further include, but is not limited to, substances necessary for co-administration of both substances.

[0057] The substances having activity on the glucagon receptor include a variety of substances, such as compounds or peptides, that have a significant level of activity on the glucagon receptor.

[0058] Although not particularly limited thereto, the substance having a significant level of activity against glucagon is not limited to native glucagon, but may also be one that exhibits in vitro activity against the glucagon receptor that is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more relative to the native ligand of the receptor (native glucagon).

[0059] Examples of the substance having activity against the glucagon receptor include, but are not limited to, natural glucagon, its agonist, or a derivative thereof.

[0060] Glucagon derivatives according to the present invention include peptides with one or more differences in amino acid sequence compared to native glucagon, peptides in which the native glucagon sequence has been altered through modification, or mimetics of native glucagon that activate the glucagon receptor in the same way as native glucagon. For example, the native glucagon derivatives are those in which one or more amino acids in native glucagon have been mutated, and the mutations may be selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof, but are not limited thereto.

[0061] Such glucagon derivatives may have an altered pI relative to native glucagon, thereby exhibiting improved physical properties. Furthermore, the glucagon derivatives may have improved solubility while retaining the activity of activating the glucagon receptor, but are not limited thereto.

[0062] The glucagon derivative may also be a non-naturally occurring one.

[0063] On the other hand, native glucagon can have the following amino acid sequence:

[0064] 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)

[0065] As used herein, the term "isoelectric point" or "pI" refers to the pH value at which a molecule such as a polypeptide or peptide has no net overall charge (0). For polypeptides containing multiple charged functional groups, the sum of these charges is zero at the pI. At a pH higher than the pI, the overall net charge of the polypeptide should be negative, and at a pH lower than the pI, the overall net charge of the polypeptide should be positive.

[0066] The pI may 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 using, for example, the pI / MW tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003).

[0067] In the present invention, the term "altered pI" means that a portion of the amino acid sequence of native glucagon is substituted with negatively and positively charged amino acid residues, resulting in a pI that is different from, i.e., is decreased or increased compared to, the pI of native glucagon. Peptides having such an altered pI are glucagon derivatives and may exhibit improved solubility and / or high stability at neutral pH. However, the present invention is not particularly limited thereto.

[0068] More specifically, the glucagon derivative may have a pI value other than that of native glucagon (6.8), more specifically, a pI value less than 6.8, specifically 6.7 or less, even more specifically 6.5 or less, or more specifically, a pI value greater than 6.8, 7 or more, even more specifically 7.5 or more, but is not limited thereto, and is included within the scope of the present invention as long as it has a pI value different from that of native glucagon. In particular, if the derivative has a pI value different from that of native glucagon and therefore exhibits improved solubility at neutral pH compared to native glucagon, and is therefore less prone to aggregation, it is particularly included within the scope of the present invention.

[0069] More specifically, the pI may be, but is not limited to, 4 to 6.5 and / or 7 to 9.5, even 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, it can exhibit improved solubility and higher stability at neutral pH compared to native glucagon. However, the pI is not limited to these.

[0070] Specifically, a derivative of native glucagon can be obtained by modifying some amino acids in the native glucagon by any one of substitution, addition, deletion and modification, or a combination of these methods.

[0071] Examples of glucagon derivatives produced by combining such 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 retain 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 multiple methods for producing derivatives.

[0072] Such modifications to produce derivatives of native glucagon also include modifications using L- or D-amino acids and / or non-naturally occurring amino acids; and / or modifications of the native sequence, such as modifications of side chain operative groups, intramolecular covalent bonds, e.g., side chain intercyclization, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. The above modifications also include substitution with non-naturally occurring compounds.

[0073] It also includes all of the additions of one or more amino acids to the amino and / or carboxy termini of native glucagon.

[0074] The substituted or added amino acids can 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 such amino acids and typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0075] Amino acid derivatives can also be obtained in a similar manner, and some examples include 4-imidazoacetic acid.

[0076] Glucagon has a pI of approximately 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 initially dissolves but precipitates by gel formation within 1 hour. Gelated glucagon consists primarily of β-sheet fibrils, and such precipitated glucagon is unsuitable for injection because it clogs the needle and clogs blood vessels when administered intravenously. To slow the precipitation process, acidic (pH 2-4) dosage forms are typically used, which maintain glucagon in a relatively aggregation-free state for a short period of time. However, because glucagon fibril formation occurs very rapidly at low pH, such acidic dosage forms must be injected immediately after preparation.

[0077] The glucagon receptor active substances of the present invention are glucagon derivatives, including those developed by altering the pI of native glucagon through substitution of negatively and positively charged amino acid residues to provide an extended profile of action, and are characterized in that such derivatives may exhibit improved solubility and / or increased stability at neutral pH due to their altered pI compared to native glucagon.

[0078] In one specific embodiment, the glucagon derivative of the present invention, which is a substance having activity against the glucagon receptor, may be a peptide comprising the amino acid sequence of the following general formula 1:

[0079] 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 46)

[0080] In the above general formula 1, X1 is tyrosine; 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 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, or cysteine, or is absent; X20 is lysine, histidine, glutamine, aspartic acid, arginine, alpha-methyl-glutamic acid, or cysteine, or is absent; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine, or is absent; X23 is isoleucine, valine, or arginine, or is absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, or 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 threonine; X30 may be cysteine ​​or absent (However, this does not include cases where the amino acid sequence of the above general formula 1 is identical to SEQ ID NO: 1 or SEQ ID NO: 12).

[0081] In addition, in the above general formula 1, X1 is tyrosine; 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, but is not limited to, cysteine ​​or may be absent (except when the amino acid sequence of General Formula 1 is identical to SEQ ID NO: 1 or SEQ ID NO: 12).

[0082] For example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, 13 to 25, 27, 29, 31, 33, and 35 to 45, and specifically may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, 13 to 25, 27, 29, 31, 33, and 35 to 45, but is not limited to this.

[0083] In addition, in the above general formula 1, X2 is Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine ​​(C); X10 is tyrosine (Y); X12 is lysine (K); X13 is tyrosine (Y); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D); X16 is glutamic acid (E) or serine (S); X17 is lysine (K), arginine (R), or cysteine ​​(C); X18 is arginine (R); X19 is alanine (A) or cysteine ​​(C); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X23 is valine (V); X24 is glutamine (Q); X27 is methionine (M); X28 is asparagine (N); X29 is threonine (T), X30 is cysteine ​​(C) or absent It may be, but is not limited to these.

[0084] For example, the peptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, but is not limited thereto.

[0085] Specifically, the peptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44, specifically, may consist (essentially) of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44, but is not limited thereto.

[0086] In addition, in the above general formula 1, In the above general formula 1 X1 is tyrosine; X2 is serine or Aib (aminoisobutyric acid); X7 is cysteine, threonine, or valine; 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 glutamic acid, lysine, arginine, cysteine, or valine; X18 is arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine or lysine; X21 is aspartic acid, glutamic acid, valine, or cysteine; X23 is valine; X24 is valine or glutamine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; X30 may be, but is not limited to, cysteine ​​or may be absent.

[0087] For example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 13 to 17, 19 to 27, 29, 31, 33, and 35 to 45, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 13 to 17, 19 to 27, 29, 31, 33, and 35 to 45, but is not limited thereto.

[0088] Also, In the above general formula 1 X1 is tyrosine; X2 is Aib (aminoisobutyric acid); X7 is cysteine, threonine, or valine; X10 is tyrosine or cysteine; X12 is lysine; 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 lysine, arginine, cysteine, or valine; X18 is arginine or cysteine; X19 is alanine or cysteine; X20 is glutamine or lysine; X21 is aspartic acid, glutamic acid, or cysteine; X23 is valine; X24 is glutamine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; X30 may be, but is not limited to, cysteine ​​or may be absent.

[0089] For example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 14, 17, 19-25, 27, 29, 31, 33, and 35-44, and specifically may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 14, 17, 19-25, 27, 29, 31, 33, and 35-44, but is not limited thereto.

[0090] In addition, in the above general formula 1 X1 is tyrosine; 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, but is not limited to, cysteine ​​or may be absent.

[0091] For example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, 13 to 15, 17, 19 to 24, 27, 29, 31, 33, and 35 to 45, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, 13 to 15, 17, 19 to 24, 27, 29, 31, 33, and 35 to 45, but is not limited thereto.

[0092] In addition, in the above 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 or arginine; X24 is glutamine or leucine; X27 is methionine; X28 is asparagine or arginine; X29 is threonine; X30 may be absent, but is not limited to this.

[0093] For example, the peptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, 31, 33, 37, and 44, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, 31, 33, 37, and 44, but is not limited thereto.

[0094] More specifically, the peptide may be a peptide comprising an amino acid sequence of the following general formula 2:

[0095] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula 2, sequence number 47)

[0096] In the above 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 may be absent.

[0097] For example, the peptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36 to 45, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36 to 45, but is not limited thereto. More specifically, the peptide is characterized by comprising or (essentially) composed of the amino acid sequence of SEQ ID NO: 20 or 37, but is not limited thereto.

[0098] Specifically, in the above general formula 2, X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine; X15 is aspartic acid; 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 glutamine; X30 may be cysteine ​​or may be absent, but is not particularly limited thereto.

[0099] For example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 to 38, 40 to 42, 44, and 45, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 to 38, 40 to 42, 44, and 45, but is not limited thereto.

[0100] Other examples of the above peptides include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11 and 13 to 45, specifically peptides that are (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11 and 13 to 45, but are not limited to these.

