Compositions comprising glucagon and dual agonists of the GLP-1 receptor and the GIP receptor and therapeutic uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to compositions comprising compounds or substances exhibiting glucagon activity and dual agonists of GLP-1 and GIP receptors, and their therapeutic uses. Background Technology

[0002] In recent years, with economic development and changes in dietary habits, the incidence of metabolic syndrome-related diseases, including obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, or liver disease, has increased rapidly. These diseases may occur individually, but are usually closely related to each other and, in most cases, occur alongside various symptoms.

[0003] Overweight and obesity increase blood pressure and cholesterol levels, which are causes or exacerbates various diseases such as heart disease, diabetes, and arthritis. Furthermore, overweight and obesity are major contributing factors to increased incidence of arteriosclerosis, hypertension, hyperlipidemia, or heart disease in children, adolescents, and adults.

[0004] Obesity is a complex disease involving mechanisms of appetite control and energy metabolism. Therefore, treatments targeting abnormal mechanisms related to appetite control and energy metabolism must be pursued simultaneously, and efforts to develop drugs that address these abnormal mechanisms continue. Through these efforts, anti-obesity drugs such as rimonabant (Sanofi-Aventis), sibutramine (Abbott), naltrexone hydrochloride and bupropion hydrochloride extended-release tablets (Contrave) (Takeda), and orlistat (Roche) have been developed. However, these drugs have drawbacks, including potentially fatal side effects or insufficient efficacy in treating obesity. For example, rimonabant has been reported to cause central nervous system side effects, sibutramine and naltrexone hydrochloride and bupropion hydrochloride extended-release tablets have shown cardiovascular side effects, and orlistat showed only about 4 kg of weight loss after one year of use.

[0005] Meanwhile, metabolic syndrome, including obesity, increases the likelihood of developing liver disease. Examples include metabolic liver disease, fatty liver, non-alcoholic fatty liver disease, steatohepatitis, and liver fibrosis. These liver diseases are increasing with the rise in obesity and diabetes populations, and the annual incidence rate in South Korea is approximately 16%. Liver disease often presents no symptoms in its early stages and is often only detected at a relatively late stage, making it a leading cause of death not only in South Korea but worldwide. Therefore, there is an urgent need to develop drugs to treat liver disease.

[0006] Meanwhile, glucagon is produced in the pancreas when blood sugar begins to drop due to medication, disease, hormone deficiency, or enzyme deficiency. Glucagon is responsible for signaling the liver to break down glycogen to release glucose and raise blood sugar levels back to normal. However, its 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 from the pancreas in a glucose concentration-dependent manner and inhibits glucagon secretion to help lower blood sugar levels. GLP-1 also acts as a satiety factor, slowing gastrointestinal digestion and delaying the time it takes for food to pass through the digestive tract, thereby reducing food intake.

[0008] Like GLP-1, GIP is a gastrointestinal hormone secreted in response to food intake. It is a hormone composed of 42 amino acids secreted by K cells in the small intestine and promotes insulin secretion from the pancreas in a glucose concentration-dependent manner, thus helping to lower blood sugar levels. GIP has been reported to increase GLP-1 activity, has anti-inflammatory effects, and improves lipid metabolism.

[0009] Therefore, research is actively underway to develop therapeutic agents for diabetes and obesity by utilizing the glycemic control and weight loss effects of GLP-1. However, GLP-1 alone has shown only a 0.5% to 1.8% reduction in HbA1c, and is therefore considered suitable for patients with HbA1c of 9% or lower (Ther Adv Endocrinol Metab. 2015 Feb; 6(1):3–18). In other words, the expected glycemic control ability is limited due to the use of GLP-1 alone.

[0010] Therefore, dual agonists capable of simultaneously activating GLP-1 and GIP receptors have been investigated, and examples of such dual agonists are described in WO 2013164483, WO 201611971, WO 2014192284, etc.

[0011] Dual agonists of glucagon, GLP-1, and GIP receptors are generally known to have opposing effects and have been used to treat different symptoms of the disease. In other words, dual agonists of glucagon, GLP-1, and GIP receptors act oppositely in the body, and there are no reports of drug therapy involving the simultaneous administration of these drugs. Furthermore, when patients are given various treatments associated with metabolic syndrome, there is a risk of side effects such as weight gain, overdose, and hypoglycemia. Summary of the Invention

[0012] [Technical Issues]

[0013] The present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic syndrome, comprising a substance exhibiting activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors.

[0014] Another object of the present invention is to provide a method for the prevention or treatment of metabolic syndrome, comprising administering to an individual in need a substance that exhibits activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors.

[0015] Another object of the present invention is to provide substances that exhibit activity against glucagon receptors and the use of dual agonists of GLP-1 and GIP receptors in the prevention or treatment of metabolic syndrome.

[0016] [Technical Solution]

[0017] One aspect of the invention is the therapeutic use of substances that exhibit activity against the glucagon receptor and combinations of dual agonists of the GLP-1 and GIP receptors.

[0018] In one embodiment, the present invention relates to a composition comprising a substance exhibiting activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors.

[0019] In the foregoing embodiments, the present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic syndrome, comprising a substance exhibiting activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors.

[0020] In one or more of the foregoing embodiments, the substance exhibiting activity against the glucagon receptor is a peptide comprising an amino acid sequence represented by Formula 1:

[0021] X1–X2–QGTF–X7–SD–X10–S–X12–X13–X14–X15–X16–X17–X18–X19–X20–X21–F –

[0022] Where X1 is tyrosine (Y);

[0023] X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine;

[0024] X7 is threonine (T), valine (V), or cysteine ​​(C);

[0025] X10 is either tyrosine (Y) or cysteine ​​(C);

[0026] X12 is either lysine (K) or cysteine ​​(C);

[0027] X13 is either tyrosine (Y) or cysteine ​​(C);

[0028] X14 is either leucine (L) or cysteine ​​(C);

[0029] X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C);

[0030] X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid or cysteine ​​(C), or it may not be present.

[0031] X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or it may not be present.

[0032] X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or it may not be present.

[0033] X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or it may not be present.

[0034] X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, or cysteine ​​(C), or it may not be present.

[0035] X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or it may not be present.

[0036] X23 is isoleucine (I), valine (V), or arginine (R), or it may not be present.

[0037] X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid, or leucine (L), or it may not be present.

[0038] X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or it may not be present.

[0039] X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or it may not be present.

[0040] X29 is threonine (T); and

[0041] X30 is either cysteine ​​(C) or absent.

[0042] (This is not included if the amino acid sequence represented by Formula 1 is the same as SEQ ID NO:1 or SEQ ID NO:12).

[0043] In the foregoing embodiments (one or more), the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following chemical formula 1:

[0044] [Chemical Formula 1]

[0045] XLF

[0046] Where X is a peptide comprising the amino acid sequence represented by Formula 1;

[0047] L is a linker containing repeating ethylene glycol units;

[0048] F is the Fc region of immunoglobulins; and

[0049] - represents the covalent bond connection between X and L, and between L and F.

[0050] In the aforementioned embodiments (one or more), in Formula 1,

[0051] X2 is Aib (aminoisobutyric acid);

[0052] X7 is threonine (T), valine (V), or cysteine ​​(C);

[0053] X10 is tyrosine (Y);

[0054] X12 is lysine (K);

[0055] X13 is tyrosine (Y);

[0056] X14 is either leucine (L) or cysteine ​​(C);

[0057] X15 is aspartic acid (D);

[0058] X16 is either glutamic acid (E) or serine (S);

[0059] X17 is lysine (K), arginine (R), or cysteine ​​(C);

[0060] X18 is arginine (R);

[0061] X19 is either alanine (A) or cysteine ​​(C);

[0062] X20 is either glutamine (Q) or lysine (K);

[0063] X21 is either aspartic acid (D) or glutamic acid (E);

[0064] X23 is valine (V);

[0065] X24 is glutamine (Q);

[0066] X27 is methionine (M);

[0067] X28 is asparagine (N);

[0068] X29 is threonine (T); and

[0069] X30 is either cysteine ​​(C) or absent.

[0070] In one or more of the foregoing embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NOs:2 to 45.

[0071] In the foregoing embodiments (one or more), the peptide comprises an amino acid sequence selected from SEQ ID NOs:7 to 11, 13 to 25, 27, 29, 31, 33, and 35 to 45.

[0072] In the foregoing embodiments (one or more), the peptide comprises an amino acid sequence selected from SEQ ID NOs:20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44.

[0073] In the aforementioned embodiments (one or more), each amino acid forms a ring 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 Formula 1.

[0074] In one or more of the foregoing embodiments, the C-terminus of the peptide is amidated.

[0075] In the aforementioned embodiments (one or more), the GLP-1 and GIP receptor dual agonist is a substance that exhibits activity against both the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic peptide) receptor.

[0076] In the aforementioned embodiments (one or more), the dual agonist of GLP-1 and GIP receptors is selected from one or more of tirzepatide, NN9709, and SAR-438335.

[0077] In the aforementioned embodiments (one or more), the chemical formula weight of the repeating unit portion of ethylene glycol in L is in the range of 1 kDa to 100 kDa.

[0078] In the aforementioned embodiments (one or more), metabolic syndrome is selected from glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease (coronary artery disease), and stroke.

[0079] In the aforementioned embodiments (one or more), liver disease is at least one disease selected from simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, liver fibrosis, cirrhosis, liver decompensation, and hepatocellular carcinoma.

[0080] In the aforementioned embodiments (one or more), cholestatic liver disease is selected from primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

[0081] In the aforementioned embodiments (one or more), the liver disease is caused by or accompanied by non-alcoholic steatohepatitis.

[0082] In the foregoing embodiments (one or more), the composition performs one or more of the following properties:

[0083] (a) Weight and fat weight loss;

[0084] (b) Improves blood lipid levels;

[0085] (c) Improves insulin sensitivity;

[0086] (d) Reduce the expression of UCP-1 and PGC-1α genes;

[0087] (e) Reduce NAS (NAFLD Activity Score); and

[0088] (f) Reduce the expression of collagen in liver tissue.

[0089] Another aspect of the invention is a pharmaceutical kit for the prevention or treatment of metabolic syndrome, comprising a substance exhibiting activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors; or a composition comprising these.

[0090] Another aspect of the invention is a method for preventing or treating metabolic syndrome, comprising administering to an individual in need a substance that exhibits activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors; or administering a composition comprising these substances.

[0091] Another aspect of the invention is the use of substances that exhibit activity against glucagon receptors and dual agonists of GLP-1 and GIP receptors; or compositions comprising these for the prevention or treatment of metabolic syndrome.

[0092] Another aspect of the invention is the use of substances that exhibit activity against glucagon receptors and dual agonists of GLP-1 and GIP receptors; or compositions comprising these, for the preparation of a medicament for the prevention or treatment of metabolic syndrome.

[0093] [Beneficial Effects]

[0094] The combination of the substance exhibiting glucagon receptor activity according to the invention and a dual agonist of GLP-1 and GIP receptors can be effectively used for the prevention or treatment of metabolic syndromes, including obesity, diabetes, and non-alcoholic steatohepatitis (NASH), and related liver diseases. Attached Figure Description

[0095] Figure 1 The results of weight changes were confirmed by combining the administration of a long-acting glucagon derivative and taripeptide (a dual agonist of GLP-1 and GIP receptors).

[0096] Figure 2 (A) and Figure 2 (B) The results of reduced fat weight and blood lipids were confirmed by the combined administration of long-acting glucagon derivatives and taripeptide (a dual agonist of GLP-1 and GIP receptors);

[0097] Figure 3 (A) and Figure 3 (B) The results of confirming increased insulin sensitivity and UCP-1 and PGC-1α gene expression by combining long-acting glucagon derivatives and taripeptide (a dual agonist of GLP-1 and GIP receptors);

[0098] Figure 4 The results of changes in NAS (NAFLD activity score) were confirmed by combining the administration of a long-acting glucagon derivative and taripeptide (a dual agonist of GLP-1 and GIP receptors); and

[0099] Figure 5The results confirmed the reduced collagen expression in liver tissue by combining the administration of a long-acting glucagon derivative and taripeptide (a dual agonist of GLP-1 and GIP receptors).

[0100] Specific details for carrying out the invention will now be described. Each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of the invention. Furthermore, the scope of the invention is not limited to the following specific description.

[0101] Furthermore, those skilled in the art will recognize many equivalents of the specific aspects of the invention described herein, or which can be determined using only conventional experiments. Moreover, these equivalents should be construed as falling within the scope of this invention.

[0102] Throughout this specification, not only are the standard single-letter and three-letter codes used for naturally occurring amino acids employed, but also the commonly accepted three-letter codes used for other amino acids, such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine) are used. Amino acids referred to herein by abbreviations according to the IUPAC-IUB nomenclature are also described.

[0103] Alanine A Arginine R

[0104] Asparagine N, aspartic acid D

[0105] Cysteine ​​C Glutamate E

[0106] Glutamine Q Glycine G

[0107] Histidine H Isoleucine I

[0108] Leucine (L-Lysine) (K-L-L)

[0109] Methionine (M) Phenylalanine (F)

[0110] Proline (P) Serine (S)

[0111] Threonine (T) and Tryptophan (W)

[0112] Tyrosine Y Valine V

[0113] One aspect of the invention provides the therapeutic use of substances that exhibit activity against glucagon receptors and combinations of dual agonists of GLP-1 and GIP receptors.

[0114] Specifically, one aspect of the invention provides a composition comprising a substance exhibiting activity against glucagon receptors; and a dual agonist of GLP-1 and GIP receptors. In one embodiment, a pharmaceutical composition for the prevention or treatment of metabolic syndrome is provided.

[0115] Pharmaceutical compositions comprising (i) a substance exhibiting activity against glucagon receptors and (ii) the dual agonist of GLP-1 and GIP receptors of the present invention may be in form a), wherein (i) the substance exhibiting activity against glucagon receptors; and (ii) the dual agonist of GLP-1 and GIP receptors may be administered as a mixture; or

[0116] Form b), wherein (i) a substance exhibiting activity against glucagon receptors; and (ii) a dual agonist of GLP-1 and GIP receptors may be administered in either form, but is not limited thereto.

[0117] For example, substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, can be formulated as a single preparation or individually. When substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, are in individual forms, they can be formulated as individual preparations and administered simultaneously, individually, sequentially, or in reverse order.

[0118] In this invention, combination administration not only means simultaneous administration, but is also understood as a dosage form in which a substance exhibiting activity against the glucagon receptor, along with a dual agonist of GLP-1 and GIP receptors, acts synergistically on an individual, such that each of these can function at a level equal to or greater than its intended function. Therefore, as used herein, the term "combination administration" is understood to refer to the simultaneous, individual, sequential, or reverse administration of a substance exhibiting activity against the glucagon receptor, along with a dual agonist of GLP-1 and GIP receptors. When the administration sequence is sequential, reverse, or individual, the order of administration is not particularly limited, but the interval between administrations of the second component is required to ensure that the beneficial effects of combination administration are not lost.