[0101] However, the peptides may have combinations that are excluded from the above categories, but the present invention is not particularly limited thereto, and unless otherwise specified in the claims, all peptides described in the claims are included in the scope of the present invention.

[0102] In addition, even if the present application describes a "peptide consisting of a specific sequence number," this does not exclude meaningless additions of sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof, as long as the peptide has the same or equivalent activity as a peptide consisting of the amino acid sequence of the sequence number.It is self-evident that even if the peptide has such additions or mutations of sequences, it falls within the scope of the present application.

[0103] The foregoing may also apply to other embodiments or aspects of the present invention, but is not intended to be limiting.

[0104] In the present invention, when comparing the sequence identity of the peptide with that of native glucagon, the peptide may have a sequence identity of at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more, but is not particularly limited thereto, and this can be easily ascertained by a person skilled in the art through a comparison of the sequence of the peptide with that of native glucagon.

[0105] The term "homology" as used herein refers to the degree of similarity between the amino acid sequence of a wild-type protein or the nucleotide sequence encoding it, and includes sequences that share the above percentage of identity with the amino acid sequence or nucleotide sequence of the present invention. Such homology can be determined by visually comparing the two sequences or by using a bioinformatics algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Automated alignment algorithms in these modules include the Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman alignment algorithms. Other useful algorithms for determining homology with sequences are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

[0106] In the present invention, the peptide may be, but is not limited to, a peptide exhibiting activity against a glucagon receptor or a glucagon derivative.

[0107] In particular, but not limited to, the peptide having a significant level of activity against the glucagon receptor may exhibit in vitro activity against the glucagon receptor of about 0.001% or more, about 0.01% or more, about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more relative to the natural ligand (native glucagon), and the range of significant activity is not limited thereto. For a method of measuring the in vitro activity of such peptides, see, but is not limited to, Example 4 of the present specification.

[0108] In the present invention, the term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about."

[0109] In particular, but without limitation, such peptides may be non-naturally occurring.

[0110] The glucagon derivatives described above may contain an intramolecular bridge (e.g., a covalent or non-covalent bridge), and specifically may have a ring-like structure, such as, but not limited to, a ring formed between the 16th and 20th amino acids of the glucagon derivative.

[0111] Non-limiting examples of such rings may include lactam bridges (or lactam rings).

[0112] The glucagon derivatives described above also include all those modified to include a ring, such as an amino acid capable of forming a ring at a desired position.

[0113] Such a ring may be formed between the amino acid side chains in the glucagon derivative, for example, a lactam ring may be formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0114] For example, a peptide comprising the amino acid sequence of general formula 1 or 2 may be, but is not limited to, one in which the amino acids in each amino acid pair 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 are substituted with glutamic acid or lysine, respectively. In the above, Xn (n is a natural number), n indicates the amino acid position from the N-terminus of the presented amino acid sequence.

[0115] Furthermore, in a peptide comprising the amino acid sequence of general formula 1 or 2, each of the amino acids in the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 may be substituted with glutamic acid or lysine to form a ring, but is not limited thereto.

[0116] Furthermore, in the above general formula 1 or 2, at least one 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) between each amino acid in each amino acid pair, but is not limited thereto.

[0117] Furthermore, in the above general formula 1 or 2, X16 may be glutamic acid, X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but is not limited thereto.

[0118] Furthermore, the peptides according to the present invention may have unmodified N- and / or C-termini, but also include those in which the N- and / or C-termini are chemically modified or protected with an organic group to protect them from in vivo protease activity and increase their stability, or modified by the addition of an amino acid to the peptide terminus, etc. When the C-terminus is unmodified, the terminus of the peptide according to the present invention has a carboxyl group, but is not particularly limited thereto.

[0119] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, and therefore, to remove such charges, the N-terminus may be acetylated and / or the C-terminus may be amidated, but is not limited thereto.

[0120] Unless otherwise specified herein, the detailed description and claims of the present invention regarding a "peptide" or a "conjugate" in which such a peptide is covalently linked to a biocompatible substance apply to the peptide or conjugate, as well as to a salt of the peptide or conjugate (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Therefore, even if the specification refers to a "peptide" or a "conjugate," the description also applies to a specific salt thereof, a specific solvate thereof, or a specific solvate of a specific salt thereof. Such a salt form may be, for example, a form using any pharmaceutically acceptable salt. The type of the salt is not particularly limited. However, a form that is safe and effective for individuals, e.g., mammals, is preferred, but is not particularly limited thereto.

[0121] The term "pharmaceutically acceptable" means, within the scope of medical judgment, a substance that can be effectively used for the desired purpose without inducing excessive toxicity, irritation, or allergic reaction.

[0122] As used herein, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0123] The term "solvate" used in the present invention refers to a complex formed between the peptide, conjugate, or salt thereof according to the present invention and a solvent molecule.

[0124] Furthermore, the glucagon derivative peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0125] Specifically, the glucagon derivative peptides of the present invention may be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, glucagon derivatives according to the present invention can be synthesized in a number of ways, including, for example, the following:

[0126] (a) synthesis of peptides stepwise or by fragment assembly by means of solid-phase or liquid-phase techniques, followed by isolation and purification of 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 in which peptide fragments are obtained by any combination of (a), (b) and (c), the fragments are then ligated to obtain a peptide, and the peptide is recovered.

[0127] 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, and expressed in the form of a fusion protein. The glucagon derivative can then be cleaved and separated from the fusion protein using a proteolytic enzyme or a compound to produce the desired glucagon derivative. For example, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme 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.

[0128] In a more specific embodiment, the peptide or glucagon derivative of the present invention, for example, a peptide comprising the amino acid sequence of general formula 1, may be in the form of a long-acting conjugate bound to a biocompatible material moiety that increases its in vivo half-life, but is not limited thereto. The biocompatible material moiety may be mixed with a carrier.

[0129] Specifically, the conjugate comprises a peptide moiety and a biocompatible material moiety covalently linked to the peptide moiety, and the peptide moiety may be the same as or contain the amino acid sequence of general formula 1.

[0130] In the present invention, the conjugate of the peptide can exhibit increased persistence of efficacy compared to a peptide not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" in the present invention.

[0131] Alternatively, such conjugates may be non-naturally occurring.

[0132] In the present invention, the term "long-acting conjugate" refers to a conjugate in which a bioactive substance (e.g., a glucagon derivative) is bound to a biocompatible substance or carrier, and which exhibits a longer duration of efficacy (e.g., an increased half-life in the body) compared to a bioactive substance not bound to a biocompatible substance or carrier. In the long-acting conjugate, the biocompatible substance or carrier may be covalently linked to the bioactive substance, but is not particularly limited thereto.

[0133] In a specific embodiment of the present invention, the long-acting conjugate of the glucagon derivative can have an increased duration of efficacy compared to native glucagon or a glucagon derivative not conjugated to a carrier.

[0134] In one embodiment of the present invention, the persistent conjugate may be a conjugate represented by the following chemical formula (1), but is not limited thereto.

[0135] XLF···(1)

[0136] wherein X is the peptide; L is a linker comprising an ethylene glycol repeat unit; F is an immunoglobulin Fc region, - represents a covalent bond between X and L, and between L and F.

[0137] In the long-acting conjugate, X may be a peptide (glucagon derivative) according to the present invention. Specifically, X may be a peptide comprising the amino acid sequence of general formula 1, or X may be a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 11, 13 to 45, or X may be a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, but is not limited thereto.

[0138] In the long-acting conjugate, F is a substance that increases the half-life of X, ie, the glucagon derivative, and corresponds to one of the components that constitute the conjugate of the present invention.

[0139] The F may be bonded to X via a covalent or non-covalent chemical bond, specifically, F and X may be bonded to each other via a covalent chemical bond via L.

[0140] As a specific example, the F may be an immunoglobulin Fc region, and more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.

[0141] In the present invention, the "immunoglobulin Fc region" refers to a region of an immunoglobulin excluding the heavy and light chain variable regions and including heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3). The immunoglobulin Fc region may be a component constituting a part of the protein conjugate of the present invention.

[0142] As used herein, the term "Fc fragment" encompasses not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as sequences that differ from the native sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native sequence.

[0143] The F may be a structure in which two polypeptide chains are linked by a disulfide bond, but is not limited to such a structure in which only one of the two chains is linked via a nitrogen atom. The linkage via a nitrogen atom can be via reductive amination to the epsilon-amino atom of lysine or the N-terminal amino group.

[0144] The reductive amination reaction refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to generate an amine, and then an amine bond is formed by a reduction reaction, and is an organic synthesis reaction widely known in the art.

[0145] In one specific example, but not limited to, the F may be linked via the nitrogen atom of the N-terminal proline.

[0146] Such an immunoglobulin Fc fragment can include, but is not limited to, a hinge portion in the heavy chain constant region.

[0147] In the present invention, the immunoglobulin Fc fragment may contain a specific hinge sequence at the N-terminus.

[0148] The term "hinge sequence" as used herein means a site located in a heavy chain that forms a dimer of immunoglobulin Fc fragments via inter-disulfide bonds.

[0149] In the present invention, the hinge sequence may be a mutated hinge sequence having the amino acid sequence shown below in which a portion of the hinge sequence is deleted to have only one cysteine ​​residue, but is not limited thereto.

[0150] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 51).

[0151] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 51 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention may be composed of 3 to 12 amino acids including only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence:

[0152] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 52), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 53), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 54), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-C Lys-Pro-Pro (SEQ ID NO: 55), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 56), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 57), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 58), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 59), Glu-Pro-Ser-Cys s-Pro (SEQ ID NO: 60), Pro-Ser-Cys-Pro (SEQ ID NO: 61), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 62), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 63), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 64), Glu-Ser -Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 65), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 66), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 67), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 68), Glu-Pro-Ser-Cys (SEQ ID NO: 69), Ser-Cys-Pro (SEQ ID NO: 70).

[0153] More specifically, the hinge sequence may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 61 (Pro-Ser-Cys-Pro) or SEQ ID NO: 70 (Ser-Cys-Pro).

[0154] The immunoglobulin Fc fragment of the present invention may be in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence, and the conjugate of chemical formula (1) of the present invention may be in a form in which one end of a linker is linked to a single chain of the dimeric immunoglobulin Fc fragment, but is not limited thereto.

[0155] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide, and may include the extreme amino terminus, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc fragment of the present invention may include, but is not limited to, a hinge sequence at the N-terminus.

[0156] Furthermore, the immunoglobulin Fc fragment of the present invention may be an extended Fc fragment that excludes only the heavy and light chain variable regions of the immunoglobulin and includes part or the entire heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), as long as it exhibits effects substantially equivalent to or improved from those of the native form. Alternatively, it may be a fragment in which a fairly long partial amino acid sequence corresponding to CH2 and / or CH3 has been deleted.

[0157] For example, the immunoglobulin Fc region of the present invention may be, but is not limited to, 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 of the CH1 domain, CH2 domain, CH3 domain, and CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0158] In one specific example, the immunoglobulin Fc region may be in a dimeric form, and one molecule of a glucagon derivative may be covalently linked to one Fc region of the dimeric form, and in this case, the immunoglobulin Fc and the glucagon derivative may be linked to each other via a non-peptidic polymer. Alternatively, two molecules of a glucagon derivative may be symmetrically linked to one Fc region of the dimeric form. In this case, the immunoglobulin Fc and the glucagon derivative or insulinotropic peptide may be linked to each other via a non-peptidic linker. However, the present invention is not limited to the above-described examples.

[0159] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also derivatives of these sequences, where one or more amino acid residues in the naturally occurring amino acid sequence have been deleted, inserted, non-conservative or conservative substitutions, or a combination thereof, resulting in a different sequence.

[0160] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as suitable sites for modification.

[0161] Various types of derivatives are possible, such as those in which disulfide bond formation sites are removed, several amino acids at the N-terminus of native Fc are removed, or a methionine residue is added to the N-terminus of native Fc. Furthermore, complement binding sites, such as Clq binding sites, may be removed to eliminate effector functions, and ADCC (antibody dependent cell-mediated cytotoxicity) sites may also be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, among others.

[0162] Amino acid exchanges in proteins and peptides that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, "The Proteins," Academic Press, New York, 1979). 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 be performed.

[0163] 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 against heat, pH, and the like of the Fc region.

[0164] Furthermore, such Fc regions may be obtained from natural forms isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or may be recombinant forms or derivatives thereof obtained from transformed animal cells or microorganisms. Here, obtaining natural forms may involve isolating whole immunoglobulins from the human or animal body and then treating them with protease. Treatment with papain results in cleavage into Fab and Fc, while treatment with pepsin results in cleavage into pF'c and F(ab)2. The resulting Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, the Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism, derived from a human.

[0165] Furthermore, the immunoglobulin Fc region may have native glycosylation, increased glycosylation compared to the native form, decreased glycosylation compared to the native form, or a form in which the glycosylation has been removed. Such increase, decrease, or removal of immunoglobulin Fc glycosylation may be achieved using conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Here, an immunoglobulin Fc region from which glycosylation has been removed from Fc has significantly reduced complement (Clq) binding ability and reduced or eliminated antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, and therefore does not induce unnecessary immune responses in vivo. In this respect, a form more suited to the original purpose as a drug carrier can be said to be a glycosylated or non-glycosylated immunoglobulin Fc region.

[0166] 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.

[0167] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as bovine, caprine, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment, of human origin.

[0168] Furthermore, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, the Fc region is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, the Fc region is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is an unglycosylated Fc region derived from human IgG4, but is not limited thereto.

[0169] In a specific embodiment, the immunoglobulin Fc fragment is a fragment of human IgG4 Fc and may be in the form of a homodimer in which two monomers are linked via a disulfide bond (inter-chain form) between the cysteines at amino acid number 3 of each monomer, and in this case, each monomer of the homodimer independently has / can have two internal disulfide bonds (intra-chain form), i.e., an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199. Each monomer may be composed of 221 amino acids, and the total number of amino acids forming the homodimer may be, but is not limited to, 442 amino acids. Specifically, the immunoglobulin Fc fragment may be a homodimer in which two monomers having the amino acid sequence of SEQ ID NO: 71 (consisting of 221 amino acids) form a disulfide bond between the cysteines at the third amino acids of each monomer, and the monomers of the homodimer may independently form an internal disulfide bond between the cysteines at positions 35 and 95 and an internal disulfide bond between the cysteines at positions 141 and 199, but this is not limited to this.

[0170] Meanwhile, in the present invention, the term "combination" refers to the formation of a dimer or multimer by a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin binding with a single-chain immunoglobulin of a different origin. That is, a dimer or multimer can be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0171] Alternatively, L may be a non-peptidic linker, for example, a linker containing ethylene glycol repeating units.

[0172] In the present invention, the term "non-peptidic linker" includes a biocompatible polymer in which two or more repeating units are bonded. The repeating units are linked to each other through any covalent bond, not a peptide bond. The non-peptidic linker may be a component that forms part of the conjugate of the present invention, and corresponds to L in the chemical formula (1). The non-peptidic linker that can be used in the present invention can be any polymer that is resistant to in vivo protease degradation. In the present invention, the non-peptidic linker can be used in combination with a non-peptidic polymer.

[0173] Although not limited thereto, the non-peptidic linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and derivatives thereof known in the art and derivatives that can be easily produced at the level of the art are also included within the scope of the present invention.

[0174] The repeating unit of the non-peptidic linker may be an ethylene glycol repeating unit. Specifically, the non-peptidic linker may contain an ethylene glycol repeating unit and a functional group used in preparing the conjugate at its terminal. The long-lasting conjugate according to the present invention may have a structure in which X and F are linked via the functional group, but is not limited to this. In the present invention, the non-peptidic linker may contain two or more functional groups, and the functional groups may be the same or different, but are not limited to this.

[0175] Specifically, the linker may be polyethylene glycol (PEG) represented by the following chemical formula (2), but is not limited thereto:

[0176] TIFF0007761557000001.tif1934...(2)

[0177] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited to these.

[0178] In the persistent conjugate, the PEG moiety may include not only the -(CH2CH2O)n- structure but also the oxygen atom between the linking element and this -(CH2CH2O)n-, but is not limited thereto.

[0179] In a specific embodiment, the conjugate may have a structure in which the glucagon derivative peptide or the peptide (X) comprising the amino acid sequence of general formula 1 and the immunoglobulin fragment (F) are covalently linked via a linker containing an ethylene glycol repeating unit, but is not limited thereto. The polyethylene glycol is a term that encompasses all forms of ethylene glycol homopolymer, PEG copolymer, and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto. Specifically, the chemical formula weight of the ethylene glycol repeating unit moiety in L may be, but is not limited to, in the range of 1 to 100 kDa.

[0180] The molecular weight of the nonpeptidic polymer is, but is not limited to, in the range of 1 to 100 kDa, specifically in the range of 1 to 20 kDa or in the range of 1 to 10 kDa. Furthermore, the nonpeptidic linker of the present invention that binds to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0181] In one specific embodiment, both ends of the non-peptide linker can be bonded to an amino or thiol group of F, for example, an immunoglobulin Fc fragment, and an amino or thiol group of X, respectively.

[0182] Specifically, the non-peptidic polymer may contain, at both ends, reactive groups capable of binding to F (e.g., an immunoglobulin Fc fragment) and X, respectively, specifically, reactive groups capable of binding to the amino group located at the N-terminus or lysine of X or F, or the thiol group of cysteine, but are not limited thereto.

[0183] Furthermore, the reactive group of the non-peptidic polymer that can be conjugated to F, e.g., an immunoglobulin Fc fragment, and X may be selected from the group consisting of, but is not limited to, an aldehyde group, a maleimide group, and a succinimide derivative.

[0184] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0185] In the above, examples of succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0186] The non-peptide linker can be linked to X and F via such a reactive group, but is not particularly limited thereto.

[0187] Furthermore, the final product generated by reductive amination with an aldehyde bond is much more stable than that linked by an amide bond. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form a covalent bond with lysine residues at high pH, ​​e.g., pH 9.0.

[0188] The reactive groups at both ends of the nonpeptidic linker may be the same or different, and may, for example, have a maleimide group at one end and an aldehyde group, propionaldehyde group, or butyraldehyde group at the other end, but are not particularly limited thereto, as long as F, specifically an immunoglobulin Fc fragment, and X are linked to each end of the nonpeptidic linker.

[0189] For example, the non-peptide 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.

[0190] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups can be activated with the 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.

[0191] In one specific embodiment, the non-peptidic polymer may be linked to a cysteine ​​residue of X, more specifically, but not limited to, the —SH group of cysteine.

[0192] Specifically, a reactive group of a non-peptidic polymer may be linked to the -SH group of the cysteine ​​residue, and all of the above applies to the reactive group. When maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of X through a thioether bond, and the aldehyde group can be linked to the -NH group of F, specifically, the -NH group of immunoglobulin Fc, through a reductive amination reaction, but this is just one example and is not limited thereto.

[0193] In addition, in the conjugate, the reactive group of the nonpeptidic polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc fragment, which is just one example.