[0119] Substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, or compositions comprising these of the present invention, may be provided in the form of kits, but are not limited thereto. As used herein, the term "kit" may include compositions according to the present invention for combined administration of substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors. Specifically, kits according to the present invention may comprise substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, formulated as a single formulation, or substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, and may additionally comprise substances required for combined administration of the two substances, but are not limited thereto.

[0120] Substances exhibiting activity against glucagon receptors include a variety of substances that show significant levels of activity against glucagon receptors, such as substances in compound or peptide form.

[0121] Although not specifically limited thereto, substances that exhibit significant levels of activity against the glucagon receptor are not only natural glucagon, but can also be substances that exhibit in vitro activity against the glucagon receptor at 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, or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

[0122] Examples of substances that exhibit activity against glucagon receptors include, but are not particularly limited to, natural glucagon, its agonists or derivatives thereof.

[0123] The glucagon derivatives according to the present invention include peptides having one or more differences in amino acid sequence compared to natural glucagon, peptides wherein the natural glucagon sequence is modified, and natural glucagon mimics capable of activating glucagon receptors like natural glucagon. For example, a derivative of natural glucagon is a derivative in which one or more amino acids are mutated from natural glucagon, and the mutation can be selected from, but is not particularly limited to, substitution, addition, deletion, modification, and any combination thereof.

[0124] These glucagon derivatives may have a modified pI relative to natural glucagon to exhibit improved physical properties. Glucagon derivatives may exhibit improved solubility while maintaining, but are not limited to, their activity in activating glucagon receptors.

[0125] Glucagon derivatives can be non-natural glucagon derivatives.

[0126] Natural glucagon may have the following amino acid sequence:

[0127] His–Ser–Gln–Gly–Thr–Phe–Thr–Ser–Asp–Tyr–Ser–Lys–Tyr–Leu–Asp–Ser–Arg–Arg–

[0128] Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1).

[0129] 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 charge (0). For polypeptides containing various charged functional groups, the sum of these charges at pI is zero. When the pH is above pI, the total net charge of the polypeptide will be negative, and when the pH is below pI, the total net charge of the polypeptide will be positive.

[0130] The pI can be determined by isoelectric point electrophoresis on a fixed pH gradient gel composed of polyacrylamide, starch, or agarose, or by estimating the pI from the amino acid sequence, for example, using the pI / MW tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003).

[0131] As used herein, the term "altered pI" refers to the substitution of certain sequences in the amino acid sequence of natural glucagon with negatively and positively charged amino acid residues to have a pI different from that of natural glucagon—that is, a pI that is reduced or increased compared to the pI of natural glucagon. Peptides with such altered pIs can exhibit improved solubility and / or high stability as glucagon derivatives at neutral pH. However, peptides are not particularly limited to this.

[0132] More specifically, glucagon derivatives may have altered pI values ​​instead of the pI value of natural glucagon (6.8), more specifically, pI values ​​less than 6.8, specifically 6.7 or less, more specifically 6.5 or less, and pI values ​​specifically greater than 6.8, 7 or more, more specifically 7.5 or more, but are not limited thereto. Any glucagon derivative having a pI value different from that of natural glucagon is included within the scope of this invention. Specifically, any glucagon derivative having a pI value different from that of natural glucagon is particularly included within the scope of this invention, thus exhibiting improved solubility compared to natural glucagon at neutral pH, and therefore exhibiting low aggregation.

[0133] More specifically, the pI value of glucagon derivatives can be 4 to 6.5, and / or 7 to 9.5, more specifically 7.5 to 9.5, more specifically 8.0 to 9.3, but is not limited thereto. In this case, the pI of the glucagon derivative is higher or lower than that of natural glucagon, and therefore, at neutral pH, the glucagon derivative may exhibit improved solubility and higher stability compared to natural glucagon. However, glucagon derivatives are not limited thereto.

[0134] Specifically, derivatives of natural glucagon can be modified by any one of the methods or any combination of the substitution, addition, deletion or modification of some amino acids in natural glucagon.

[0135] Examples of glucagon derivatives prepared by combination of these methods include, but are not limited to, one or more amino acid sequences having an amino acid sequence different from that of natural glucagon, deamination at N-terminal amino acid residues, and peptides that activate glucagon receptors. Natural glucagon derivatives used in this invention can be prepared by combining various methods for preparing derivatives.

[0136] Such modifications used in the preparation of natural glucagon derivatives include all modifications using L- or D-amino acids, and / or unnatural amino acids; and / or modifications of the natural sequence, such as modifications of side-chain functional groups and intramolecular covalent bonds (e.g., interchain ring formation), methylation, acylation, ubiquitination, phosphorylation, aminohexylation, and biotinylation. These modifications include all substitutions with unnatural compounds.

[0137] Modifications include all modifications in which one or more amino acids are added to the amino and / or carboxyl termini of natural glucagon.

[0138] As substitutes or additions, not only the 20 amino acids commonly found in human proteins can be used, but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized by commercial peptide synthesis companies such as American Peptide or Bachem in the United States, or Anygen in South Korea, and can be purchased from them.

[0139] Amino acid derivatives can also be produced in the same way, and to name just a few examples, 4-imidazolidineacetic acid and analogues can be used.

[0140] Glucagon has a pI of approximately 7, making it insoluble in solutions at physiological pH (pH 4 to pH 8) and prone to precipitation at neutral pH. In aqueous solutions at pH 3 or lower, glucagon initially dissolves but precipitates within one hour due to gel formation. Gelated glucagon consists primarily of β-lamellae fibrils, and therefore precipitated glucagon is unsuitable for use as an injectable formulation because the gel can clog blood vessels when administered intravenously or via a needle. To delay the precipitation process, acidic formulations (pH 2 to pH 4) are typically used, which allow glucagon to remain in a relatively non-aggregated state for a short period. However, because fibril formation of glucagon occurs very rapidly at low pH values, such acidic formulations need to be injected immediately after preparation.

[0141] Substances exhibiting activity against the glucagon receptor of the present invention are glucagon derivatives, including those developed by altering the pI of natural glucagon through the substitution of negatively and positively charged amino acid residues, thereby creating glucagon derivatives with extended action profiles. Compared to natural glucagon at neutral pH, these derivatives have altered pIs and therefore may exhibit improved solubility and / or higher stability.

[0142] As a specific aspect, a glucagon derivative, which is a substance that exhibits activity against the glucagon receptor of the present invention, may be a peptide comprising the amino acid sequence represented by Formula 1 below.

[0143] X1–X2–QGTF–X7–SD–X10–S–X12–X13–X14–X15–X16–X17–X18–X19–X20–X21–F –

[0144] In Equation 1,

[0145] X1 can be tyrosine;

[0146] X2 can be α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine, or D-serine;

[0147] X7 can be threonine, valine, or cysteine;

[0148] X10 can be tyrosine or cysteine;

[0149] X12 can be lysine or cysteine;

[0150] X13 can be tyrosine or cysteine;

[0151] X14 can be leucine or cysteine;

[0152] X15 can be aspartic acid, glutamic acid, or cysteine;

[0153] X16 can be glutamic acid, aspartic acid, serine, α-methylglutamic acid, cysteine, or it may not be present.

[0154] X17 can be aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or it may not be present.

[0155] X18 can be alanine, aspartic acid, glutamic acid, arginine, valine, or cysteine, or it may not be present.

[0156] X19 can be alanine, arginine, serine, valine, or cysteine, or it may not be present.

[0157] X20 can be lysine, histidine, glutamine, aspartic acid, arginine, α-methylglutamic acid, or cysteine, or it may not be present.

[0158] X21 can be aspartic acid, glutamic acid, leucine, valine, or cysteine, or it may not be present.

[0159] X23 can be isoleucine, valine, or arginine, or it may not be present.

[0160] X24 can be valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methylglutamic acid, or leucine, or it may not exist.

[0161] X27 can be isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or it may not be present.

[0162] X28 can be glutamine, lysine, asparagine, or arginine, or it may not be present.

[0163] X29 could be threonine; and

[0164] X30 may be cysteine ​​or may not be present (provided that the amino acid sequence represented by Formula 1 is identical to SEQ ID NO:1 or SEQ ID NO:12, in which case it is not included).

[0165] In Equation 1,

[0166] X1 can be tyrosine;

[0167] X2 can be serine or Aib (aminoisobutyric acid);

[0168] X7 can be threonine, valine, or cysteine;

[0169] X10 can be tyrosine or cysteine;

[0170] X12 can be lysine or cysteine;

[0171] X13 can be tyrosine or cysteine;

[0172] X14 can be leucine or cysteine;

[0173] X15 can be aspartic acid or cysteine;

[0174] X16 can be glutamic acid, serine, or cysteine;

[0175] X17 can be aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine;

[0176] X18 can be aspartic acid, glutamic acid, arginine, or cysteine;

[0177] X19 can be alanine or cysteine;

[0178] X20 can be glutamine, aspartic acid, lysine, or cysteine;

[0179] X21 can be aspartic acid, glutamic acid, leucine, valine, or cysteine;

[0180] X23 can be isoleucine, valine, or arginine;

[0181] X24 can be valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine;

[0182] X27 can be isoleucine, valine, alanine, methionine, glutamine, or arginine;

[0183] X28 can be glutamine, lysine, asparagine, or arginine;

[0184] X29 could be threonine; and

[0185] X30 may be cysteine ​​or absent, but glucagon derivatives are not limited to this (provided that when the amino acid sequence represented by Formula 1 is the same as SEQ ID NO:1 or SEQ ID NO:12, it is not included).

[0186] For example, a peptide may include, but is not limited to, an amino acid sequence selected from SEQ ID NOs:7 to 11, SEQ ID NOs:13 to 25, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 45, and specifically (substantially) may consist of an amino acid sequence selected from, but is not limited to, SEQ ID NOs:7 to 11, SEQ ID NOs:13 to 25, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 45.

[0187] In Equation 1,

[0188] X2 can be Aib (aminoisobutyric acid);

[0189] X7 can be threonine (T), valine (V), or cysteine ​​(C);

[0190] X10 can be tyrosine (Y);

[0191] X12 can be lysine (K);

[0192] X13 can be tyrosine (Y);

[0193] X14 can be leucine (L) or cysteine ​​(C);

[0194] X15 can be aspartic acid (D);

[0195] X16 can be glutamic acid (E) or serine (S);

[0196] X17 can be lysine (K), arginine (R), or cysteine ​​(C);

[0197] X18 can be arginine (R);

[0198] X19 can be either alanine (A) or cysteine ​​(C);

[0199] X20 can be glutamine (Q) or lysine (K);

[0200] X21 can be aspartic acid (D) or glutamic acid (E);

[0201] X23 can be valine (V);

[0202] X24 can be glutamine (Q);

[0203] X27 can be methionine (M);

[0204] X28 can be asparagine (N);

[0205] X29 can be threonine (T); and

[0206] X30 can be cysteine ​​(C) or absent, but glucagon derivatives are not limited to this.

[0207] For example, a peptide may include an amino acid sequence selected from SEQ ID NOs:20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42 and 44, and may specifically (mainly) consist of an amino acid sequence selected from SEQ ID NOs:20, 22, 23, 27, 33, 35, 38, 40, 41, 42 and 44, but is not limited thereto.

[0208] Specifically, the peptide may include, but is not limited to, amino acid sequences selected from SEQ ID NOs:20, 22, 23, 27, 33, 37, 38 and 44, and may (mainly) consist of amino acid sequences selected from SEQ ID NOs:20, 22, 23, 27, 33, 37, 38 and 44.

[0209] In Equation 1,

[0210] X1 can be tyrosine;

[0211] X2 can be serine or Aib (aminoisobutyric acid);

[0212] X7 can be cysteine, threonine, or valine;

[0213] X10 can be tyrosine or cysteine;

[0214] X12 can be lysine or cysteine;

[0215] X13 can be tyrosine or cysteine;

[0216] X14 can be leucine or cysteine;

[0217] X15 can be aspartic acid or cysteine;

[0218] X16 can be glutamic acid, serine, or cysteine;

[0219] X17 can be glutamic acid, lysine, arginine, cysteine, or valine;

[0220] X18 can be arginine or cysteine;

[0221] X19 can be either alanine or cysteine;

[0222] X20 can be glutamine or lysine;

[0223] X21 can be aspartic acid, glutamic acid, valine, or cysteine;

[0224] X23 could be valine;

[0225] X24 can be valine or glutamine;

[0226] X27 can be methionine;

[0227] X28 can be asparagine or arginine;

[0228] X29 could be threonine; and

[0229] X30 can be cysteine ​​or absent, but glucagon derivatives are not limited to this.

[0230] For example, a peptide may include an amino acid sequence selected from SEQ ID NO:11, SEQ ID NOs:13 to 17, SEQ ID NOs:19 to 27, SEQ ID NOs:29, 31, 33, and SEQ ID NOs:35 to 45, and may specifically consist of (mainly) an amino acid sequence selected from SEQ ID NO:11, SEQ ID NOs:13 to 17, SEQ ID NOs:19 to 27, SEQ ID NOs:29, 31, 33, and SEQ ID NOs:35 to 45, but is not limited thereto.

[0231] In Equation 1,

[0232] X1 can be tyrosine;

[0233] X2 can be Aib (aminoisobutyric acid);

[0234] X7 can be cysteine, threonine, or valine;

[0235] X10 can be tyrosine or cysteine;

[0236] X12 can be lysine;

[0237] X13 can be tyrosine or cysteine;

[0238] X14 can be leucine or cysteine;

[0239] X15 can be aspartic acid or cysteine;

[0240] X16 can be glutamic acid, serine, or cysteine;

[0241] X17 can be lysine, arginine, cysteine, or valine;

[0242] X18 can be arginine or cysteine;

[0243] X19 can be either alanine or cysteine;

[0244] X20 can be glutamine or lysine;

[0245] X21 can be aspartic acid, glutamic acid, or cysteine;

[0246] X23 could be valine;

[0247] X24 can be glutamine;

[0248] X27 can be methionine;

[0249] X28 can be asparagine or arginine;

[0250] X29 could be threonine; and

[0251] X30 can be cysteine ​​or absent, but glucagon derivatives are not limited to this.

[0252] For example, a peptide may include an amino acid sequence selected from SEQ ID NOs:11, 14, 17, SEQ ID NOs:19 to 25, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 44, and specifically may (mainly) consist of an amino acid sequence selected from SEQ ID NOs:11, 14, 17, SEQ ID NOs:19 to 25, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 44, but is not limited thereto.