[0194] Furthermore, the conjugates described above may have an increased duration of effect compared to native glucagon or compared to X in which F is not modified, and such conjugates include not only the above-described forms but also forms encapsulated in biodegradable nanoparticles, etc.

[0195] Meanwhile, the GLP-1 receptor and GIP receptor dual agonist of the present invention includes various substances, for example, compounds or peptides, that have significant levels of activity at the GLP-1 receptor and the GIP receptor. In this application, the GLP-1 receptor and GIP receptor dual agonist may be used interchangeably as a "GLP-1 / GIP dual agonist" or a "dual agonist."

[0196] Specifically, dual agonists are characterized by having one or more of the following activities i) to ii), specifically significant activities:

[0197] i) activation of the GLP-1 receptor; and ii) activation of the GIP receptor.

[0198] Although not particularly limited thereto, the GLP-1 (Glucagon-like peptide-1) receptor and GIP (Glucose-dependent insulinotropic polypeptide) receptor dual agonist is a substance having a significant level of activity against the receptors, and may exhibit in vitro activity against not only native GLP-1 and GIP but also GLP-1 receptor and GIP receptor that is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more relative to the native ligands of the receptors (native GLP-1 and GIP), and the range of a significant increase is included without limitation.

[0199] The in vitro activity of such dual agonists can be measured by measuring various in vitro activities known in the art, and examples thereof can be found in Example 2 of the present specification, but are not limited thereto.

[0200] In particular, but without limitation, such dual agonists may be non-naturally occurring.

[0201] Examples of substances active against the GLP-1 receptor and GIP receptor include, but are not limited to, naturally occurring GLP-1 and GIP, agonists thereof, and derivatives thereof. The "derivatives" are as defined above.

[0202] In particular, but not limited to, the GLP-1 receptor and GIP receptor dual agonist may be a peptide having activity at the GLP-1 receptor and the GIP receptor.

[0203] Specifically, the GLP-1 receptor and GIP receptor dual agonist of the present invention is a derivative of GLP-1 or GIP that exhibits activity at the GLP-1 receptor and the GIP receptor, and any GLP-1 receptor and GIP receptor dual agonist known in the art may be included within the scope of the present invention without limitation. The GLP-1 receptor and GIP receptor dual agonist of the present invention may be a commercially available product or one produced by a method known in the art, as long as it exhibits activity at the GLP-1 receptor and the GIP receptor. Examples of GLP-1 receptor and GIP receptor dual agonists include, but are not limited to, tirzepatide, NN9709, and SAR-438335.

[0204] Tirzepatide, an example of a GLP-1 receptor and GIP receptor dual agonist according to the present invention, is L-tyrosyl-2-methylalanyl-L-α-glutamylglycyl-L-threonyl-Lphenylalanyl-L-threonyl-L-seryl-L-α-aspartyl-L-tyrosyl-Lseryl-L-isoleucyl-2-methylalanyl-L-leucyl-L-α-aspartyl-Llysyl-L-isoleucyl-L-alanyl-L-glutaminyl-N It is known as 6-[(22S)-22,42-dicarboxy-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazadotetracontan-1-oyl]-L-lysyl-L-alanyl-L-phenylalanyl-Lvalyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-isoleucyl-Lalanylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-Lprolyl-L-prolyl-L-prolyl-L-serinamide (CAS# 2023788-19-2) and has the following sequence:

[0205] Y X EGTFTSDY SI X LDKIAQ K AFVQWLIAGG PSSGAPPP S (SEQ ID NO: 50)

[0206] where: X , K and S are residues that have been modified as follows:

[0207] JPEG0007761557000002.jpg51165

[0208] Meanwhile, although not particularly limited thereto, the GLP-1 receptor and GIP receptor dual agonist of the present invention may have a terminally modified form, specifically, one terminal of the dual agonist may be acylated or amidated, but is not limited thereto.

[0209] Depending on the length, the peptide of the present invention can be synthesized by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0210] Specifically, the peptides of the present invention may be produced by standard synthetic methods, recombinant expression systems, or any of a variety of methods known in the art. Thus, peptides according to the present invention can be synthesized in a number of ways, including, for example, the following:

[0211] (a) synthesis of peptides by means of solid-phase or solution-phase techniques, either stepwise or by fragment assembly, followed by isolation and purification of 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), subsequently ligating the fragments to obtain a peptide, and recovering the peptide.

[0212] As a more specific example, a polynucleotide encoding a peptide can be prepared through genetic engineering, and then transformed into a host cell to produce the desired peptide.

[0213] Although not particularly limited thereto, the composition of the present invention may include glucagon derivativehas an in vitro activity of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the activity of the native ligand of the glucagon receptor, and in the case of a GLP-1 receptor and GIP receptor dual agonist, The GLP-1 activity of the in vitro activity of the natural ligand of the receptor is about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more The GIP receptor activity may be, but is not limited to, about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more. derivativehas an in vitro activity of about 0.1% to about 300% or less, about 2% to about 300% or less, about 3% to about 300% or less, about 4% to about 300% or less, about 5% to about 300% or less, about 6% to about 300% or less, about 7% to about 300% or less, about 8% to about 300% or less, about 9% to about 300% or less, or about 10% to about 300% or less of the activity of a natural ligand of the glucagon receptor. % or less, about 20% to about 300%, about 30% to about 300%, about 40% to about 300%, about 50% to about 300%, about 60% to about 300%, about 70% to about 300%, about 80% to about 300%, about 90% to about 300%, and about 100% to about 300%; in the case of a GLP-1 receptor and GIP receptor dual agonist, The GLP-1 activity in vitro relative to the activity of the natural ligand for the receptor is about 0.1% to about 200%; about 2% to about 200%; about 3% to about 200%; about 4% to about 200%; about 5% to about 200%; about 6% to about 200%; about 7% to about 200%; about 8% to about 200%; about 9% to about 200%; about 10% to about 200%; about 20% to about 200%; about 30% to about 200%; about 40% to about 200%; about 50% to about 200%; about 60% to about 200%; about 70% to about 200%; about 80% to about 200%; about 90% to about 200%; and about 100% to about 200%. In particular, the GIP receptor activity may be, but is not limited to, about 0.1% to about 300%; about 2% to about 300%; about 3% to about 300%; about 4% to about 300%; about 5% to about 300%; about 6% to about 300%; about 7% to about 300%; about 8% to about 300%; about 9% to about 300%; about 10% to about 300%; about 20% to about 300%; about 30% to about 300%; about 40% to about 300%; about 50% to about 300%; about 60% to about 300%; about 70% to about 300%; about 80% to about 300%; about 90% to about 300%; or about 100% to about 300%.

[0214] The composition of the present invention can be used to prevent or treat metabolic syndrome.

[0215] In the present invention, the term "prevention" means any action that suppresses or delays the onset of a target disease, such as metabolic syndrome, by administering (i) a substance having glucagon activity, and (ii) a GLP-1 receptor and GIP receptor dual agonist; or a composition containing the same; and "treatment" means any action that improves or provides benefit to the symptoms of a target disease, such as metabolic syndrome, by administering (i) a substance having glucagon activity, and (ii) a GLP-1 receptor and GIP receptor dual agonist; or a composition containing the same.

[0216] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition is not particularly limited, but may be any common route by which the composition can reach its target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0217] Substances with glucagon activity and dual agonists of the GLP-1 and GIP receptors can be used to prevent or treat metabolic syndrome.

[0218] The combined administration of (i) a substance having glucagon activity and (ii) a dual agonist of the GLP-1 receptor and GIP receptor of the present invention is effective not only in preventing weight gain, promoting weight loss, reducing overweight, and treating obesity, including morbid obesity (e.g., by regulating appetite, feeding, food intake, calorie intake, and / or energy expenditure), but also in diseases including obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. The combined administration of (i) a substance having glucagon activity and (ii) a dual agonist of the GLP-1 receptor and GIP receptor of the present invention can also be effective in treating metabolic syndrome or obesity-related liver disease, and can be used as a drug to treat related diseases and conditions, including, but not limited to, these. The substances having glucagon activity and dual agonists of the GLP-1 receptor and GIP receptor of the present invention can also be used to treat metabolic syndrome-related diseases other than obesity, such as impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis due to dyslipidemia, atherosclerosis, coronary heart disease, and stroke, etc. However, the effects of the substances having glucagon activity and dual agonists of the GLP-1 receptor and GIP receptor of the present invention on these pathological conditions may be mediated in whole or in part through the weight-related effects described above, or may be independent thereof.

[0219] The term "metabolic syndrome" as used herein refers to a condition in which various diseases resulting from chronic metabolic disorders occur singly 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, liver disease, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

[0220] In the present invention, the term "obesity" refers to a state in which there is an excess of adipose tissue in the body, and is defined as a body mass index (body weight (kg) divided by the square of height (m)) of 25 or greater. Obesity is typically induced by energy imbalance, which occurs when nutrients are consumed in excess relative to energy expenditure over a long period of time. Obesity is a metabolic disease that affects the entire body, and increases the likelihood of developing diabetes and hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular disease, and in some cases is associated with the development of cancer. Treatment of obesity can be achieved through, but is not limited to, the reduction of body weight and body fat.