[0253] In Equation 1,

[0254] X1 may be tyrosine;

[0255] X2 can be serine or Aib (aminoisobutyric acid);

[0256] X7 could be threonine, valine, or cysteine;

[0257] X10 can be tyrosine or cysteine;

[0258] X12 can be lysine or cysteine;

[0259] X13 can be tyrosine or cysteine;

[0260] X14 can be leucine or cysteine;

[0261] X15 can be aspartic acid or cysteine;

[0262] X16 can be glutamic acid, serine, or cysteine;

[0263] X17 can be aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine;

[0264] X18 can be aspartic acid, glutamic acid, arginine, or cysteine;

[0265] X19 can be either alanine or cysteine;

[0266] X20 can be glutamine, aspartic acid, or lysine;

[0267] X21 can be aspartic acid or glutamic acid;

[0268] X23 may be valine;

[0269] X24 can be valine or glutamine;

[0270] X27 can be isoleucine or methionine;

[0271] X28 can be asparagine or arginine;

[0272] X29 may be threonine; and

[0273] X30 can be cysteine ​​or absent, but glucagon derivatives are not limited to this.

[0274] For example, a peptide may include, but is not limited to, amino acid sequences selected from SEQ ID NOs:7 to 11, SEQ ID NOs:13 to 15, SEQ ID NO:17, SEQ ID NOs:19 to 24, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 45, and specifically may (mainly) consist of amino acid sequences selected from SEQ ID NOs:7 to 11, SEQ ID NOs:13 to 15, SEQ ID NO:17, SEQ ID NOs:19 to 24, SEQ ID NOs:27, 29, 31, 33, and SEQ ID NOs:35 to 45.

[0275] In Equation 1,

[0276] X1 can be tyrosine;

[0277] X2 can be Aib (aminoisobutyric acid);

[0278] X7 can be threonine;

[0279] X10 can be tyrosine;

[0280] X12 can be lysine;

[0281] X13 can be tyrosine;

[0282] X14 can be leucine;

[0283] X15 can be aspartic acid or cysteine;

[0284] X16 can be glutamic acid, serine, or cysteine;

[0285] X17 can be lysine or arginine;

[0286] X18 can be arginine;

[0287] X19 can be alanine;

[0288] X20 can be glutamine, cysteine, or lysine;

[0289] X21 can be aspartic acid, cysteine, valine, or glutamic acid;

[0290] X23 can be valine or arginine;

[0291] X24 can be glutamine or leucine;

[0292] X27 can be methionine;

[0293] X28 can be asparagine or arginine;

[0294] X29 could be threonine; and

[0295] X30 may not exist, but glucagon derivatives are not limited to it.

[0296] For example, a peptide may include an amino acid sequence selected from SEQ ID NOs:14, 16, 18, 19, 25, 31, 33, 37 and 44, and may specifically consist of (mainly) an amino acid sequence selected from SEQ ID NOs:14, 16, 18, 19, 25, 31, 33, 37 and 44, but is not limited thereto.

[0297] More specifically, a peptide can be a peptide comprising an amino acid sequence represented by Formula 2 below:

[0298] Y–Aib–QGTF–X7–SD–X10–S–X12–Y–L–X15–X16–X17–R–A–X20–X21–F–V–X24–W–L–M–N–T–X30 (Formula 2, SEQ ID NO: 47)

[0299] In Equation 2,

[0300] X7 can be threonine, valine, or cysteine;

[0301] X10 can be tyrosine or cysteine;

[0302] X12 can be lysine or cysteine;

[0303] X15 can be aspartic acid or cysteine;

[0304] X16 can be glutamic acid or serine;

[0305] X17 can be lysine or arginine;

[0306] X20 can be glutamine or lysine;

[0307] X21 can be aspartic acid or glutamic acid;

[0308] X24 can be valine or glutamine; and

[0309] X30 can be cysteine ​​or it may not be present.

[0310] For example, a peptide may include, but is not limited to, amino acid sequences selected from SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NOs:36 to 45. More specifically, a peptide may include, or be (mainly) composed of, the corresponding amino acid sequences. However, peptides are not limited to these.

[0311] Specifically, in Equation 2,

[0312] X7 can be threonine, valine, or cysteine;

[0313] X10 can be tyrosine or cysteine;

[0314] X12 can be lysine;

[0315] X15 can be aspartic acid;

[0316] X16 can be glutamic acid or serine;

[0317] X17 can be lysine or arginine;

[0318] X20 can be glutamine or lysine;

[0319] X21 can be aspartic acid or glutamic acid;

[0320] X24 can be glutamine; and

[0321] X30 can be cysteine ​​or absent, but peptides are not particularly limited to this.

[0322] For example, a peptide may include an amino acid sequence selected from SEQ ID NOs:36 to 38, SEQ ID NOs:40 to 42, SEQ ID NO:44 and SEQ ID NO:45, and specifically may (mainly) consist of an amino acid sequence selected from SEQ ID NOs:36 to 38, SEQ ID NOs:40 to 42, SEQ ID NO:44 and SEQ ID NO:45, but is not limited thereto.

[0323] As another example of a peptide, a peptide may be a peptide comprising an amino acid sequence selected from SEQ ID NOs:2 to 11 and SEQ ID NOs:13 to 45, and specifically may (mainly) consist of an amino acid sequence selected from SEQ ID NOs:2 to 11 and SEQ ID NOs:13 to 45, but is not limited thereto.

[0324] However, peptides may exist in combinations other than those listed above, but are not particularly limited thereto, and all peptides described in the claims are included within the scope of the invention unless otherwise stated in the claims.

[0325] Furthermore, even though it is described herein as a “peptide consisting of a specific SEQ ID NO”, when the activity is the same as or corresponding to the activity of a peptide consisting of the amino acid sequence of the corresponding SEQ ID NO, the peptide does not exclude the addition of a meaningless sequence before or after the amino acid sequence of the corresponding SEQ ID NO, or a mutation that may occur naturally, or a silent mutation thereof, and when such a sequence addition or mutation is present, the peptide obviously also falls within the scope of this application.

[0326] The above description can be applied to other embodiments or other aspects of the present invention, but the present invention is not limited thereto.

[0327] In this invention, 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 with natural glucagon, but the sequence identity is not particularly limited thereto, and can be readily determined by those skilled in the art through sequence comparison between the peptide and natural glucagon.

[0328] As used herein, the term "homology" refers to the degree of similarity to the amino acid sequence or nucleotide sequence encoding a wild-type protein, and includes sequences having the aforementioned percentage or more of the same sequence as the amino acid or base sequence of the present invention. This homology can also be determined by visual comparison of two sequences, however, it can also be determined using bioinformatics algorithms that analyze the degree of homology by comparing the sequences to be compared side-by-side. Homology between two amino acid sequences can be expressed as a percentage. Useful automated algorithms are provided in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (GeneticsComputer Group, Madison, WI, USA). The automated alignment algorithms in the modules include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman alignment algorithms. Algorithms and homology determinations for other useful sequences, including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W, are automatically operated in the software.

[0329] In this invention, the peptide may be a peptide or a glucagon derivative that exhibits activity against the glucagon receptor, but is not limited thereto.

[0330] Although not specifically limited thereto, peptides exhibiting significant activity levels 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, but including but not limited to the range of significant activity shown. As a method for measuring such in vitro activity of the peptide, reference can be made to Example 4 of this specification, but the method is not specifically limited thereto.

[0331] As used herein, the term “about” means all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​that are equal to or similar to the value following the term “about”.

[0332] Although not particularly limited to this, peptides can be non-naturally occurring peptides.

[0333] The aforementioned glucagon derivatives may include intramolecular bridges (e.g., covalent or non-covalent bridges), and specifically may be in the form of rings. For example, a glucagon derivative may be in the form of a ring formed between the 16th and 20th amino acids of the glucagon derivative, but is not particularly limited thereto.

[0334] Non-limiting examples of rings may include lactam bridges (or lactam rings).

[0335] Glucagon derivatives include all glucagon derivatives modified to include amino acids capable of forming a ring at a target position to include the ring.

[0336] Such a ring can be formed between the amino acid side chains of a glucagon derivative, and can be, for example, in the form of a lactam ring formed between a lysine side chain and a glutamic acid side chain, but is not particularly limited thereto.

[0337] For example, a peptide comprising an amino acid sequence represented by Formula 1 or Formula 2 may be, but is not limited to, a peptide in which each of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in Formula 1 or Formula 2 is replaced by glutamic acid or lysine. In Xn (n is a natural number), n represents the position of the amino acid from the N-terminus of the presented amino acid sequence.

[0338] A peptide comprising an amino acid sequence represented by Formula 1 or Formula 2 may be, but is not limited to, a peptide in which each amino acid 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 is replaced by a glutamic acid or lysine that is capable of forming a ring.

[0339] The peptide can be, but is not limited to, a peptide in which a ring (e.g., a lactam ring) is formed between the respective amino acids of at least one of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in Formula 1 or Formula 2.

[0340] The peptide can be, but is not limited to, a peptide in which X16 is glutamic acid, X20 is lysine, and the side chains of X16 and X20 form a lactam ring.

[0341] The peptides according to the invention may have unmodified N-termini and / or C-termini, but to protect the peptide from in vivo proteases and increase stability, the N-termini, C-termini, and / or analogs may be chemically modified or protected by organic groups, or amino acids may be added to the ends of the peptide, and modified forms of these forms are also included within the scope of the peptides according to the invention. When the C-terminus is unmodified, the peptides according to the invention have a carboxyl group at the end, but are not particularly limited thereto.

[0342] Specifically, in the case of chemically synthesized peptides, since the N-terminus and C-terminus are charged, the N-terminus can be acetylated and / or the C-terminus can be amidated to remove the charge, but the N-terminus and C-terminus are not particularly limited thereto.

[0343] Unless otherwise stated in this specification, the detailed description of the "peptide" or "conjugate" in which a peptide is covalently linked to a biocompatible substance according to the invention applies not only to the respective peptide or conjugate, but also to the range of salts (e.g., pharmaceutically acceptable salts of the peptide) or solvates thereof, including all forms of the respective peptide or conjugate. Therefore, even if only the "peptide" or "conjugate" is described in the specification, the description applies to its specific salt, its specific solvate, and the specific solvate of the specific salt. For example, such salt form can be any form in which a pharmaceutically acceptable salt is used. The type of salt is not particularly limited. However, the salt is preferably in a form that is safe and effective for individuals (e.g., mammals), but is not particularly limited thereto.

[0344] The term "pharmaceutical acceptable" refers to a substance that, within the scope of pharmacological judgment, can be effectively used for the desired purpose without causing excessive toxicity, irritation, allergic reactions, etc.

[0345] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids may include hydrochloric acid, hydrobromic 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, and benzenesulfonic acid. Salts derived from suitable bases may include salts of alkali metals (such as sodium and potassium), salts of alkaline earth metals (such as magnesium), ammonium salts, and the like.

[0346] As used herein, the term "solvent" refers to a solvate in which a peptide or conjugate or its salt according to the invention forms a complex with a solvent molecule.

[0347] The glucagon derivative peptides of the present invention can be synthesized by methods known in the art, for example, using an automated peptide synthesizer according to their length, or they can also be produced by genetic engineering techniques.

[0348] Specifically, the glucagon derivative peptides of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other methods in the art. Therefore, the glucagon derivatives according to the present invention can be synthesized by a variety of methods including, for example, the following:

[0349] (a) A method for synthesizing peptides stepwise or by fragment assembly via solid-phase or liquid-phase methods, and for isolating and purifying the final peptide product; or

[0350] (b) A method for expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from a host cell culture; or

[0351] (c) A method for in vitro cell-free expression of a nucleic acid construct encoding a peptide and recovery of the expression product; or

[0352] A method for obtaining peptide fragments by any combination of methods (a), (b), and (c), then linking the peptide fragments to obtain the peptide, and recovering the peptide.

[0353] As a more concrete example, a fusion gene encoding a fusion protein comprising a fusion partner and a glucagon derivative can be prepared by genetic manipulation, transformed into a host cell, expressed as a fusion protein, and then the glucagon derivative can be cleaved and isolated from the fusion protein using a proteolytic enzyme or compound, thereby producing the desired glucagon derivative. For this purpose, for example, a DNA sequence encoding amino acid residues that can be cleaved by proteolytic enzymes such as factor Xa or enterokinase, CNBr, or compounds such as hydroxylamine can be inserted between the fusion partner and the polynucleotide encoding the glucagon derivative.

[0354] More specifically, the peptides or glucagon derivatives according to the invention, such as peptides comprising an amino acid sequence represented by Formula 1, may be, but are not limited to, long-acting conjugates bound to a biocompatible moiety that increases its in vivo half-life. The biocompatible moiety may be used interchangeably with the carrier.

[0355] 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 sequence as the amino acid sequence represented by Formula 1, or include the amino acid sequence therein.

[0356] In this invention, peptide conjugates exhibit increased efficacy persistence compared to unbound peptides. In this invention, such conjugates are referred to as "long-acting conjugates".

[0357] Such conjugates can be non-naturally occurring conjugates.

[0358] As used herein, the term "long-acting conjugate" refers to a conjugate in which a biocompatible moiety or carrier is bound to a physiologically active substance (e.g., a glucagon derivative), and exhibits increased duration of efficacy (e.g., increased in vivo half-life) compared to the physiologically active substance without the bound biocompatible moiety or carrier. In long-acting conjugates, the biocompatible moiety or carrier may be covalently linked to the physiologically active substance, but is not particularly limited thereto.

[0359] In certain embodiments of the invention, long-acting conjugates of glucagon derivatives may exhibit increased potency durability compared to unbound natural glucagon or glucagon derivatives.

[0360] In one embodiment of the present invention, the long-acting conjugate may be a conjugate represented by the following chemical formula 1, but is not limited thereto:

[0361] [Chemical Formula 1]

[0362] XLF

[0363] Where X is the aforementioned peptide;

[0364] L is a linker containing repeating ethylene glycol units;

[0365] F is the Fc region of immunoglobulins; and

[0366] - represents the covalent bond connection between X and L, and between L and F.

[0367] In the long-acting conjugate, X can be a peptide (glucagon derivative) according to the present invention. Specifically, X can be a peptide comprising an amino acid sequence represented by Formula 1, or X can be a peptide comprising an amino acid sequence comprising any one of SEQ ID NOs:2 to 11 and SEQ ID NOs:13 to 45, or X can be a peptide comprising an amino acid sequence selected from SEQ ID NOs:20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42 and 44, but is not limited thereto.

[0368] In the long-acting conjugate, F is X, that is, a substance capable of increasing the half-life of glucagon derivatives, and corresponds to a component constituting a portion of the conjugate of the present invention.

[0369] F can bind to X through covalent or non-covalent chemical bonds, specifically via L binding to X through a covalent chemical bond.

[0370] As a specific example, F can be the Fc region of an immunoglobulin, and more specifically, the Fc region of an immunoglobulin can be derived from IgG, but F is not particularly limited to this.