[0221] In the present invention, the term "hyperlipidemia" refers to a condition in which lipids, such as free cholesterol, cholesterol esters, phospholipids, and triglycerides, are abnormally elevated in the blood. While hyperlipidemia generally does not itself exhibit specific symptoms, excess lipids in the blood adhere to the walls of blood vessels, reducing their size and causing atherosclerosis through an inflammatory response. This can lead to coronary heart disease, cerebrovascular disease, and peripheral vascular occlusion. Furthermore, excess lipids in the blood can accumulate in liver tissue, resulting in fatty liver. Fatty liver, as defined above, refers to a condition in which fat accounts for more than 5% of the liver's weight and can be induced by not only excessive fat intake but also alcohol consumption. Treatment of hyperlipidemia can involve, but is not limited to, improving blood lipid levels.

[0222] In the present invention, the term "diabetes" refers to a metabolic disease characterized by insufficient insulin secretion or impaired insulin function, characterized by hyperglycemia (high blood glucose levels). Recently, the incidence of diabetes has been increasing explosively due to the rise in obesity rates, particularly abdominal obesity. If chronic hyperglycemia is not properly treated, it can lead to various pathological symptoms, including retinal disease, renal dysfunction, neuropathy, vascular disease-related stroke, kidney and heart disease, diabetic foot ulcers, and an increased risk of cardiovascular disease. Diabetes can be treated by improving blood glucose levels through improved insulin sensitivity, but this is not limited to these.

[0223] The metabolic syndrome described above is closely related to liver diseases that lead to fat accumulation in liver tissue and the resulting inflammation and fibrosis, and examples of the metabolic syndrome can include various liver diseases.

[0224] Therefore, the composition of the present invention may be used for the prevention or treatment of liver diseases. Specifically, a composition according to the present invention comprising (i) a substance having glucagon activity and (ii) a GLP-1 receptor and GIP receptor dual agonist exhibits the effect of suppressing and ameliorating inflammation and / or fibrosis in liver tissue, and may be used for the prevention or treatment of liver diseases, but is not limited thereto.

[0225] In the present invention, "liver disease" refers to a disease that develops in the liver, and may include, but is not limited to, metabolic liver disease. Representative examples of liver diseases include simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, hepatic fibrosis, cirrhosis, liver failure, and liver cancer. As long as abnormalities occur in the liver tissue and function, they may be considered liver diseases according to the present invention. In addition to alcohol consumption, drugs, and viral infections, liver inflammation can also be caused by obesity, metabolic disorders, etc., and it is known that the progression and chronicity of liver inflammation can lead to diseases such as cirrhosis and liver cancer.

[0226] Specifically, the composition according to the present invention comprising (i) a substance having glucagon activity and (ii) a dual agonist of GLP-1 receptor and GIP receptor can exhibit preventive or therapeutic effects against liver diseases associated with or caused by metabolic syndrome.

[0227] The liver diseases for which the composition according to the present invention comprising (i) a substance having glucagon activity and (ii) a GLP-1 receptor and GIP receptor dual agonist has a therapeutic effect may include, but are not limited to, metabolic liver diseases, which are diseases caused by abnormal chemical reactions in the body that interfere with the body's metabolism, and include simple steatosis, fatty liver, steatohepatitis, non-alcoholic fatty liver disease, etc.

[0228] In the present invention, "nonalcoholic fatty liver disease (NAFLD)" refers to a case in which fatty liver is present despite no history of alcohol consumption and no relationship to alcohol intake. Fatty liver refers to the abnormal deposition of triglycerides within hepatocytes, which differs from normal cases. A normal liver is composed of approximately 5% adipose tissue, with triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters being the main components of fat. However, once fatty liver develops, most components are replaced by triglycerides, and fatty liver is diagnosed when the amount of triglycerides is 5% or more of the liver weight. Fatty liver is caused by impaired fat metabolism within hepatocytes or defects in the process of transporting excess fat, and is primarily caused by impaired fat metabolism in the liver. The majority of the fat accumulated in the fatty liver may be triglycerides.

[0229] Non-alcoholic fatty liver disease refers to a group of diseases including simple steatosis, which is characterized by excessive accumulation of fat in hepatocytes, non-alcoholic fatty liver, and non-alcoholic steatohepatitis (NASH), which is accompanied by hepatocyte necrosis, inflammation, and fibrosis, but is not limited thereto as long as it can be treated with the composition of the present invention. Non-alcoholic fatty liver disease according to the present invention may be accompanied by non-alcoholic steatohepatitis, but is not limited thereto.

[0230] Furthermore, the liver disease for which the composition comprising (i) a substance having glucagon activity and (ii) a GLP-1 receptor and GIP receptor dual agonist according to the present invention has a therapeutic effect may be, but is not limited to, liver inflammation. In the present invention, "liver inflammation" refers to a disease that causes inflammation in the liver, which is the most common cause of liver disease. It is classified into acute hepatitis and chronic hepatitis depending on the cause and symptoms. The main causes include viruses, alcohol, drugs, immune disorders, and metabolic disorders.

[0231] The composition according to the present invention, which comprises (i) a substance having glucagon activity and (ii) a dual agonist of GLP-1 receptor and GIP receptor, not only has the effect of reducing liver inflammation itself, but also has an effect on diseases that accompany or are caused by liver inflammation, such as hepatitis, non-alcoholic steatohepatitis, and hepatic fibrosis.

[0232] In the present invention, "non-alcoholic steatohepatitis" is a type of non-alcoholic fatty liver disease and a representative example of a liver disease accompanied by hepatocyte necrosis, inflammation, and fibrosis. A composition according to the present invention comprising (i) a substance having glucagon activity and (ii) a GLP-1 receptor and GIP receptor dual agonist can suppress liver inflammation and fibrosis and exhibit an effect on non-alcoholic steatohepatitis, specifically, but not limited to, non-alcoholic steatohepatitis accompanied by fatty liver, liver fibrosis, or cirrhosis; or liver cancer caused by non-alcoholic steatohepatitis.

[0233] Specifically, the composition of the present invention comprising (i) a substance having glucagon activity and (ii) a dual agonist of the GLP-1 receptor and the GIP receptor exhibits a NAS (NAFLD activity score) reducing effect, which indicates a therapeutic effect against nonalcoholic steatohepatitis.

[0234] In the present invention, "liver fibrosis" refers to the result of the injury recovery process following repeated liver injury, resulting in the formation of excessive fibrous connective tissue in organs and tissues during the reparative and reactive process. Chronic and deepening liver inflammation is known to be one of the causes of liver fibrosis. Unlike cirrhosis, liver fibrosis is reversible, composed of thin fibrils, and lacks nodule formation. Once the cause of liver injury is eliminated, liver fibrosis may return to normal. However, repeated progression of this process leads to increased crosslinking within the extracellular matrix (ECM), resulting in irreversible liver cirrhosis with nodules. The composition according to the present invention may exhibit preventive or therapeutic effects on liver fibrosis, specifically, but not limited to, liver fibrosis associated with nonalcoholic steatohepatitis.

[0235] Specifically, the composition of the present invention comprising (i) a substance having glucagon activity and (ii) a dual agonist of the GLP-1 receptor and the GIP receptor can be effective against liver fibrosis, specifically, it may reduce collagen expression in liver tissue and prevent or treat liver fibrosis.

[0236] In the present invention, "cholestasis" refers to a condition in which the flow of bile from the liver to the duodenum is slowed or blocked, and "cholestasis liver disease" refers to a condition in which bile formation in the liver is disrupted by conditions such as various diseases, extended jugular vein feeding, or the side effects of certain medications (e.g., some antibiotics). Common symptoms of cholestasis include fatigue, pruritus (itching), jaundice, and xanthomas (the accumulation of cholesterol-rich substances under the skin). The effects of cholestasis are extreme and widespread, leading to the progression of liver disease to systemic disease, liver failure, and the need for liver transplantation. Causes of cholestatic liver disease include acute hepatitis and inflammation of the bile ducts.

[0237] The cholestatic liver diseases include, but are not limited to, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), and Alagille syndrome (AS).

[0238] Primary biliary cirrhosis (PBC), also known as primary biliary cholangitis (PBC), is a chronic cholestatic liver disease of unknown etiology. Progressive bile duct damage due to portal and periportal inflammation can lead to progressive fibrosis and eventual cirrhosis. To date, immunological, genetic, and environmental factors have been identified as potential causes of the disease. PBC primarily affects middle-aged women, and initial symptoms may include fatigue, itching, or unexplained hyperlipidemia.

[0239] To date, primary biliary sclerosis is known to be an immune-mediated disease. Specifically, immunohistochemical staining of T lymphocytes in portal and periportal areas reveals CD4-positive and CD8-negative T cells. Abnormal suppressor T cell activity has also been reported in asymptomatic first-grade relatives of affected individuals. Interleukins have been reported to play a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis (GJ Webb et al., J. Autoimmunity, 2015 Nov; 64:42-52).

[0240] The treatment of PBC is bile acid therapy using ursodeoxycholic acid (UDSA) and obeticholic acid (OCA). The mechanism of action of both drugs in PBC is related to their ability to activate FXR and TGFR-5, exerting anti-inflammatory effects. However, approximately 40% of patients treated with UDCA did not achieve a satisfactory biochemical response.

[0241] Primary sclerosing cholangitis (PSC) is a chronic, progressive cholestatic liver disease caused by inflammation and fibrosis of the intrahepatic and extrahepatic biliary tract of unknown etiology. Specifically, as the disease progresses as an inflammatory disease of the bile duct and biliary tract, fibrosis occurs, causing the bile duct wall to thicken and narrow or become strictured. Although the cause is still unknown, it is suspected to involve a complex mix of factors, including genetic and environmental factors, and associated immune responses.

[0242] If blood liver function tests reveal elevated alkaline phosphatase levels, elevated aminotransferase levels, and gammaglobulinemia, the patient is diagnosed with primary sclerosing cholangitis.