[0371] As used herein, the term "immunoglobulin Fc region" refers to a site comprising heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), excluding the heavy and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component constituting a part of the protein conjugate of this invention.

[0372] As used herein, the term Fc fragment includes not only the natural sequence obtained by papain digestion of immunoglobulins, but also its derivatives, such as those in which one or more amino acid residues in the natural sequence are altered by deletion, insertion, non-conservative or conserved substitution, or a combination thereof to form a sequence different from the natural sequence.

[0373] F is a structure in which two polypeptide chains are linked by disulfide bonds, and it can be a structure in which only one of the two chains is linked by a nitrogen atom, but is not limited to this. The linkage by a nitrogen atom can be achieved by reductive amination of the ε-amino atom of lysine or the N-terminal amino group.

[0374] Reductive amination is a reaction in which the amino or amino group of a reactant reacts with the aldehyde (i.e., the functional group capable of reductive amination) of another reactant to produce an amine, and then forms an amine bond through a reduction reaction. Reductive amination is a well-known organic synthesis reaction in the art.

[0375] As an implementation method, F can be linked to the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0376] This immunoglobulin Fc fragment may include, but is not limited to, the hinge region in the constant region of the heavy chain.

[0377] In this invention, the immunoglobulin Fc fragment may include a specific hinge sequence at its N-terminus.

[0378] As used herein, the term "hinge sequence" refers to a site on the heavy chain that forms a dimer of the immunoglobulin Fc fragment via an inter-disulfide bond.

[0379] In this invention, the hinge sequence can be mutated to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto:

[0380] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys–Pro–Ser–Cys–Pro (SEQ ID NO: 51).

[0381] By deleting the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO:51, the hinge sequence may consist of only one cysteine ​​residue. The hinge sequence of the present invention may consist of 3 to 12 amino acids comprising only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence:

[0382] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Pro–Ser–Cys–Pro (SEQ ID NO: 52),

[0383] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys–Pro–Ser–Pro (SEQ ID NO: 53),

[0384] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys–Pro–Ser (SEQ ID NO: 54),

[0385] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys–Pro–Pro (SEQ ID NO: 55),

[0386] Lys–Tyr–Gly–Pro–Pro–Cys–Pro–Ser (SEQ ID NO: 56),

[0387] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys (SEQ ID NO: 57),

[0388] Glu–Lys–Tyr–Gly–Pro–Pro–Cys (SEQ ID NO: 58),

[0389] Glu–Ser–Pro–Ser–Cys–Pro (SEQ ID NO:59),

[0390] Glu–Pro–Ser–Cys–Pro (SEQ ID NO: 60),

[0391] Pro–Ser–Cys–Pro (SEQ ID NO:61),

[0392] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Ser–Cys–Pro (SEQ ID NO: 62),

[0393] Lys–Tyr–Gly–Pro–Pro–Pro–Ser–Cys–Pro (SEQ ID NO: 63),

[0394] Glu–Ser–Lys–Tyr–Gly–Pro–Ser–Cys–Pro (SEQ ID NO: 64),

[0395] Glu–Ser–Lys–Tyr–Gly–Pro–Pro–Cys (SEQ ID NO: 65),

[0396] Lys–Tyr–Gly–Pro–Pro–Cys–Pro (SEQ ID NO: 66),

[0397] Glu–Ser–Lys–Pro–Ser–Cys–Pro (SEQ ID NO: 67),

[0398] Glu–Ser–Pro–Ser–Cys–Pro (SEQ ID NO: 68),

[0399] Glu–Pro–Ser–Cys (SEQ ID NO:69), or

[0400] Ser-Cys-Pro (SEQ ID NO:70).

[0401] More specifically, the hinge sequence may include, 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).

[0402] Due to the presence of the hinge sequence, the immunoglobulin Fc fragment of the present invention can be in the form of a dimer formed by two molecules of the immunoglobulin Fc chain, and the conjugate represented by chemical formula 1 of the present invention can be in the form of a linker having one end attached to a chain of the dimer immunoglobulin Fc fragment, but is not limited thereto.

[0403] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide and may include the last amino terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the last amino terminus. The immunoglobulin Fc fragments of the present invention may include a hinge sequence at the N-terminus, but are not limited thereto.

[0404] The immunoglobulin Fc fragment of the present invention may be an extended Fc fragment comprising part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), except for the heavy and light chain variable regions of the immunoglobulin, provided that its effect is substantially equivalent to or enhanced than that of the native type. The immunoglobulin Fc fragment may be a fragment in which some relatively long amino acid sequences corresponding to CH2 and / or CH3 are removed.

[0405] For example, the immunoglobulin Fc region of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of one or more of the CH1, CH2, CH3, or CH4 domains with an immunoglobulin hinge region (or a portion of the hinge region); or 6) a dimer of each domain of the heavy chain constant region with the light chain constant region. However, the immunoglobulin Fc region is not limited to these.

[0406] In one embodiment, the immunoglobulin Fc region can be in dimer form, and a molecule of a glucagon derivative can be covalently linked to one of the dimer Fc regions. In this case, the immunoglobulin Fc and the glucagon derivative can be linked to each other via a non-peptide polymer. Two molecules of the glucagon derivative may also be symmetrically bound to one of the dimer Fc regions. In this case, the immunoglobulin Fc and the glucagon derivative or insulin secretion peptide can be linked to each other via a non-peptide linker. However, the immunoglobulin Fc region is not limited to the examples described above.

[0407] The immunoglobulin Fc region of the present invention comprises the natural amino acid sequence and its sequence derivatives. An amino acid sequence derivative refers to one or more amino acid residues in the natural amino acid sequence that have different sequences through deletion, insertion, non-conservative or conserved substitution, or a combination thereof.

[0408] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are important for binding, are known to be suitable sites for modification.

[0409] Various types of derivatives are possible—where sites capable of forming inter-disulfide bonds are removed, some amino acids are removed from the N-terminus of the native Fc, or methionine residues are added to the N-terminus of the native Fc. To eliminate effector function, complement-binding sites (e.g., C1q binding sites) or ADCC (antibody-dependent cell-mediated cytotoxicity) sites can be removed. Techniques for preparing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[0410] Amino acid exchanges in proteins or peptides that do not alter their overall molecular activity are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges occur between amino acid residues such as 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 can be made through phosphorylation, sulfation, acrylamide, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0411] The aforementioned Fc derivatives can exhibit biological activity equivalent to that of the Fc region of the present invention, as well as enhanced structural stability of the Fc region against heat, pH, etc.

[0412] This Fc region can be obtained from naturally occurring types isolated from animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or it can be a recombinant or a derivative thereof obtained from transformed animal cells or microorganisms. Here, the method for obtaining the Fc region from the naturally occurring type can be a method in which the whole immunoglobulin is isolated from a human or animal and then treated with a protease. When treated with papain, the whole immunoglobulin is cleaved into Fab and Fc, while when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc and pF'c can be separated by size exclusion chromatography or similar methods. In a more specific embodiment, the Fc region is a recombinant immunoglobulin Fc region obtained from a human Fc region derived from microorganisms.

[0413] The Fc region of immunoglobulins can possess native glycans, glycans with increased glycans compared to the native type, glycans with decreased glycans compared to the native type, or deglycosylated forms. Conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms, can be used to increase, decrease, or remove these immunoglobulin Fc glycans. Here, immunoglobulin Fc regions with glycans removed from the Fc region do not induce unwanted immune responses in vivo because their binding affinity to complement (c1q) is significantly reduced, and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated. In this respect, deglycosylated or aglycosylated immunoglobulin Fc regions are more suitable as drug carriers for the intended purpose.

[0414] In this invention, "deglycosylation" refers to the removal of the Fc region of a sugar by an enzyme, and "non-glycosylation" refers to the unglycosylated Fc region produced from a prokaryote, in a more specific embodiment, Escherichia coli (E. coli).

[0415] The immunoglobulin Fc region may be of human or animal origin (e.g., cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs), and in a more specific embodiment, is of human origin.

[0416] The immunoglobulin Fc region can be derived from IgG, IgA, IgD, IgE, or IgM, or combinations thereof, or hybrids thereof. In a more specific embodiment, the immunoglobulin Fc region is derived from the most abundant IgG or IgM in human blood, and in yet another more specific embodiment, it is derived from IgG known to enhance the half-life of ligand-binding proteins. In still a more specific embodiment, the immunoglobulin Fc region is the IgG4 Fc region. In the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0417] In a specific embodiment, the immunoglobulin Fc fragment, as a fragment of human IgG4 Fc, can be in the form of a homodimer, wherein two monomers are linked by a disulfide bond (interchain form) between cysteine ​​residues, where cysteine ​​is the third amino acid of each monomer. In this case, each monomer of the homodimer independently has / may have an internal disulfide bond between cysteine ​​residues at positions 35 and 95 and an internal disulfide bond between cysteine ​​residues at positions 141 and 199 (i.e., two disulfide bonds (intrachain form)). The number of amino acids in each monomer can be 221, and the amino acids forming the homodimer can consist of a total of 442 amino acids, but the number of amino acids is not limited to this. Specifically, in the immunoglobulin Fc fragment, two monomers having the amino acid sequence SEQ ID NO:71 (consisting of 221 amino acids) form a homodimer through an inter-disulfide bond between cysteine ​​residues, where cysteine ​​is the third amino acid in each monomer. The monomers of the homodimer can each independently form a disulfide bond between cysteine ​​residues at positions 35 and 95, as well as between cysteine ​​residues at positions 141 and 199. However, the immunoglobulin Fc fragment is not limited to this.

[0418] In this invention, "combination" means that when dimers or multimers are formed, polypeptides encoding the Fc region of single-chain immunoglobulins of the same origin form bonds with single-chain polypeptides of different origins. In other words, dimers or multimers can be prepared from two or more fragments selected from IgG Fc, IgA Fc, IgMFc, IgD Fc, and IgE Fc fragments.

[0419] L can be a non-peptide linker, such as a linker containing ethylene glycol repeating units.

[0420] In this invention, a "non-peptide-based linker" comprises a biocompatible polymer in which two or more repeating units are bonded. The repeating units are bonded to each other by any covalent bond other than a peptide bond. The non-peptide-based linker can be a component constituting a conjugate portion of this invention and corresponds to L in Formula 1. Any polymer resistant to proteolytic enzymes in vivo can be used without limitation as a non-peptide-based linker that can be used in this invention. In this invention, the non-peptide-based linker is interchangeable with the non-peptide-based polymer.

[0421] Although not particularly limited thereto, non-peptide linkers may be linkers containing ethylene glycol repeating units (e.g., polyethylene glycol), and derivatives thereof known in the art, as well as derivatives that can be readily prepared at the level of expertise in the art, are also included within the scope of this invention.

[0422] The repeating unit of the non-peptide linker can be an ethylene glycol repeating unit. Specifically, the non-peptide linker may include an ethylene glycol repeating unit and a functional group at the end for preparing the conjugate. The long-acting conjugate according to the invention may be in the form where X and F are linked by functional groups, but is not limited thereto. In the invention, the non-peptide linker may include two, three or more functional groups, and the respective functional groups may be the same or different from each other, but the non-peptide linker is not limited thereto.

[0423] Specifically, the linker can be polyethylene glycol (PEG) represented by the following chemical formula 2, but is not limited to:

[0424] [Chemical Formula 2]

[0425]

[0426] Where n = 10 to 2400, n = 10 to 480, or n = 50 to 250, but not limited to these.

[0427] The PEG moiety in long-acting conjugates can include not only –(CH2CH2O) n –The structure may also include insertion into connecting elements and –(CH2CH2O). n The oxygen atom between – but not limited to.

[0428] In specific embodiments, the conjugate may have, but is not limited to, a structure in which a glucagon derivative peptide or a peptide (X) comprising an amino acid sequence represented by Formula 1 is covalently linked to an immunoglobulin fragment (F) via a linker containing a glycol repeating unit. Polyethylene glycol is a term that encompasses, but is not particularly limited to, all forms of glycol homopolymers, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG). Specifically, the chemical weight of the glycol repeating unit portion in L may be in the range of 1 kDa to 100 kDa, but is not limited thereto.

[0429] The molecular weight of the non-peptide polymer is in the range of 1 kDa to 100 kDa, specifically in the range of 1 kDa to 20 kDa, or in the range of 1 kDa to 10 kDa, but is not limited thereto. As the non-peptide linker of the present invention that binds to the polypeptide corresponding to F, not only a single polymer can be used, but also a combination of different types of polymers can be used.

[0430] In a particular embodiment, the two ends of the non-peptide linker may be bound to the amine or thiol group of F, for example, the immunoglobulin Fc fragment and the amine or thiol group of X.

[0431] Specifically, non-peptide polymers may include reactive groups capable of binding to F (e.g., immunoglobulin Fc fragments) and X, specifically including, but not limited to, reactive groups capable of binding to an amino group located at the N-terminus or lysine residue, or a thiol group located at both the X and F ends of a cysteine ​​residue.

[0432] The reactive groups that can bind to non-peptide polymers of F (e.g., immunoglobulin Fc fragments and X) can be selected from, but are not limited to, aldehyde groups, maleimide groups, and succinimide derivatives.

[0433] In the above text, examples of aldehyde groups include propionaldehyde or butyraldehyde, but aldehyde groups are not limited to these.

[0434] In the above text, succinimide derivatives may include succinimide valerate, succinimide methyl butyrate, succinimide methyl propionate, succinimide butyrate, succinimide propionate, N-hydroxysuccinimide, hydroxysuccinimide, succinimide carboxymethyl or succinimide carbonate, but the succinimide derivatives are not limited to these.

[0435] Non-peptide linkers can be attached to X and F via such reactive groups, but are not particularly limited thereto.

[0436] The final product generated by aldehyde reductive amination is much more stable than the final product generated by amide linkage. The reactive aldehyde group reacts selectively with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH (e.g., pH 9.0).

[0437] The reactive groups at both ends of a non-peptide linker can be the same or different from each other. For example, a non-peptide linker can have a maleimide group at one end and an aldehyde, propionaldehyde, or butyraldehyde group at the other end. However, non-peptide linkers are not particularly limited to this, as long as F (specifically, the immunoglobulin Fc fragment) and X can bind to each end of the non-peptide linker.

[0438] For example, one end of a non-peptide linker may include a maleimide group as a reactive group, while the other end may include an aldehyde group, a propionaldehyde group, or a butyraldehyde group.

[0439] When using polyethylene glycol with hydroxyl reactive groups at both ends as a non-peptide polymer, the long-acting protein conjugates of the present invention can be prepared by activating hydroxyl groups into various reactive groups using known chemical reactions, or by using commercially available polyethylene glycol with modified reactive groups.

[0440] In a specific embodiment, the non-peptide polymer may be linked to the cysteine ​​residue of X, more specifically the -SH group of cysteine, but is not limited thereto.

[0441] Specifically, the reactive group of the non-peptide polymer can be linked to the -SH group of the cysteine ​​residue, and all the above descriptions apply to the reactive group. When using maleimide-PEG-aldehyde, the maleimide group can be linked to the -SH group of X via a thioether bond, and the aldehyde group can be linked to F (specifically, the -NH2 group of immunoglobulin Fc) via a reductive amination reaction, but the conjugate is not limited to this, and this corresponds to one example.

[0442] In conjugates, the reactive group of a non-peptide polymer can be linked to -NH2 at the N-terminus of an immunoglobulin Fc fragment, however this corresponds to one instance.

[0443] Compared to natural glucagon or to unmodified X, the above conjugates exhibit enhanced efficacy durability, and such conjugates include not only the above forms but also forms encapsulated in biodegradable nanoparticles.

[0444] The GLP-1 and GIP receptor dual agonists of the present invention include various substances, such as compounds or peptides, that exhibit significant levels of activity against both GLP-1 and GIP receptors. The term "GLP-1 and GIP receptor dual agonist" is used interchangeably with "GLP-1 / GIP dual agonist" or "dual agonist".

[0445] Specifically, the dual agonist has one or more of the following activities i) to ii), particularly significant activities:

[0446] i) Activation of GLP-1 receptor; and ii) Activation of GIP receptor.

[0447] Although not particularly limited thereto, dual agonists of GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic peptide) receptors are substances that exhibit significant levels of activity against the receptors and may exhibit in vitro activity against natural GLP-1 and GIP, as well as against GLP-1 and GIP receptors, at levels of 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, 100% or more, but not limited to significantly increased ranges.

[0448] The in vitro activity of such dual agonists can be measured by various in vitro activity measurements known in the art, and Example 2 of this specification may be referred to as an example, but the method is not particularly limited thereto.

[0449] Although not particularly limited to this, dual agonists can be non-natural dual agonists.

[0450] Examples of substances that exhibit activity against GLP-1 and GIP receptors include, but are not particularly limited to, natural GLP-1 and GIP, their agonists, or derivatives thereof. “Derivatives” are defined as described above.

[0451] Although not particularly limited to this, dual agonists of GLP-1 and GIP receptors can be peptides that exhibit activity against both GLP-1 and GIP receptors.

[0452] Specifically, the GLP-1 and GIP receptor dual agonist of the present invention is a derivative of GLP-1 or GIP that exhibits activity against both GLP-1 and GIP receptors, and any GLP-1 and GIP receptor dual agonist known in the art is unrestrictedly included within the scope of the present invention. As long as the GLP-1 and GIP receptor dual agonist according to the present invention exhibits activity against both GLP-1 and GIP receptors, it can be a commercially available dual agonist or prepared by methods known in the art. Examples of GLP-1 and GIP receptor dual agonists include, but are not limited to, taripipride, NN9709, and SAR-438335.

[0453] Examples of dual GLP-1 and GIP receptor agonists according to the present invention, such as teriparatide, are known as L-tyrosyl-2-methylalanyl-L-α-glutamylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-α-aspartyl-L-tyrosyl-L-seryl-L-isoleucyl-2-methylalanyl-L-leucyl-L-α-aspartyl-L-lysyl-L-isoleucyl-L-glutamine-N 6 -[(22S)-22,42-dicarboxy-10,19,24-trioxy-3,6,12,15-tetraoxy-9,18,23-tris(2-azaadamantane)-N-oxygenolecithin-1-oyl]-L-lysyl-L-alanyl-L-phenylalanyl-L-valine-L-glutamine-L-tryptophanyl-L-leucyl-L-isoleucyl-L-alanylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl-L-prolyl-L-seryl-L-seramide (CAS#2) 023788-19-2)(L-tyrosyl-2-methylalanyl-L-α-glutamylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-α-aspart yl-L-tyrosyl-L-seryl-L-isoleucyl-2-methylalanyl-L-leucyl-L-α-aspartyl-L-lysyl-L-isoleucyl-L-alanyl-L-glutaminyl-N 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-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-isoleucyl-L-alanylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl-L-prolyl-L-prolyl-L-serinamide), and is a substance having the following sequence:

[0454] Y X EGTFTSDY SI X LDKIAQ K AFVQWLIAGG PSSGAPPP S (SEQ ID NO:50)

[0455] in, X , K and S Each is a residue modified as follows

[0456]

[0457] Although not particularly limited thereto, the GLP-1 and GIP receptor dual agonist of the present invention may be in a terminally modified form, specifically in a form in which one end of the dual agonist is acylated or amidated, but is not limited thereto.

[0458] The peptides of the present invention can be synthesized by methods known in the art, for example, using an automated peptide synthesizer depending on their length, or they can also be produced by genetic engineering techniques.

[0459] Specifically, the peptides of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other methods in the art. Therefore, the peptides according to the present invention can be synthesized by a variety of methods, including, for example, the following:

[0460] (a) A method for synthesizing peptides stepwise or by fragment assembly via solid-phase or liquid-phase methods, and for isolating and purifying the final peptide product; or

[0461] (b) A method for expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from a host cell culture; or

[0462] (c) Perform in vitro cell-free expression of the nucleic acid construct encoding the peptide and recover the expression product; or

[0463] A method for obtaining peptide fragments by any combination of methods (a), (b), and (c), then linking the peptide fragments to obtain the peptide, and recovering the peptide.

[0464] As a more concrete example, polynucleotides encoding peptides can be prepared through genetic manipulation and transformed into host cells, which can then produce the desired peptides.

[0465] Although not particularly limited thereto, in the compositions of the present invention, the in vitro activity of the glucagon derivative compared to the natural ligand activity of the glucagon receptor can be 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; and in the case of dual agonists of GLP-1 and GIP receptors, the in vitro activity of GLP-1 compared to the natural ligand activity of the GLP-1 receptor can be 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, or more. The in vitro activity of GIP may be more than, 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, compared to the natural ligand activity of the GIP receptor; but the in vitro activity is not limited to these. Although not particularly limited thereto, in the compositions of the present invention, the in vitro activity of glucagon derivatives may be about 0 compared to the activity of natural ligands of glucagon receptors.1% or more and about 300% or less, about 2% or more and about 300% or less, about 3% or more and about 300% or less, about 4% or more and about 300% or less, about 5% or more and about 300% or less, about 6% or more and about 300% or less, about 7% or more and about 300% or less, about 8% or more and about 300% or less, about 9% or more and about 300% or less, about 10% or more and about 300% or less, about 20% or more and about 300% or less. Approximately 30% more and approximately 300% or less, approximately 40% more and approximately 300% or less, approximately 50% or more and approximately 300% or less, approximately 60% more and approximately 300% or less, approximately 70% or more and approximately 300% or less, approximately 80% or more and approximately 300% or less, approximately 90% or more and approximately 300% or less, or approximately 100% or more and approximately 300% or less; and in the case of dual agonists of GLP-1 and GIP receptors, compared with the natural ligand activity of the GLP-1 receptor, GLP The in vitro activity of -1 can be about 0.1% or more and about 200% or less, about 2% or more and about 200% or less, about 3% or more and about 200% or less, about 4% or more and about 200% or less, about 5% or more and about 200% or less, about 6% or more and about 200% or less, about 7% or more and about 200% or less, about 8% or more and about 200% or less, about 9% or more and about 200% or less, about 10% or more and about 200% or less, about 20% or more More than and about 200% or less, about 30% or more and about 200% or less, about 40% or more and about 200% or less, about 50% or more and about 200% or less, about 60% or more and about 200% or less, about 70% or more and about 200% or less, about 80% or more and about 200% or less, about 90% or more and about 200% or less, or about 100% or more and about 200% or less; and the in vitro activity of GIP may be about 0 compared to the natural ligand activity of the GIP receptor.1% or more and about 300% or less, about 2% or more and about 300% or less, about 3% or more and about 300% or less, about 4% or more and about 300% or less, about 5% or more and about 300% or less, about 6% or more and about 300% or less, about 7% or more and about 300% or less, about 8% or more and about 300% or less, about 9% or more and about 300% or less, about 10% or more and about 300% or less, about 2 0% or more and about 300% or less, about 30% or more and about 300% or less, about 40% or more and about 300% or less, about 50% or more and about 300% or less, about 60% or more and about 300% or less, about 70% or more and about 300% or less, about 80% or more and about 300% or less, about 90% or more and about 300% or less, or about 100% or more and about 300% or less, but in vitro activity is not limited to these.

[0466] The compositions of the present invention can be used to prevent or treat metabolic syndrome.

[0467] As used herein, the term “prevention” means any action that inhibits or delays the onset of a target disease (e.g., metabolic syndrome) by administering (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors; or a composition containing these; and the term “treatment” means any action that improves or benefits the symptoms of a target disease (e.g., metabolic syndrome) by administering (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors; or a composition containing these.

[0468] As used herein, the term “administration” means the introduction of a predetermined substance into a patient by any suitable method, and the route of administration of the composition is not particularly limited thereto. The composition may be administered via any general route by which the composition can achieve its target in the body, and administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0469] Substances exhibiting glucagon activity, as well as dual agonists of GLP-1 and GIP receptors, can be used to prevent or treat metabolic syndrome.

[0470] The combined administration of (i) the substance exhibiting glucagon activity and (ii) the dual agonist of GLP-1 and GIP receptors of the present invention not only prevents weight gain, promotes weight loss, reduces excess weight, or shows effects on obesity, including pathological obesity (e.g., by regulating appetite, eating, food intake, calorie intake, and / or energy expenditure), but is also effective for diseases including obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. The combined administration of (i) the substance exhibiting glucagon activity and (ii) the dual agonist of GLP-1 and GIP receptors of the present invention can also be effective for metabolic syndrome or obesity-related liver disease, and the substance exhibiting glucagon activity and the dual agonist of GLP-1 and GIP receptors can be used as a medicine for treating related diseases and health conditions, not limited to these. The glucagon-active substances of the present invention, as well as the dual agonists of GLP-1 and GIP receptors, can also be used to treat metabolic syndromes other than obesity, i.e., related diseases such as impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis caused by dyslipidemia, atherosclerosis, coronary heart disease (CHD), and stroke. However, the effects of the glucagon-active substances and the dual agonists of GLP-1 and GIP receptors of the present invention on these conditions can be mediated, in whole or in part, through the aforementioned weight-related effects, or independently.

[0471] As used herein, the term "metabolic syndrome" refers to the symptoms of various diseases caused by chronic metabolic disorders, either alone or in a complex manner. Specifically, diseases corresponding to metabolic syndrome include, but are not limited to, impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, liver disease, arteriosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease (CHD), and stroke.

[0472] In this invention, the term "obesity" refers to a state of excessive body fat, and is defined as a body mass index (BMI, weight (kg) divided by the square of height (m)) of 25 or higher. Obesity is typically caused by an energy imbalance, resulting from a long-term intake of nutrients that is excessive compared to energy expenditure. Obesity is a metabolic disease affecting the whole body, increasing the risk of diabetes and hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular disease, and in some cases, it is also associated with cancer. Treatment for obesity can be achieved through weight loss and body fat reduction, but is not limited to these methods.

[0473] As used herein, the term "hyperlipidemia" refers to a condition in which there is an abnormal increase in lipids such as free cholesterol, cholesterol esters, phospholipids, and triglycerides in the blood. Hyperlipidemia itself usually does not present with specific symptoms, but excess lipids in the blood adhere to the walls of blood vessels, narrowing their size and leading to atherosclerosis through inflammatory responses. This can result in coronary heart disease, cerebrovascular disease, and peripheral vascular occlusion. Excess lipids in the blood accumulate in liver tissue, which can lead to fatty liver disease. In the above context, fatty liver refers to a condition in which fat constitutes more than 5% of the liver's weight and may be caused by alcohol consumption and excessive fat intake. Treatment for hyperlipidemia can be achieved by improving blood lipid levels, but is not limited to this.

[0474] As used in this article, the term "diabetes" refers to a metabolic disorder characterized by high blood sugar, such as insufficient insulin secretion or inability of insulin to function properly, and is marked by elevated blood glucose levels. Recently, the incidence of diabetes has exploded due to rising rates of obesity, particularly abdominal obesity. If chronic hyperglycemia is left untreated, it can lead to various pathological conditions in the body. Typically, it increases the risk of retinopathy, renal insufficiency, neuropathy, stroke due to vascular disorders, kidney or heart disease, diabetic foot ulcers, and cardiovascular disease. Treatment for diabetes can improve blood sugar levels by improving insulin sensitivity, but is not limited to this.

[0475] The aforementioned metabolic syndrome is closely associated with liver diseases, including the accumulation of fat in liver tissue and the resulting inflammation and fibrosis, and instances of metabolic syndrome may include a variety of liver diseases.

[0476] Therefore, the compositions of the present invention can have preventive or therapeutic uses for liver diseases. Specifically, the compositions of the present invention comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors can have preventive or therapeutic uses for liver diseases by exhibiting effects of inhibiting and improving inflammation and / or fibrosis in liver tissue, but are not limited thereto.

[0477] As used herein, the term "liver disease" refers to a disease occurring in the liver and may include, but is not limited to, metabolic liver diseases. Representative examples of liver diseases include simple steatosis, non-alcoholic fatty liver disease, hepatitis, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, cirrhosis, decompensated liver disease, and hepatocellular carcinoma, and any abnormality occurring in the tissues and function of the liver can be considered a liver disease according to the present invention. In addition to alcohol consumption, drugs, and viral infections, obesity, metabolic disorders, etc., can lead to liver inflammation, and diseases such as cirrhosis and hepatocellular carcinoma are known to occur depending on the progression and duration of liver inflammation.

[0478] Specifically, the composition according to the invention comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors may exhibit preventive or therapeutic effects on liver diseases associated with or caused by metabolic syndrome.

[0479] The liver disease for which the composition of the present invention, comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors, exhibits therapeutic effects can be metabolic liver disease, but is not limited thereto. Metabolic liver disease is a disease caused by abnormal chemical reactions in the body that interfere with the body's metabolism, and includes simple steatosis, fatty liver, steatohepatitis, and non-alcoholic fatty liver disease.

[0480] In this invention, "non-alcoholic fatty liver disease (NAFLD)" refers to fatty liver disease present in individuals with no history of alcohol consumption or unrelated to alcohol consumption. Fatty liver disease is a condition characterized by the abnormal deposition of triglycerides in hepatocytes, contrary to normal conditions. A normal liver consists of approximately 5% adipose tissue, and triglycerides, fatty acids, phospholipids, cholesterol, and cholesterol esters are the main components of fat. Once fatty liver disease develops, most of these components are replaced by triglycerides, and a diagnosis of fatty liver disease is made when the amount of triglycerides reaches 5% or more of the liver's weight. Fatty liver disease is caused by impaired fat metabolism or defects in the process of transporting excessive fat within hepatocytes, primarily due to impaired fat metabolism in the liver. The majority of the fat accumulated in fatty liver disease can be triglycerides.