[0243] No clear treatment for PSC has yet been reported, and liver transplantation is the only treatment that can fundamentally cure the disease.

[0244] Therefore, there is still a need to develop drugs that can treat PBS and PSC while ensuring patient convenience and without side effects.

[0245] The "liver cirrhosis" of the present invention is a chronic disease that develops through repeated hepatocyte regeneration and fibrous tissue growth, and is pathologically accompanied by necrosis, inflammation, and fibrosis. It ultimately progresses to complications of liver cirrhosis, such as liver failure, or liver cancer, leading to death. In particular, since liver fibrosis has no noticeable symptoms in the early stages and is only detected at a fairly advanced stage, it is necessary to quickly treat liver fibrosis, which is a state before it evolves into liver cirrhosis. The composition of the present invention may exhibit preventive or therapeutic effects on liver cirrhosis, specifically, liver cirrhosis associated with non-alcoholic steatohepatitis, but is not limited thereto.

[0246] "Liver decompensation" in the present invention refers to a condition in which the liver is unable to perform normal physiological functions such as protein synthesis and metabolic functions due to weakened liver function caused by liver damage or liver disease such as viral hepatitis, cirrhosis, drugs, or alcohol. It is classified as acute liver failure or chronic liver failure depending on the rate of progression, and is known to cause various complications. The composition according to the present invention exhibits effects such as inhibiting inflammation and fibrosis, and therefore can be effective in preventing or treating liver failure.

[0247] "Hepatocellular carcinoma" in the present invention refers to a malignant tumor derived from hepatocytes. It can be divided into primary hepatic cancer (hepatocellular carcinoma), which arises from hepatic cells themselves, and metastatic hepatic cancer, which occurs when cancer from other tissues metastasizes to the liver. Approximately 90% of liver cancers are primary hepatic cancers. Known causes include hepatitis, chronic liver disease, alcohol, smoking, and obesity. The composition according to the present invention can exhibit preventive or therapeutic effects against liver cancer, specifically, liver cancer caused by non-alcoholic steatohepatitis, but is not limited to these.

[0248] In the examples of the present invention, high-fat diet-induced obesity mice and CD-HFD (choline-deficient, high-fat, high-cholesterol) diet-induced mouse models were used. The CD-HFD diet-induced model has high fat and cholesterol content, and long-term intake of the diet can induce fatty liver and steatohepatitis. Choline deficiency is known to further exacerbate steatohepatitis and even induce fibrosis.

[0249] In the examples of the present invention, the effects of the composition of the present invention comprising (i) a substance having glucagon activity and (ii) a GLP-1 receptor and GIP receptor dual agonist were confirmed in each of the above models, suggesting that the composition is useful for the prevention or treatment of liver diseases such as metabolic syndrome, hepatic fibrosis, simple steatosis, fatty liver, and non-alcoholic steatohepatitis.

[0250] Compositions according to the present invention may exhibit one or more of the following properties, but are not limited to:

[0251] (a) Reduction in body weight and fat mass; (b) improvement in blood lipid levels; (c) improved insulin sensitivity; (d) decreased UCP-1 and PGC-1α gene expression; (e) reduction in NAS (NAFLD Activity Score); and (f) Decreased collagen expression in liver tissue.

[0252] The composition according to the present invention can be characterized by not causing weight gain, which is a side effect of existing drugs for treating liver diseases, or by causing a relatively low level of weight gain.

[0253] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. The term "pharmaceutically acceptable" as used herein means a sufficient amount to exhibit a therapeutic effect and not cause side effects, and can be easily determined by those skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity to the drug, administration route, administration method, administration frequency, treatment period, and drugs used in combination or concomitantly.

[0254] The pharmaceutical composition of the present invention comprising a substance having glucagon activity and a GLP-1 receptor and GIP receptor dual agonist may further comprise a pharmaceutically acceptable carrier. The carrier is not particularly limited to, but may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavorings, etc. for oral administration, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. for injection, and bases, excipients, lubricants, preservatives, etc. for topical administration.

[0255] The composition of the present invention may be prepared in various dosage forms by mixing with the above-mentioned pharmaceutically acceptable carriers. 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-dose forms. It may also be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0256] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. Furthermore, fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, preservatives, etc. may also be included.

[0257] 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.

[0258] Furthermore, the composition may be formulated into a unit dosage form suitable for administration into the body of a patient by a method conventional in the pharmaceutical field, specifically, into a dosage form useful for administering peptide pharmaceuticals, and administered orally or parenterally using an administration method conventionally used in the art, including, but not limited to, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes.

[0259] In addition, the above-mentioned substances having glucagon activity and GLP-1 receptor and GIP receptor dual agonists are used by mixing them with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and to increase stability and absorbability, 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 are used.

[0260] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined depending on the type of drug as an active ingredient, as well as various 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.

[0261] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the above-mentioned ingredient (active ingredient) in an amount of 0.01 to 99% weight to volume.

[0262] The total effective amount of the composition of the present invention can 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 vary in the content of the active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the substance having glucagon activity and the GLP-1 receptor and GIP receptor dual agonist of the present invention may be about 0.0001 μg to 500 mg per kg of patient body weight per day.

[0263] Specifically, the composition of the present invention may contain the substance having glucagon activity at 0.15 to 2.5, 0.19 to 2.25, 0.25 to 1.5, or 0.37 to 1.12 nmol / kg and the GLP-1 receptor and GIP receptor dual agonist at 2.0 to 35, 2.08 to 31.16, or 10.39 to 31.16 nmol / kg; more specifically, the composition may contain the substance having glucagon activity at 0.37, 0.75, or 1.12 nmol / kg and the GLP-1 receptor and GIP receptor dual agonist at 10.39, 20.77, or 31.16 nmol / kg, but is not limited to these. Furthermore, the composition of the present invention may contain the substance having glucagon activity:GLP-1 receptor and GIP receptor dual agonist at a molar ratio of 1:0.1 to 1:500, 1:0.5 to 1:250, 1:0.9 to 1:167, or 1:10 to 1:56.2, but is not limited thereto.

[0264] However, the effective dose of the peptide is determined by taking into consideration various factors such as the administration route of the pharmaceutical composition and the number of treatments, as well as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate, and taking these factors into consideration, a person skilled in the art would be able to determine an appropriate effective dose depending on the 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, or administration method, as long as it exhibits the effects of the present invention.

[0265] The pharmaceutical composition of the present invention has excellent in vivo durability and potency, and can be administered less frequently and less frequently than other drugs, but is not particularly limited thereto.

[0266] In particular, the pharmaceutical compositions of the present invention contain as an active ingredient a glucagon derivative having an altered pI compared to native glucagon, and therefore exhibit improved solubility and / or high stability at neutral pH, making them useful for producing stable glucagon dosage forms for the treatment of target diseases, including metabolic syndrome.

[0267] The pharmaceutical composition for preventing or treating metabolic syndrome or the therapy for preventing or treating metabolic syndrome may contain a compound or substance having therapeutic activity against metabolic syndrome in addition to the substance having activity against the glucagon receptor and / or the GLP-1 receptor and GIP receptor dual agonist, and the therapy may include the additional use of the compound or substance.

[0268] Another aspect of the present invention provides a method for preventing or treating metabolic syndrome, comprising administering to an individual in need thereof a substance having activity at the glucagon receptor; and a GLP-1 receptor and GIP receptor dual agonist.

[0269] The substance having activity against the glucagon receptor, the GLP-1 receptor and GIP receptor dual agonist, the composition containing the same, and the metabolic syndrome, prevention and treatment are as described above.

[0270] In the present invention, the individual is an individual suspected of having metabolic syndrome, and the individual suspected of having metabolic syndrome refers to mammals including rats, livestock, and the like, including humans, who are suffering from or may suffer from the disease. and GLP-1 receptor and GIP receptor dual agonist Alternatively, individuals who can be treated with the composition containing the composition include, without limitation, the following: Furthermore, by administering the pharmaceutical composition of the present invention to an individual suspected of having metabolic syndrome, the individual can be effectively treated; metabolic syndrome is as described above.

[0271] The method of the present invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising (i) a substance active at the glucagon receptor and (ii) a dual agonist of the GLP-1 and GIP receptors. The method of the present invention may involve administering (i) a substance active at the glucagon receptor and (ii) a dual agonist of the GLP-1 and GIP receptors in a single formulation, or may involve administering separate formulations simultaneously, separately, sequentially, or in reverse order, but is not limited thereto.

[0272] The appropriate total daily dose will be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the time and route of administration, the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concomitantly with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0273] Specifically, the substance having activity against the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist may each be administered in an amount of about 0.0001 mg to 500 mg per kg of patient body weight per day, and when both substances are used in combination, the total amount may be administered in an amount of about 0.0001 mg to 1000 mg per kg of patient body weight per day, but is not limited to this.

[0274] Furthermore, the substance having activity against the glucagon receptor and the GLP-1 receptor and GIP receptor dual agonist to be administered in combination can be administered at a molar ratio of 1:0.01 to 1:100, but is not limited thereto.

[0275] Specifically, the substance having glucagon activity may be administered at 0.15 to 2.5, 0.19 to 2.25, 0.25 to 1.5, or 0.37 to 1.12 nmol / kg, and the GLP-1 receptor and GIP receptor dual agonist may be administered at 2.0 to 35, 2.08 to 31.16, or 10.39 to 31.16 nmol / kg; more specifically, the substance having glucagon activity may be administered at 0.37, 0.75, or 1.12 nmol / kg, and the GLP-1 receptor and GIP receptor dual agonist may be administered at 10.39, 20.77, or 31.16 nmol / kg, but is not limited to this. Alternatively, the substance having glucagon activity and the GLP-1 receptor and GIP receptor dual agonist may be administered at a molar ratio of 1:0.1 to 1:500, 1:0.5 to 1:250, 1:0.9 to 1:167, or 1:10 to 1:1:56.2, but is not limited to these.

[0276] Meanwhile, the method for preventing or treating metabolic syndrome may be a combination therapy that further includes administering one or more compounds or substances having therapeutic activity against metabolic syndrome in addition to (i) a substance having activity against the glucagon receptor and (ii) a GLP-1 receptor and GIP receptor dual agonist, but is not limited thereto.

[0277] Another embodiment of the present invention provides use of a composition comprising (i) a substance active at the glucagon receptor and (ii) a GLP-1 receptor and GIP receptor dual agonist for the prevention or treatment of metabolic syndrome.

[0278] Another aspect of the present invention provides use of a composition comprising (i) a substance having activity at the glucagon receptor, and (ii) a GLP-1 receptor and GIP receptor dual agonist, for the manufacture of a medicament for the prevention or treatment of metabolic syndrome.

[0279] The present invention will be described in more detail with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0280] Example 1: Production of cell lines that exhibit a cAMP response to glucagon PCR was performed using a portion corresponding to the ORF in the cDNA of the human glucagon receptor gene (OriGene Technologies, Inc., USA) as a template and forward and reverse primers of SEQ ID NOs: 48 and 49 below, which contain an EcoRI cleavage site and an XhoI cleavage site, respectively.

[0281] The PCR reaction consisted of denaturation at 95°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 68°C for 30 seconds, repeated 30 times. The amplified PCR product was electrophoresed on a 1.0% agarose gel, and a 450 bp band was isolated.

[0282] Forward primer (SEQ ID NO: 48): 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' Reverse primer (SEQ ID NO: 49): 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3'

[0283] The PCR product was cloned into a known animal cell expression vector, xoGc / dhfr, to prepare a recombinant vector, x0GC / GCGR.

[0284] The recombinant vector xOGC / GCGR was transformed into CHO DG44 cells cultured in 10% FBS-containing DMEM / F12 medium using Lipofectamine, followed by selective culture in a selective medium containing 1 mg / mL G418 and 10 nM methotrexate. Monoclonal cell lines were selected by limiting dilution, and a cell line showing a superior concentration-dependent cAMP response to glucagon was finally selected.

[0285] Example 2: Synthesis of glucagon derivatives To develop glucagon derivatives with improved physical properties, the amino acid sequence of natural glucagon of SEQ ID NO: 1 was substituted with negatively and positively charged amino acid residues to synthesize the glucagon derivatives shown in Table 1 below. The relative in vitro activities listed therein were measured by the method described in Example 4 below.

[0286] [Table 1] TIFF0007761557000004.tif141167

[0287] In the sequences listed in Table 1 above, the amino acid marked X represents the unnatural amino acid aminoisobutyric acid (Aib), the underline in the amino acid symbol represents the formation of a lactam ring between the side chains of the underlined amino acid pair, and "-" represents the absence of an amino acid residue at that position. In addition, "-" in the column for whether or not ring formation occurs indicates that the sequence does not form a ring.

[0288] Example 3: pI measurement of glucagon derivatives To confirm the improved physical properties of the glucagon derivative synthesized in Example 2 above, 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).

[0289] As shown in Table 1 above, native glucagon of SEQ ID NO: 1 has a pI of 6.8, while some glucagon derivatives according to the present invention have a pI in the range of about 4 to 6. Such glucagon derivatives have a lower or higher pI than native glucagon, and therefore may exhibit improved solubility and higher stability at neutral pH, etc., compared to native glucagon.

[0290] The glucagon derivatives according to the present invention can improve patient compliance when used as therapeutic agents for target diseases such as metabolic syndrome, and are suitable for combined administration with other anti-obesity therapeutic agents or anti-diabetic agents. They can be useful as therapeutic agents for metabolic syndromes including obesity, diabetes, nonalcoholic steatohepatitis (NASH), dyslipidemia, and coronary heart disease.

[0291] Example 4: Measurement of cAMP activity of glucagon derivatives The activity of the glucagon derivative synthesized in Example 2 was measured using the cell line having the human glucagon receptor produced in Example 1. Specifically, the transformed cell line was subcultured three or four times a week, and then cultured in a 384-well plate with 6 × 10 cells per well. 3The subcultured cell lines were divided into aliquots and cultured for 24 hours. Native glucagon was suspended at 200 nM and glucagon derivatives 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). The solutions were serially diluted 10 times, 4-fold, and added to the cells using a cAMP assay kit (LANCE cAMP 384 kit, PerkinElmer). The fluorescence intensity was measured. The highest fluorescence intensity was set at 100%, and the EC of the glucagon derivatives was calculated from this value. 50 The values ​​were calculated and compared with those of natural glucagon, and the results are shown in Table 1 above.

[0292] Example 5: Preparation of a conjugate containing a glucagon derivative and immunoglobulin Fc (glucagon derivative-immunoglobulin Fc region conjugate) A representative glucagon derivative with a pI value of 6-7 and an in vitro activity of 200% or greater, as prepared in Example 2, was selected and used to prepare a conjugate. Specifically, a 10 kDa PEG bearing a maleimide group and an aldehyde group at each end, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), was used to pegylate the cysteine ​​residue of the glucagon derivative. 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 50 mM Tris buffer (pH 7.5) containing 20-60% isopropanol. After completion of the reaction, the reaction mixture was applied to SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative mono-pegylated at the cysteine ​​residue.

[0293] Next, the purified mono-pegylated glucagon derivative and immunoglobulin Fc (homodimer of SEQ ID NO: 71) 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 mixture was incubated in 100 mM potassium phosphate buffer (pH 6.0) supplemented with 10-50 mM sodium cyanoborohydride (reducing agent) and 10-20% isopropanol. After the reaction was completed, the reaction mixture 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.

[0294] Meanwhile, the immunoglobulin Fc is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 71 (consisting of 221 amino acids) through a disulfide bond between the cysteines at the third amino acids of each monomer, and the monomers of the homodimer independently form internal disulfide bonds between the cysteines at the 35th and 95th positions and between the cysteines at the 141st and 199th positions.

[0295] After preparation, the purity was determined to be 95% or higher by reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography.

[0296] Herein, a conjugate in which a glucagon derivative and an immunoglobulin Fc are linked via PEG is named a "long-acting glucagon derivative conjugate" or a "long-acting glucagon derivative," and these terms may be used interchangeably in the present application.

[0297] Example 6: Production of cell lines that exhibit cAMP response to GLP-1 and GIP Recombinant vectors for expressing the human GLP-1 receptor and the human GIP receptor were constructed using the expression vector X0GC / dhfr, and then transformed into CHO DG44 cells using Lipofectamine. These cells were then selectively cultured in a selective medium containing G418 and methotrexate. Monoclonal cell lines were then selected by limiting dilution, and cell lines showing excellent concentration-dependent cAMP responses to GLP-1 and GIP were finally selected.

[0298] Example 7: Preparation of acylated GLP-1 receptor and GIP receptor dual agonists Tirzepatide, a dual agonist active at both the GLP-1 receptor and the GIP receptor, was synthesized, and its production method was described in detail in WO2016-111971 A1.

[0299] Example 8: Measurement of cAMP activity of acylated GLP-1 receptor and GIP receptor dual agonists The activity of the dual agonist synthesized in Example 7 was measured in the cell line expressing the human GLP-1 receptor and the human GIP receptor produced in Example 6.

[0300] Specifically, the transformed cell line of Example 6 was subcultured three or four times a week, and then cultured in a 384-well plate at 6 × 10 cells per well. 3The subcultured cell lines were divided into aliquots and cultured for 24 hours. Native GLP-1 and GIP were suspended at 200 nM, and the dual agonists 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). The solutions were serially diluted 10 times, 4-fold, and added to the cells using a cAMP assay kit (LANCE cAMP 384 kit, PerkinElmer). The fluorescence intensity was then measured. The fluorescence intensity of the highest concentration of the native substance used in each cell line was set at 100%, and the EC values ​​of the dual agonists were calculated from this. 50 The values ​​were calculated and compared with those of natural GLP-1 and GIP. The results are shown in Table 2 below.

[0301] [Table 2]

[0302] Experimental Example 1: Comparison of the effects of long-acting glucagon derivatives and dual agonists on reducing body weight and fat mass, improving blood lipid levels, improving insulin sensitivity, and increasing energy metabolism genes in high-fat diet-induced obese mice, and confirmation of additional efficacy by combined administration High-fat diet-induced obese mice, a widely used animal model of obesity, were used in this study. Mice weighed approximately 50-55 g before administration. Mice were housed seven animals per group and had free access to water during the study period. Lights were off from 6 PM to 6 AM.

[0303] The high-fat diet-fed test groups were: Group 1: vehicle-free control group (5 ml / kg, injected once every other day); Group 2: GLP-1 analogue obesity treatment Saxenda (registered trademark) 50 nmol / kg (injected twice daily); Group 3: long-acting glucagon derivative 2.0 nmol / kg (injected once every other day); Group 4: dual agonist (tirzepatide) 20 nmol / kg (injected once every other day); and Group 5: dual agonist 20 nmol / kg (injected once every other day) combined with long-acting glucagon derivative 2.0 nmol / kg (injected once every other day). All administrations were subcutaneously administered.