[0481] Nonalcoholic fatty liver disease refers to, but is not limited to, a group of diseases including, simple steatosis with excessive fat accumulation only in hepatocytes, nonalcoholic fatty liver, and nonalcoholic steatohepatitis (NASH) with hepatocyte necrosis, inflammation, and fibrosis, as long as it is treated with the compositions of the present invention. Nonalcoholic fatty liver disease according to the present invention may be accompanied by nonalcoholic steatohepatitis, but is not limited thereto.

[0482] The liver disease for which the composition of the present invention, comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors, exhibits therapeutic effects can be liver inflammation, but is not limited thereto. In this invention, "liver inflammation" refers to a disease causing inflammation of the liver, which is the leading cause of liver disease, and is classified into acute hepatitis and chronic hepatitis based on etiology and symptoms. Liver inflammation is primarily caused by viruses, alcohol, drugs, immune abnormalities, and metabolic diseases.

[0483] The composition of the present invention comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors not only has the effect of reducing inflammation of the liver itself, but also exhibits the effect on diseases accompanied by or caused by liver inflammation (e.g., hepatitis, non-alcoholic steatohepatitis, and liver fibrosis).

[0484] In this invention, "non-alcoholic steatohepatitis" is a type of non-alcoholic fatty liver disease and a representative example of liver disease accompanied by hepatocellular necrosis, inflammation, and fibrosis. The composition according to the invention, comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors, can exert an effect on non-alcoholic steatohepatitis by inhibiting liver inflammation and fibrosis, specifically on non-alcoholic steatohepatitis with fatty liver, liver fibrosis, or cirrhosis; or on hepatocellular carcinoma caused by non-alcoholic steatohepatitis, but is not limited thereto.

[0485] Specifically, the composition of the present invention comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors exhibits a reduction in NAS (NAFLD activity score), which implies a therapeutic effect on non-alcoholic steatohepatitis.

[0486] In this invention, "liver fibrosis" refers to the excessive formation of fibrous connective tissue in an organ or tissue during the wound healing process of repeated liver injury, during repair or reaction. Chronicity and exacerbation of liver inflammation are known to be contributing factors. Unlike cirrhosis, liver fibrosis is known to be reversible, composed of thin fibrous tissue, and without nodule formation; the liver can return to a normal state once the cause of liver injury is eliminated. However, when this liver fibrosis process is repeated, cross-linking between extracellular matrix (ECM) cells increases, and liver fibrosis progresses to irreversible nodular cirrhosis. The compositions according to the invention can exhibit preventative or therapeutic effects on liver fibrosis, specifically liver fibrosis accompanied by non-alcoholic steatohepatitis, but are not limited thereto.

[0487] Specifically, the composition of the present invention comprising (i) a substance exhibiting glucagon activity and (ii) a dual agonist of GLP-1 and GIP receptors can exhibit an effect on liver fibrosis, specifically by preventing or treating liver fibrosis by reducing collagen expression in liver tissue.

[0488] In this invention, "cholestasis" refers to a condition in which the flow of bile from the liver to the duodenum is slowed or obstructed, and "cholestatic liver disease" refers to conditions in which the formation of bile within the liver is interfered with by conditions such as various diseases, nutrient supply from dilated jugular veins, or the side effects of certain medications (e.g., some antibiotics). Common signs of cholestasis include fatigue, itching, jaundice, and xanthelasma (the deposition of cholesterol-rich substances under the skin). The effects of cholestasis are severe and widespread, leading to the progression of liver disease into systemic illness, liver decompensation, and the need for liver transplantation. Causes of cholestatic liver disease include acute hepatitis and cholangitis.

[0489] Cholestatic liver diseases can include, but are not limited to, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), and Alagille syndrome (AS).

[0490] Primary biliary cirrhosis, also known as primary biliary cholangitis (PBC), is a chronic cholestatic liver disease of unknown etiology. Progressive damage to the bile ducts due to inflammation of the portal and periportal vessels can lead to progressive fibrosis and eventually cirrhosis. To date, immune, genetic, and environmental factors are considered potential causes of the disease. Primary biliary cirrhosis primarily affects middle-aged women, and early symptoms may include fatigue, itching, or unexplained hyperlipidemia.

[0491] PBC is known to be an immune-mediated primary cholecystitis. Specifically, immunohistochemical staining of T lymphocytes in the portal and periportal regions reveals CD4-positive and CD8-negative T cells. Abnormal suppressor T cell activity has been reported in asymptomatic first-degree relatives of affected individuals. Interleukins have been reported to play a role in the pathogenesis of PBC by contributing to alterations in immune function and fibrosis (GJWebb et al., J. Autoimmunity, 2015 Nov; 64:42–52).

[0492] Treatment for PBC involves bile acid therapy using ursodeoxycholic acid (UDCA) and obeticholic acid (OCA). The mechanism of action of these two drugs in PBC is related to their ability to activate FXR and TGFR-5 to exert anti-inflammatory effects. However, in patients treated with UDCA, approximately 40% do not achieve a sufficient biochemical response.

[0493] Primary sclerosing cholangitis (PSC) is a chronic, progressive cholestatic liver disease caused by inflammation and fibrosis of the intrahepatic / extrahepatic bile ducts of unknown etiology. Specifically, PSC is an inflammatory disease of the bile ducts and biliary tracts, and as the disease progresses, fibrosis occurs, and the bile duct walls thicken and narrow, leading to biliary strictures. Although the etiology of PSC is unknown, it is estimated that a complex interplay of factors, including genetic factors, environmental factors, and associated immune responses, contributes.

[0494] Primary sclerosing cholangitis is diagnosed when elevated levels of alkaline phosphatase, transaminase, and gamma globulin are observed in liver function tests performed using blood.

[0495] Treatment options for PSC have not been clearly reported, and liver transplantation is the only treatment that can fundamentally cure PSC.

[0496] Therefore, there is still a need to develop drugs that can treat PBS and PSC without side effects, while ensuring patient convenience.

[0497] The "cirrhosis" of this invention is a chronic disease that occurs during repeated episodes of hepatocyte regeneration and increased fibrosis, pathologically characterized by necrosis, inflammation, and fibrosis, and ultimately leads to cirrhotic complications (such as liver decompensation) and diseases (such as hepatocellular carcinoma), resulting in death. Cirrhosis is particularly asymptomatic in its early stages and is therefore often diagnosed only at a relatively late stage. Rapid treatment of liver fibrosis, a condition that precedes the development of cirrhosis, is necessary. The compositions according to the invention can exhibit preventive or therapeutic effects on cirrhosis, specifically cirrhosis accompanied by non-alcoholic steatohepatitis, but are not limited thereto.

[0498] The "hepatic decompensation" of this invention refers to a condition in which liver function is weakened due to liver damage or liver disease (such as viral hepatitis, cirrhosis, drugs, or alcohol), and the liver is unable to perform its normal physiological functions of protein synthesis and metabolism. Hepatic decompensation is classified into acute or chronic hepatic decompensation based on its rate of progression and is known to cause various complications. The compositions according to the invention exhibit effects such as inhibiting inflammation and fibrosis, and therefore can exhibit preventive or therapeutic effects against hepatic decompensation.

[0499] The "hepatocellular carcinoma" of this invention refers to a malignant tumor originating from hepatocytes, and can be divided into primary liver cancer (hepatocellular carcinoma) that begins in the hepatocytes themselves and metastatic liver cancer that spreads to the liver from cancer that originated in another tissue, with 90% or more of liver cancers being primary liver cancer. Besides hepatitis and chronic liver disease, alcohol, smoking, and obesity are known to be major causes of liver cancer. The compositions according to the invention can exhibit preventive or therapeutic effects against hepatocellular carcinoma, specifically hepatocellular carcinoma caused by non-alcoholic steatohepatitis, but are not limited thereto.

[0500] In examples of this invention, obese mice induced by a high-fat diet and mouse models induced by CD-HFD (choline deficiency and high-fat, high-cholesterol diet) were used. The CD-HFD diet-induced model has high fat and cholesterol levels, and therefore, prolonged intake may lead to fatty liver and steatohepatitis. Choline deficiency is known to exacerbate this steatohepatitis and even lead to fibrosis.

[0501] In examples of the invention, the effects of the compositions comprising (i) substances exhibiting glucagon activity and (ii) dual agonists of GLP-1 and GIP receptors of the invention have been examined in each model, and the results indicate that the compositions may be used for the prevention or treatment of metabolic syndromes and liver diseases such as liver fibrosis, simple steatosis, fatty liver, and non-alcoholic steatohepatitis.

[0502] The composition according to the invention may be a composition exhibiting one or more of the following properties, but is not limited thereto:

[0503] (a) Weight and fat loss;

[0504] (b) Improves blood lipid levels;

[0505] (c) Improves insulin sensitivity;

[0506] (d) Reduce the expression of UCP-1 and PGC-1α genes;

[0507] (e) Reduce NAS (NAFLD Activity Score); and

[0508] (f) Reduce the expression of collagen in liver tissue.

[0509] The compositions according to the invention may have no or relatively low levels of weight gain, which is a side effect of existing liver disease treatments.

[0510] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable carriers, excipients, or diluents. As used herein, the term “pharmaceutically acceptable” means an amount sufficient to demonstrate a therapeutic effect without causing side effects, and can be readily determined by those skilled in the art based on factors known in the medical field, such as the type of disease, the patient’s age, weight, health, sex, and sensitivity to the drug, route of administration, method of administration, frequency of administration, treatment duration, and combination or simultaneous use of drugs.

[0511] The pharmaceutical compositions of the present invention, comprising substances exhibiting glucagon activity and dual agonists of GLP-1 and GIP receptors, may further comprise pharmaceutically acceptable carriers. Carriers are not particularly limited thereto, but may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, fragrances, etc., for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., for injection; and alkalis, excipients, lubricants, preservatives, etc., may be used for topical administration.

[0512] The formulation of the composition of the present invention can be prepared in various forms by mixing with the aforementioned pharmaceutically acceptable carrier. For example, when administered orally, the composition can be prepared as tablets, lozenges, capsules, elixirs, suspensions, syrups, rice paper wafers, etc.; and in the case of injection, the composition can be prepared as single-dose ampoules or various dosage forms. Furthermore, the composition can be formulated as solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0513] Examples of suitable carriers, excipients, and diluents for formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil. The formulation may further contain fillers, anticoagulants, lubricants, wetting agents, fragrances, preservatives, etc.

[0514] The pharmaceutical compositions of the present invention may be selected from any one of the following formulations: tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations, and suppositories.

[0515] According to conventional methods in the pharmaceutical field, the composition may be formulated into a unit dosage form suitable for administration to a patient, specifically into a dosage form useful for the administration of peptide drugs, and administered via oral or parenteral routes (including skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, pulmonary, percutaneous, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, vaginal, or rectal routes) using methods of administration commonly used in the art, but the formulation and administration methods are not limited thereto.

[0516] Substances exhibiting glucagon activity, as well as dual agonists of GLP-1 and GIP receptors, can be used in combination with a variety of pharmaceutically acceptable carriers (e.g., saline or organic solvents), and carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers can be used as pharmaceutical agents to increase stability or absorption.

[0517] The dosage and amount of the pharmaceutical composition of the present invention are determined together with the type of drug as the active ingredient and several related factors (such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease).

[0518] Although not particularly limited thereto, the pharmaceutical compositions of the present invention may contain 0.01% to 99% of a component (active ingredient) by weight or volume.

[0519] The total effective amount of the composition of the present invention can be administered to the patient as a single dose, or it can be administered via a fractionated treatment regimen of multiple doses over a long period of time. Depending on the severity of the disease, the pharmaceutical composition of the present invention may contain varying amounts of the active ingredient. Specifically, the preferred total dose of the substance exhibiting glucagon activity and the dual agonist of GLP-1 and GIP receptors of the present invention may be from about 0.0001 μg to 500 mg per kg of patient body weight per day.

[0520] Specifically, the compositions of the present invention may comprise 0.15 nmol / kg to 2.5 nmol / kg, 0.19 nmol / kg to 2.25 nmol / kg, 0.25 nmol / kg to 1.5 nmol / kg, 0.37 nmol / kg to 1.12 nmol / kg of a substance exhibiting glucagon activity, and 2.0 nmol / kg to 35 nmol / kg, 2.08 nmol / kg to 31.16 nmol / kg, 10.39 nmol / kg to 31.16 nmol / kg of a dual agonist of GLP-1 and GIP receptors; more specifically, 0.37 nmol / kg, 0.75 nmol / kg, or 1.12 nmol / kg of a substance exhibiting glucagon activity, and 10.39 nmol / kg, 20.77 nmol / kg, or 31.16 nmol / kg of a dual agonist of GLP-1 and GIP receptors, but are not limited thereto. The compositions of the present invention may comprise, but are not limited thereto, a substance exhibiting glucagon activity and a dual agonist of GLP-1 and GIP receptors in 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.

[0521] However, the dosage of the peptide as an effective dose for the patient is determined by considering various factors, such as the severity of the disease and the patient's age, weight, health status, sex, diet, and excretion rate, as well as the route of administration and number of treatments of the pharmaceutical composition. With this in mind, those skilled in the art will be able to determine the appropriate effective dose for the specific use of the composition according to the invention. The pharmaceutical compositions according to the invention are not particularly limited in their formulation, route of administration, and method of administration, provided that the effects of the invention are demonstrated.

[0522] The pharmaceutical compositions of the present invention can have excellent duration and potency of in vivo efficacy, and can have fewer administrations and lower administration frequency compared to other drugs, but are not particularly limited thereto.

[0523] Specifically, the pharmaceutical compositions of the present invention comprise a glucagon derivative as an active ingredient, which has a modified pI compared to natural glucagon, thus exhibiting improved solubility and / or high stability at neutral pH, and is therefore useful for preparing stable glucagon formulations for treating target diseases including metabolic syndrome.

[0524] In pharmaceutical compositions for the prevention or treatment of metabolic syndrome, and in therapies for the prevention or treatment of metabolic syndrome, in addition to substances that exhibit activity against glucagon receptors and / or dual agonists of GLP-1 and GIP receptors, the pharmaceutical composition may also contain compounds or substances that exhibit therapeutic activity against metabolic syndrome, and the therapy may include further uses of the compounds or substances.

[0525] Another aspect of the invention provides a method for the prevention or treatment of metabolic syndrome, comprising administering to an individual in need a substance that exhibits activity against glucagon receptors and a dual agonist of GLP-1 and GIP receptors.

[0526] Substances exhibiting activity against glucagon receptors, dual agonists of GLP-1 and GIP receptors, compositions containing these, metabolic syndrome, prevention, and treatment as described above.