[0304] The experiment was completed on the 21st day. During the experiment, changes in the body weight of the mice in each group were measured every two days. After the experiment was completed, fat mass, blood lipid levels, HOMA-IR, and the expression of genes related to energy metabolism in the tissues were confirmed.

[0305] As shown in Figure 1, the weight change measurements showed that the group administered with only the long-acting glucagon derivative (2.0 nmol / kg, administered once every other day) and the group administered with the combination of the long-acting glucagon derivative (2.0 nmol / kg, administered once every other day) and the dual agonist (tirzepatide) 20 nmol / kg (injected once every other day) lost weight by -34.92% and -51.16%, respectively, compared to before administration, demonstrating superior weight loss efficacy in the combination group compared to the group administered with only the long-acting glucagon derivative. This effect was superior to the weight loss efficacy of the control group (Vehicle), the GLP-1 analogue obesity drug Saxenda (registered trademark), and the dual agonist (tirzepatide), which were 2.22%, -14.73%, and -19.39%, respectively.

[0306] As can be seen in Figures 2(A) and (B), significant decreases in fat mass and blood lipid levels were observed along with weight loss, and HOMA-IR measurements confirmed improved insulin sensitivity as shown in Figure 3(A). Furthermore, as can be seen in Figure 3(B), increased expression of energy metabolism-related genes (UCP-1, a marker related to thermoenergy production, and PGC-1α, a marker related to mitochondrial biogenesis) in adipose tissue was also confirmed.

[0307] Experimental Example 2: Effects of long-acting glucagon derivatives and dual agonists on NASH and fibrosis in mice with choline deficiency and high-fat, high-cholesterol diet-induced NASH and fibrosis To confirm the efficacy of the combined use of the long-acting glucagon derivatives prepared according to the present invention and the GLP-1 / GIP dual agonist (tirzaptide) in improving NASH and fibrosis, a CD-HFD (choline-deficient, high-fat, high-cholesterol) diet mouse model was used.

[0308] Mice were induced on a CD-HFD diet for 8 weeks and divided into three groups: a vehicle control group, a group treated with a long-acting glucagon analog (1.3 nmol / kg, Q2D, subcutaneous), a group treated with a dual agonist (73 nmol / kg, Q2D, subcutaneous), and a group treated with a combination of a long-acting glucagon analog and a dual agonist. These groups underwent repeated administration for 6 weeks. A negative control group was treated with a vehicle-administered normal diet. After 6 weeks of repeated administration, liver tissue was collected from each mouse at autopsy, and the NASH and fibrosis improvement effects were evaluated by measuring NAS (NAFLD activity score) by H&E staining and collagen expression by quantitative PCR.

[0309] As a result, it was confirmed that repeated administration of a long-acting glucagon derivative or dual agonist for 6 weeks significantly reduced NAS compared to the CD-HFD diet and vehicle control groups.

[0310] Furthermore, it was confirmed that co-administration of a long-acting glucagon derivative and a dual agonist resulted in a significant additional reduction in NASH (Figure 4), confirming that the NASH-improving efficacy of a long-acting glucagon derivative and a GLP-1 / GIP dual agonist can be further improved through co-administration.

[0311] Furthermore, we confirmed that collagen expression in liver tissue, which was increased in the CD-HFD and vehicle groups, was significantly reduced by administration of a long-acting glucagon derivative or dual agonist, and that this reduction was further improved by concomitant administration of a long-acting glucagon derivative or dual agonist (Figure 5).

[0312] We confirmed that the combined administration of a long-acting glucagon derivative and a GLP-1 / GIP dual agonist can significantly improve NASH and liver fibrosis in CD-HFD mice through the effects of reducing NAS and collagen expression in liver tissue.

[0313] All statistical analyses were performed using one-way ANOVA to compare the vehicle group (control group) and the test group, and t-tests to compare the additive efficacy of the combined administration group compared to the single administration group (*~***p<0.05~0.001 vs. vehicle control group).

[0314] These results suggest that long-acting glucagon derivatives exhibit superior weight loss efficacy compared to other obesity treatments, such as GLP-1 analogs and dual agonists. Furthermore, co-administration of long-acting glucagon derivatives with a GLP-1 receptor and GIP receptor dual agonist not only enhances obesity treatment efficacy through additional weight loss, but also improves insulin sensitivity and blood glucose levels. Furthermore, in addition to these obesity and blood glucose improvement effects, the co-administration of long-acting glucagon derivatives with a GLP-1 receptor and GIP receptor dual agonist also demonstrated superior efficacy in liver diseases such as nonalcoholic steatohepatitis and resulting liver fibrosis through reductions in NAS and collagen expression in liver tissue.

[0315] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and all modifications and variations derived from the equivalent concepts thereof, rather than the above detailed description. Another aspect of the present invention may be as follows. [1] A pharmaceutical composition for preventing or treating metabolic syndrome, comprising (i) a substance active against a glucagon receptor and (ii) a GLP-1 receptor and GIP receptor dual agonist, The substance having activity against the glucagon receptor is 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 46) In the above formula, X1 is tyrosine (Y); X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine; X7 is threonine (T), valine (V), or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X13 is tyrosine (Y) or cysteine ​​(C); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C); X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methyl-glutamic acid, or cysteine ​​(C), or is absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), alpha-methyl-glutamic acid, or cysteine ​​(C), or is absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methyl-glutamic acid, or leucine (L), or is absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X29 is threonine (T), X30 is cysteine ​​(C) or absent. (However, this does not include cases where the amino acid sequence of the above general formula 1 is identical to SEQ ID NO: 1 or SEQ ID NO: 12). [2] The composition according to [1] above, wherein the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following chemical formula (1): [ka] In this case, X is a peptide containing the amino acid sequence of the above general formula 1; L is a linker comprising an ethylene glycol repeat unit; F is an immunoglobulin Fc region, - represents a covalent bond between X and L, and between L and F. [3] In the above general formula 1, X2 is Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine ​​(C); X10 is tyrosine (Y); X12 is lysine (K); X13 is tyrosine (Y); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D); X16 is glutamic acid (E) or serine (S); X17 is lysine (K), arginine (R), or cysteine ​​(C); X18 is arginine (R); X19 is alanine (A) or cysteine ​​(C); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X23 is valine (V); X24 is glutamine (Q); X27 is methionine (M); X28 is asparagine (N); X29 is threonine (T); X30 is cysteine ​​(C), The composition described in [1] or [2] above. [4] The composition according to [1], wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, and 13 to 25, 27, 29, 31, 33, and 35 to 45. [5] The composition described in [3], wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44. [6] The composition according to [1], wherein in at least one amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in General Formula 1, the respective amino acids form a ring. [7] The composition described in [1], wherein the C-terminus of the peptide is amidated. [8] The composition described in [1], wherein the GLP-1 receptor and GIP receptor dual agonist is a peptide having activity against the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic polypeptide) receptor. [9] The composition described in [1], wherein the GLP-1 receptor and GIP receptor dual agonist is one or more selected from the group consisting of tirzepatide, NN9709, and SAR-438335.

[10] The composition according to [2] above, wherein the chemical formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

[11] The composition described in [1], wherein the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

[12] The composition according to

[11] , wherein the liver disease is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, hepatic fibrosis, cirrhosis, liver failure, and liver cancer.

[13] The composition described in

[12] above, wherein the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

[14] The composition according to

[12] , wherein the liver disease is caused by or is accompanied by non-alcoholic steatohepatitis.

[15] The composition according to [1], wherein the composition exhibits one or more of the following properties: (a) Reduction in body weight and fat mass; (b) improvement in blood lipid levels; (c) improved insulin sensitivity; (d) decreased UCP-1 and PGC-1α gene expression; (e) reduction in NAS (NAFLD Activity Score); and (f) Decreased collagen expression in liver tissue.

Claims

1. A pharmaceutical composition for preventing or treating metabolic syndrome, comprising (i) a substance having activity against the glucagon receptor and (ii) a GLP-1 receptor and GIP receptor dual agonist, the substance having activity against the glucagon receptor is a peptide comprising the amino acid sequence of SEQ ID NO: 37; The composition, wherein the GLP-1 receptor and GIP receptor dual agonist is tirzepatide.

2. The composition according to claim 1, wherein the peptide is in the form of a persistent conjugate, and the persistent conjugate is represented by the following chemical formula (1): 【Chemistry 1】 wherein X is a peptide comprising the amino acid sequence of SEQ ID NO: 37; L is a linker comprising an ethylene glycol repeat unit; F is an immunoglobulin Fc region, - represents a covalent bond between X and L, and between L and F.

3. The composition of claim 1 , wherein the C-terminus of the peptide is amidated.

4. 2. The composition according to claim 1, wherein the GLP-1 receptor and GIP receptor dual agonist is a peptide having activity at the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic polypeptide) receptor.

5. The composition according to claim 2, wherein the chemical formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

6. 2. The composition of claim 1, wherein the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke.

7. 7. The composition according to claim 6, wherein the liver disease is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, hepatic fibrosis, liver cirrhosis, liver failure, and liver cancer.

8. 8. The composition according to claim 7, wherein the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

9. 8. The composition of claim 7, wherein the liver disease is caused by or associated with non-alcoholic steatohepatitis.

10. The composition of claim 1 , wherein the composition exhibits one or more of the following properties: (a) reduction in body and fat mass; (b) improvement of blood lipid levels; (c) improving insulin sensitivity; (d) decreased UCP-1 and PGC-1α gene expression; (e) a decrease in NAS (NAFLD Activity Score); and (f) Decreased collagen expression in liver tissue.

11. 10. Use of the composition of claim 1 for the manufacture of a medicament for preventing or treating metabolic syndrome.

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