[0527] In this invention, an individual is an individual suspected of having metabolic syndrome, and an individual suspected of having metabolic syndrome refers to mammals such as mice and livestock, including humans who have or may have the disease, and includes, but is not limited to, individuals who can be treated with a composition containing a glucagon derivative and a dual agonist of GLP-1 and GIP receptors, or a composition containing a glucagon derivative and a dual agonist of GLP-1 and GIP receptors of this invention. An individual can be effectively treated by administering the pharmaceutical composition of this invention to an individual suspected of having metabolic syndrome. Metabolic syndrome is as described above.

[0528] The method of the present invention may include administering a pharmaceutical composition comprising a pharmaceutically effective amount of (i) a substance exhibiting activity against glucagon receptors and (ii) a dual agonist of GLP-1 and GIP receptors. The method according to the invention may be, but is not limited to, administering (i) the substance exhibiting activity against glucagon receptors and (ii) the dual agonist of GLP-1 and GIP receptors as a single formulation; or administering (i) the substance exhibiting activity against glucagon receptors and (ii) the dual agonist of GLP-1 and GIP receptors as separate formulations simultaneously, separately, sequentially, or in reverse order.

[0529] The treating physician can determine the appropriate daily dose within a reasonable medical judgment range, and it can be administered once or divided into several doses. However, for the purposes of this invention, the specific therapeutically effective dose for a particular patient is preferably applied differently based on various factors and similar factors known in the medical field, including the type and extent of the response to be achieved, the specific composition including whether other preparations are optionally used, the patient's age, weight, general health, sex and diet, time of administration, route of administration, secretion rate of the composition, treatment duration, and drugs used together or simultaneously with the specific composition.

[0530] Specifically, substances exhibiting activity against glucagon receptors, as well as dual agonists of GLP-1 and GIP receptors, may each be administered at a dose of approximately 0.0001 mg to 500 mg per kg of patient body weight per day. When these two substances are used in combination, the total dose may be approximately 0.0001 mg to 1000 mg per kg of patient body weight per day, but the dose is not limited to this.

[0531] The combination of substances that exhibit activity against glucagon receptors and dual agonists of GLP-1 and GIP receptors can be administered in molar ratios from 1:0.01 to 1:100, but the molar ratio is not limited to this.

[0532] Specifically, substances exhibiting glucagon activity can be administered at doses of 0.15 nmol / kg to 2.5 nmol / kg, 0.19 nmol / kg to 2.25 nmol / kg, 0.25 nmol / kg to 1.5 nmol / kg, or 0.37 nmol / kg to 1.12 nmol / kg, and dual agonists of GLP-1 and GIP receptors can be administered at doses of 2.0 nmol / kg to 35 nmol / kg, 2.08 nmol / kg to 31.16 nmol / kg, or 10.39 nmol / kg to 31.16 nmol / kg; and more specifically, substances exhibiting glucagon activity can be administered at doses of 0.37 nmol / kg, 0.75 nmol / kg, or 1.12 nmol / kg, and dual agonists of GLP-1 and GIP receptors can be administered at doses of 10.39 nmol / kg, 20.77 nmol / kg, or 31.16 nmol / kg, but the dosage is not limited thereto. Substances exhibiting glucagon activity and dual agonists of GLP-1 and GIP receptors can be administered at molar ratios 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 the molar ratios are not limited thereto.

[0533] Although not particularly limited thereto, methods for the prevention or treatment of metabolic syndrome may be combination therapy, which further includes, in addition to the administration of (i) a substance that exhibits activity against glucagon receptors and (ii) a dual agonist of GLP-1 and GIP receptors, the administration of a compound or substance that exhibits therapeutic activity against one or more metabolic syndromes.

[0534] Another aspect of the invention provides the use of a composition comprising (i) a substance exhibiting activity against glucagon receptors and (ii) a dual agonist of GLP-1 and GIP receptors for the prevention or treatment of metabolic syndrome.

[0535] Another aspect of the invention provides the use of a composition comprising (i) a substance exhibiting activity against glucagon receptors and (ii) a dual agonist of GLP-1 and GIP receptors in the preparation of a medicament for the prevention or treatment of metabolic syndrome.

[0536] The invention will be described in more detail below by way of examples. However, these examples are for exemplary purposes and the scope of the invention is not limited to these examples.

[0537] Example 1: A cell line that generates a cAMP response to glucagon

[0538] PCR was performed using the portion of the human glucagon receptor gene cDNA corresponding to the ORF as a template, and forward and reverse primers of SEQ ID NO:48 and SEQ ID NO:49, which respectively include EcoRI and XhoI cleavage sites.

[0539] The PCR reaction was then denatured at 95°C for 60 seconds, annealed at 55°C for 60 seconds, and extended at 68°C for 30 seconds, repeated 30 times. Electrophoresis was performed on a 1.0% agarose gel, and the amplified PCR product was obtained by eluting the 450 bp band.

[0540] Forward primer (SEQ ID NO:48):

[0541] 5′-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3′

[0542] Reverse primer (SEQ ID NO:49):

[0543] 5′-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3′

[0544] The PCR product was cloned into the known animal cell expression vector x0GC / dhfr to prepare the recombinant vector x0GC / GCGR.

[0545] The recombinant vector x0GC / GCGR, prepared thereby, was transformed into CHO DG44 cells cultured in DMEM / F12 medium containing 10% FBS using lipofectamine, and selectively cultured in selective medium containing 1 mg / mL G418 and 10 nM methotrexate. Monoclonal cell lines were thus selected by limiting dilution. Ultimately, cell lines exhibiting excellent concentration-dependent cAMP responses to glucagon were selected.

[0546] Example 2: Synthesis of glucagon derivatives

[0547] To develop glucagon derivatives exhibiting improved physical properties, the amino acid sequence of natural glucagon (SEQ ID NO: 1) was substituted with negatively and positively charged amino acid residues to synthesize glucagon derivatives as shown in Table 1 below. The relative in vitro activities described herein were measured using the methods described in Example 4 below.

[0548] [Table 1]

[0549] Amino acid sequences of natural glucagon and glucagon derivatives

[0550]

[0551]

[0552] In Table 1, the amino acids indicated by X represent the non-natural amino acid aminoisobutyric acid (Aib). An underline under the amino acid symbol indicates the formation of a lactam ring between the side chains of the corresponding underlined amino acid pair, while a "-" indicates that there is no amino acid residue at that position. In the columns indicating the presence or absence of ring formation, a "-" indicates that no ring has formed in the sequence.

[0553] Example 3: Measurement of pI of glucagon derivatives

[0554] To verify the improved physical properties of the glucagon derivative synthesized in Example 2, the pI was estimated from the amino acid sequence using the pI / Mw tool (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003) on the ExPASy server.

[0555] As shown in Table 1 above, the pI of natural glucagon in SEQ ID NO:1 is 6.8, while the pI of some glucagon derivatives according to the present invention is in the range of about 4 to 6. These glucagon derivatives have pIs lower or higher than that of natural glucagon, and therefore exhibit improved solubility and higher stability compared to natural glucagon at neutral pH and similar pH conditions.

[0556] When such glucagon derivatives according to the invention are used as therapeutic agents for target diseases such as metabolic syndrome, patient compliance can be increased, and these glucagon derivatives are suitable for combination with other anti-obesity or diabetes drugs and can be effectively used as therapeutic agents for metabolic syndromes (including obesity, diabetes, non-alcoholic steatohepatitis (NASH), dyslipidemia, and coronary heart disease).

[0557] Example 4: Measurement of cAMP activity of glucagon derivatives

[0558] The activity of the glucagon derivative synthesized in Example 2 was measured in cell lines containing the human glucagon receptor generated in Example 1. Specifically, the transformed cell lines were passaged 3 or 4 times per week, and then the passaged cell lines were cultured at 6 × 10⁶ cells per well. 3Cells were seeded in 384-well plates and cultured for 24 hours. Natural glucagon was suspended at 200 nM and glucagon derivatives at 1600 nM in HBSS (Hanks 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-piperazine ethanesulfonic acid), and then serially diluted 4-fold 10 times. The solutions were then added to the cultured cell lines using a cAMP assay kit (LANCE cAMP 384 kit, PerkinLemer), and fluorescence values ​​were measured. The highest fluorescence value was selected as 100% after measurement, from which the EC50 of the glucagon derivatives was calculated. 50 The value was compared with the EC50 of natural glucagon. 50 The values ​​were compared. Table 1 above shows the results.

[0559] Example 5: Preparation of a conjugate containing glucagon derivative and immunoglobulin Fc (glucagon derivative-immunoglobulin Fc region conjugate)

[0560] The glucagon derivatives prepared in Example 2 with a pI value of 6 to 7 and an in vitro activity of 200% or higher were selected as representative glucagon derivatives for preparing conjugates. Specifically, for the polyethylene glycolation of a 10 kDa PEG (i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan)) with maleimide and aldehyde groups at both ends, respectively, with cysteine ​​residues of the glucagon derivative, the molar ratio of the glucagon derivative to maleimide-PEG-aldehyde was set to 1:1 to 5, and the protein concentration was set to 3 mg / mL to 10 mg / mL. The reaction was carried out at low temperature for 1 to 3 hours. During this time, 20% to 60% isopropanol was added to a 50 mM Tris buffer (pH 7.5) for the reaction. After the reaction was completed, the reaction solution was applied to SP agarose gels (GE Healthcare, USA) to purify the cysteine-monopolyglycolated glucagon derivative.

[0561] Next, the purified polyethylene glycolated glucagon derivative and immunoglobulin Fc (a homodimer of SEQ ID NO:71) were reacted at 4°C to 8°C for 12 to 18 hours with a molar ratio of 1:2 to 10 and a protein concentration of 10 mg / mL to 50 mg / mL. The reaction was carried out in an environment where 10 mM to 50 mM sodium cyanoborohydride and 10% to 20% isopropanol were added as reducing agents to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was complete, the reaction solution was applied to a butyl agarose 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.

[0562] In immunoglobulin Fc, two monomers having the amino acid sequence SEQ ID NO:71 (consisting of 221 amino acids) form a homodimer through an inter-disulfide bond between cysteine ​​residues—cysteine ​​being the third amino acid in each monomer—and each monomer of the homodimer independently forms a disulfide bond between cysteine ​​residues at positions 35 and 95 and between cysteine ​​residues at positions 141 and 199.

[0563] After preparation, the purity was 95% or higher as analyzed by reversed-phase chromatography, size exclusion chromatography and ion exchange chromatography.

[0564] Here, the conjugates of glucagon derivatives and immunoglobulin Fc linked via PEG are named “long-acting glucagon derivative conjugates” or “long-acting glucagon derivatives”, and these conjugates are used interchangeably in this document.

[0565] Example 6: Generating cell lines that show cAMP response to GLP-1 and GIP

[0566] Recombinant vectors expressing human GLP-1 and human GIP receptors were constructed using the expression vector X0GC / dhfr. These vectors were then transformed into CHO DG44 cells using liposomes and selectively cultured in a selective medium containing G418 and methotrexate. Monoclonal cell lines were then selected by limiting dilution. Cell lines exhibiting excellent concentration-dependent cAMP responses to GLP-1 and GIP were ultimately selected.

[0567] Example 7: Preparation of a dual agonist of acylated GLP-1 and GIP receptors

[0568] Taripeptide, a dual agonist exhibiting activity against both GLP-1 and GIP receptors, was synthesized and its preparation was performed in accordance with a known method (WO 2016-111971 A1).

[0569] Example 8: Measurement of cAMP activity of a dual agonist of acylated GLP-1 and GIP receptors

[0570] The activity of the dual agonist synthesized in Example 7 was measured in cell lines expressing the human GLP-1 receptor and human GIP receptor generated in Example 6.

[0571] Specifically, the transformed cell line of Example 6 was passaged 3 or 4 times per week, and then the passaged cell line was cultured at 6 × 10⁶ cells per well. 3 Cells were seeded in 384-well plates and cultured for 24 hours. Natural GLP-1 and GIP were suspended at 200 nM and the dual agonist at 1600 nM in HBSS (Hanks 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-piperazine ethanesulfonic acid), and then serially diluted 4-fold 10 times. This was applied to a cAMP assay kit (LANCE-cAMP 384 kit, Perkinlemer) and added to each cultured cell line, and fluorescence values ​​were measured. Subsequently, the fluorescence value of the highest concentration of the natural substance used in each cell line was selected as 100%, from which the EC50 of the dual agonist was calculated. 50 The value was compared with the EC values ​​of natural GLP-1 and GIP. 50 The values ​​were compared. Table 2 below shows the results.

[0572] [Table 2]

[0573]

[0574]

[0575] Experimental Example 1: Comparing the effects of long-acting glucagon derivatives and dual agonists on weight and fat loss, improved blood lipid levels, improved insulin sensitivity, and increased expression of energy metabolism-related genes in high-fat diet-induced obese mice, and verifying the effects of combined administration on other factors.

[0576] This study used obese mice induced by a high-fat diet, a widely used animal model of obesity. The mice weighed approximately 50-55g before administration. During the study, seven mice were housed and had free access to water. The lights were turned off from 6 PM to 6 AM.

[0577] In the experimental groups fed a high-fat diet, Group 1 received a vehicle (5 mL / kg, injected every two days) without a long-acting glucagon derivative – the control group (vehicle). Group 2 received a GLP-1 analog anti-obesity drug at 50 nmol / kg. Group 3 received a long-acting glucagon derivative at 2.0 nmol / kg (injected every two days), Group 4 received a dual agonist (Tagristolide) at 20 nmol / kg (injected every two days), and Group 5 received a combination of a dual agonist at 20 nmol / kg (injected every two days) and a long-acting glucagon derivative at 2.0 nmol / kg (injected every two days). All cases were administered via subcutaneous injection.

[0578] The experiment ended on day 21. During the experiment, the body weight of mice in each group was measured every two days. After the experiment, tissue fat weight, blood lipid levels, HOMA-IR, and the expression of energy metabolism-related genes were measured.

[0579] As a result of measuring changes in body weight, from Figure 1 It can be seen that the body weight of the long-acting glucagon derivative (2.0 nmol / kg, administered every two days) alone group and the combination group of long-acting glucagon derivative (2.0 nmol / kg, administered every two days) and dual agonist (Tagristolide) (20 nmol / kg, injected every two days) changed by -34.92% and -51.16% respectively compared with the pre-treatment levels. The weight loss effect in the combination group was greater than that in the long-acting glucagon derivative alone group, and this effect was superior to that in the control group (transporter) and GLP-1 analog anti-obesity drugs. The weight loss effects were 2.22%, -14.73%, and -19.39% in the dual agonist (Taripipride).

[0580] from Figure 2 (A) and Figure 2 As shown in (B), it is confirmed that with weight loss, both fat mass and blood lipid levels decrease significantly. Figure 3 (A) As an example, HOMA-IR measurements confirmed that insulin sensitivity was also improved. Furthermore, as... Figure 3 (B) Example also confirmed increased expression of energy metabolism-related genes (UCP-1, a marker associated with heat production, and PGC-1α, a marker associated with mitochondrial biosynthesis) in adipose tissue.

[0581] Experimental Example 2: Effects of long-acting glucagon derivatives and dual agonists on improving NASH and fibrosis in mice with choline deficiency and high-fat, high-cholesterol diet-induced NASH and fibrosis.

[0582] To verify the efficacy of the combined administration of the long-acting glucagon derivative and GLP-1 / GIP dual agonist (Taripatide) prepared in the examples for improving NASH and fibrosis, a CD-HFD (choline deficiency and high-fat, high-cholesterol diet) mouse model was used.

[0583] Mice induced by CD-HFD for 8 weeks were randomly assigned to four groups: a vector control group, a long-acting glucagon derivative (1.3 nmol / kg, Q2D, subcutaneous) administration group, a dual agonist (73 nmol / kg, Q2D, subcutaneous) administration group, and a combination of long-acting glucagon derivative and dual agonist administration group. The administration was repeated for 6 weeks. A group of mice given the vector on a normal diet served as a negative control. After 6 weeks of repeated administration, liver tissue from each mouse was necropsy performed, and the efficacy in improving NASH and fibrosis was evaluated by measuring NAS (NAFLD activity score) by H&E staining and collagen expression by quantitative PCR.

[0584] Therefore, it was confirmed that when long-acting glucagon derivatives or dual agonists were repeatedly administered for 6 weeks, NAS was significantly reduced compared with the CD-HFD diet and the carrier control group.

[0585] Furthermore, it was confirmed that when a long-acting glucagon derivative and a dual agonist were administered in combination, the reduction in other NAS was significant. Figure 4 This study confirmed that the efficacy of combining long-acting glucagon derivatives and GLP-1 / GIP dual agonists in improving NASH can be further enhanced.

[0586] Furthermore, collagen expression in liver tissue is increased in the CD-HFD diet, and is significantly reduced in the carrier control group by administration of either a long-acting glucagon derivative or a dual agonist. This reduction is further enhanced by the combined administration of a long-acting glucagon derivative and a dual agonist. Figure 5 ).

[0587] We confirmed that the combined administration of a long-acting glucagon derivative and a GLP-1 / GIP dual agonist can effectively improve NASH and liver fibrosis in CD-HFD mice by reducing NAS and collagen expression in liver tissue.

[0588] For statistical processing of all cases, one-way ANOVA was used to compare the carrier group (control group) and the experimental group, and t-tests were used to compare other efficacy of the combination administration group and the single administration group (* to *** p < 0.05 to 0.001 vs. carrier control group).

[0589] These results indicate that long-acting glucagon derivatives exhibit greater weight loss effects than GLP-1 analogs and dual agonists alone, which are also used as other anti-obesity drugs. Furthermore, when long-acting glucagon derivatives are administered in combination with dual agonists of GLP-1 and GIP receptors, enhanced effects on obesity treatment, not only through other weight loss mechanisms, but also through improved insulin sensitivity, have been anticipated. In addition to these effects on improving obesity and blood glucose, the combination of long-acting glucagon derivatives and dual agonists of GLP-1 and GIP receptors has been confirmed to provide excellent efficacy against liver diseases such as non-alcoholic steatohepatitis or its fibrotic effects, through reduced NAS and collagen expression in liver tissue.

[0590] Based on the foregoing description, those skilled in the art will understand that the present invention may be implemented in different specific forms without altering its technical spirit or essential characteristics. Therefore, it should be understood that the above embodiments are not restrictive, but exemplary in all respects. The scope of this disclosure is defined by the appended claims rather than the foregoing description; therefore, all changes and modifications falling within the scope of the claims or their equivalents are intended to be covered by the claims. <110> Hanmi Pharmaceutical Co., Ltd. <120> Combinations of glucagon and GLP-1 / GIP receptor agonists, and their therapeutic uses. <130> OPA20169 <150> KR 10-2019-0123250 <151> 2019-10-04 <160> 71 <170> KoPatentIn 3.0 <210> 1 <211> 29 <212> PRT <213> Homo sapiens <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 2 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 2 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Asp Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 3 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Glu Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 4 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 4 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Asp Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 5 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 5 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Glu Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 6 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 6 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Glu Ala Asp Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 7 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <400> 7 Tyr Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Glu Ala Asp Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 8 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 8 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Asp Ala Gln Asp Phe Val Gln Trp Leu Ile Asn Thr 20 25 <210> 9 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 9 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Asp Ala Gln Asp Phe Val Val Trp Leu Ile Asn Thr 20 25 <210> 10 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 10 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Cys Asp Ala Asp Asp Phe Val Val Trp Leu Ile Asn Thr 20 25 <210> 11 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 11 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Cys Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 12 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 12 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 13 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 13 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Cys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 14 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 14 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 15 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 15 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Cys Glu 1 5 10 15 Lys Arg Ala Gln Asp Phe Val Val Trp Leu Met Asn Thr 20 25 <210> 16 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 16 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Arg Arg Ala Gln Val Phe Val Gln Trp Leu Met Arg Thr 20 25 <210> 17 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 17 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Val Arg Ala Gln Asp Phe Val Gln Trp Leu Met Arg Thr 20 25 <210> 18 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 18 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Cys Asp Phe Arg Leu Trp Leu Met Asn Thr 20 25 <210> 19 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 19 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Cys Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 20 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 20 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> twenty one <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> twenty one Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Cys Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> twenty two <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> twenty two Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Cys Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> twenty three <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> twenty three Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> twenty four <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> twenty four Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 25 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 25 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Cys Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 26 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 26 Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 27 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 27 Tyr Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 28 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 28 Trp Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 29 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 29 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 30 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 30 Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 31 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (17) (21) <223> The 17th and 21st amino acids form a ring. <400> 31 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 32 <211> 28 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (15)..(19) <223> The 15th and 19th amino acids form a ring. <400> 32 Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Cys 1 5 10 15 Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 33 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 33 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 34 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 34 Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu 1 5 10 15 Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 35 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 35 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu 1 5 10 15 Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 36 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 36 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 37 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 37 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 38 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 38 Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 39 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 39 Tyr Xaa Gln Gly Thr Phe Val Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 40 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 40 Tyr Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 41 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The 16th and 20th amino acids form a ring. <400> 41 Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 42 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 42 Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 43 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 43 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 44 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 44 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 45 <211> 29 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 45 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Cys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 46 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is α-methylglutamic acid, aminoisobutyric acid (Aib), D-alanine, glycine, Sar (N-methylglycine), serine, or D-serine. <220> <221> MISC_FEATURE <222> (7) <223> Xaa is threonine, valine, or cysteine. <220> <221> MISC_FEATURE <222> (10) <223> Xaa is tyrosine or cysteine. <220> <221> MISC_FEATURE <222> (12) <223> Xaa is either lysine or cysteine. <220> <221> MISC_FEATURE <222> (13) <223> Xaa is tyrosine or cysteine. <220> <221> MISC_FEATURE <222> (14) <223> Xaa is leucine or cysteine. <220> <221> MISC_FEATURE <222> (15) <223> Xaa is aspartic acid, glutamic acid, or cysteine. <220> <221> MISC_FEATURE <222> (16) <223> Xaa is glutamic acid, aspartic acid, serine, α-methylglutamic acid, or cysteine, or it may not exist. <220> <221> MISC_FEATURE <222> (17) <223> Xaa may be aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or it may not be present. <220> <221> MISC_FEATURE <222> (18) <223> Xaa can be alanine, aspartic acid, glutamic acid, arginine, valine, or cysteine, or it may not be present. <220> <221> MISC_FEATURE <222> (19) <223> Xaa can be alanine, arginine, serine, valine, or cysteine, or it may not be present. <220> <221> MISC_FEATURE <222> (20) <223> Xaa may be lysine, histidine, glutamine, aspartic acid, arginine, α-methylglutamic acid, or cysteine, or may not be present. <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa can be aspartic acid, glutamic acid, leucine, valine, or cysteine, or it may not be present. <220> <221> MISC_FEATURE <222> (twenty three) <223> Xaa may be isoleucine, valine, or arginine, or it may not exist. <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa can be valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methylglutamic acid, or leucine, or it may not be present. <220> <221> MISC_FEATURE <222> (27) <223> Xaa may be isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or it may not be present. <220> <221> MISC_FEATURE <222> (28) <223> Xaa may be glutamine, lysine, asparagine, or arginine, or it may not be present. <220> <221> MISC_FEATURE <222> (30) <223> Xaa may be cysteine ​​or it may not be present. <400> 46 Tyr Xaa Gln Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Xaa Xaa Thr Xaa 20 25 30 <210> 47 <211> 30 <212> PRT <213> Artificial sequence <220> <223> glucagon derivatives <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (7) <223> Xaa is threonine, valine, or cysteine. <220> <221> MISC_FEATURE <222> (10) <223> Xaa is tyrosine or cysteine. <220> <221> MISC_FEATURE <222> (12) <223> Xaa is either lysine or cysteine. <220> <221> MISC_FEATURE <222> (15) <223> Xaa is aspartic acid or cysteine. <220> <221> MISC_FEATURE <222> (16) <223> Xaa is glutamic acid or serine. <220> <221> MISC_FEATURE <222> (17) <223> Xaa is either lysine or arginine. <220> <221> MISC_FEATURE <222> (20) <223> Xaa is glutamine or lysine. <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa is aspartic acid or glutamic acid. <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa is valine or glutamine. <220> <221> MISC_FEATURE <222> (30) <223> Xaa may be cysteine ​​or it may not be present. <400> 47 Tyr Xaa Gln Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Tyr Leu Xaa Xaa 1 5 10 15 Xaa Arg Ala Xaa Xaa Phe Val Xaa Trp Leu Met Asn Thr Xaa 20 25 30 <210> 48 <211> 34 <212> DNA <213> Artificial sequence <220> <223> forward primer <400> 48 cagcgacacc gaccgtcccc ccgtacttaa ggcc 34 <210> 49 <211> 32 <212> DNA <213> Artificial sequence <220> <223> reverse primer <400> 49 ctaaccgact ctcggggaag actgagctcg cc 32 <210> 50 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Tareparaide <220> <221> MISC_FEATURE <222> (2) <223> Xaa is 2-methylalanine (Aib). <220> <221> MISC_FEATURE <222> (13) <223> Xaa is 2-methylalanine (Aib). <220> <221> MISC_FEATURE <222> (20) <223> Xaa is N6-{N-(eicosanoyl)-g-Glu-bis[iminobis(ethoxy)acetyl]}-lysine <220> <221> MISC_FEATURE <222> (39) <223> Xaa is L-seramide <400> 50 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 51 <211> 12 <212> PRT <213> Homo sapiens <400> 51 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 52 Glu Ser Lys Tyr Gly Pro Pro Pro Ser Cys Pro 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 53 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Pro 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 54 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 55 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 1 5 10 <210> 56 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 56 Lys Tyr Gly Pro Pro Cys Pro Ser 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 57 Glu Ser Lys Tyr Gly Pro Pro Cys 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 58 Glu Lys Tyr Gly Pro Pro Cys 1 5 <210> 59 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 59 Glu Ser Pro Ser Cys Pro 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 60 Glu Pro Ser Cys Pro 1 5 <210> 61 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 61 Pro Ser Cys Pro 1 <210> 62 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 62 Glu Ser Lys Tyr Gly Pro Pro Ser Cys Pro 1 5 10 <210> 63 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 63 Lys Tyr Gly Pro Pro Pro Ser Cys Pro 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 64 Glu Ser Lys Tyr Gly Pro Ser Cys Pro 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 65 Glu Ser Lys Tyr Gly Pro Pro Cys 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 66 Lys Tyr Gly Pro Pro Cys Pro 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 67 Glu Ser Lys Pro Ser Cys Pro 1 5 <210> 68 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 68 Glu Ser Pro Ser Cys Pro 1 5 <210> 69 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 69 Glu Pro Ser Cys 1 <210> 70 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Variations of the hinge area <400> 70 Ser Cys Pro 1 <210> 71 <211> 221 <212> PRT <213> Homo sapiens <400> 71 Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 1 5 10 15 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20 25 30 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 35 40 45 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 55 60 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 65 70 75 80 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 85 90 95 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 100 105 110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120 125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 130 135 140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 145 150 155 160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 165 170 175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185 190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200 205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220

Claims

1. The use of a pharmaceutical composition comprising (i) a substance exhibiting activity against a glucagon receptor and (ii) a dual agonist of GLP-1 and GIP receptors in the preparation of a medicament for the prevention or treatment of metabolic syndrome, wherein the substance exhibiting activity against the glucagon receptor is a peptide, the peptide comprising an amino acid sequence selected from SEQ ID NO: 20, 21, 22, 23, 24, 27, 29, 33, 37, 38, and 44, and The GLP-1 and GIP receptor dual agonist mentioned above is taripipride. The metabolic syndrome mentioned above is selected from glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, and liver disease, and The peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following chemical formula 1: [Chemical Formula 1] XLF Where X is a peptide comprising an amino acid sequence selected from SEQ ID NO: 20, 21, 22, 23, 24, 27, 29, 33, 37, 38 and 44; L is a linker containing repeating ethylene glycol units; F stands for the Fc region of immunoglobulins. The immunoglobulin Fc region mentioned therein is the IgG4 Fc region; and - represents the covalent bond connection between X and L, and between L and F.

2. The application according to claim 1, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO: 20 to 24, 27, 29 and 33.

3. The application according to claim 1, wherein the peptide comprises an amino acid sequence selected from SEQ ID NO: 20, 22, 23, 27, 33, 37, 38 and 44.

4. The application according to claim 1, wherein the C-terminus of the peptide is amidated.

5. The application according to claim 1, wherein the GLP-1 and GIP receptor dual agonist is a peptide that exhibits activity against both the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic peptide) receptor.

6. The application according to claim 1, wherein the chemical formula weight of the repeating unit portion of ethylene glycol in L is in the range of 1 kDa to 100 kDa.

7. The application according to claim 1, wherein the liver disease is selected from at least one disease selected from liver inflammation, cholestatic liver disease, liver fibrosis, cirrhosis and hepatocellular carcinoma.

8. The application according to claim 1, wherein the liver disease is non-alcoholic steatohepatitis (NASH).

9. The application according to claim 1, wherein the liver disease is at least one disease selected from simple steatosis and non-alcoholic fatty liver disease.

10. The application according to claim 7, wherein the cholestatic liver disease is selected from primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

11. The application according to claim 7, wherein the liver disease is caused by or accompanied by non-alcoholic steatohepatitis.

12. The application according to claim 1, wherein the composition performs one or more of the following properties: (a) Weight and fat mass reduction; (b) Improve blood lipid levels; (c) Improves insulin sensitivity; (d) Reduce the expression of UCP-1 and PGC-1α genes; (e) Reduce NAFLD activity scores (NAS); and (f) Reduce collagen expression in liver tissue.

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