Long-acting conjugate of a glucagon / GLP-1 / GIP receptor triple agonist

Abstract

The invention title of the present invention is a long-acting conjugate of a glucagon / GLP-1 / GIP receptor triple agonist. The present invention relates to a long-acting conjugate of a triple agonist that is active against all of the glucagon, GLP-1, and GIP receptors and its uses.

Description

[0001] This application is a divisional application. The filing date of the original application is December 30, 2016, the application number is 2016800823398 (PCT / KR2016 / 015555), and the invention title is "Long-acting Conjugates of Glucagon / GLP-1 / GIP Receptor Triple Agonists". Technical Field

[0002] The present invention relates to long-acting conjugates of triple agonists that are active against all of the glucagon, GLP-1, and GIP receptors, and uses thereof. Background Art

[0003] Obesity and diabetes including type 2 diabetes are representative metabolic diseases that occur in modern society. These diseases are considered factors threatening health in the world and the economic costs associated with the incidence of these diseases are currently increasing rapidly.

[0004] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are representative gastrointestinal hormones and neuronal hormones and are substances involved in controlling blood sugar levels according to food intake. Glucagon is a peptide hormone secreted by the pancreas and is involved in controlling blood sugar levels together with the above two substances.

[0005] GLP-1 is a hormone secreted by the small intestine stimulated by food intake. GLP-1 promotes insulin secretion in the pancreas and inhibits glucagon secretion in a blood sugar-dependent manner, thereby helping to reduce blood sugar levels. In addition, GLP-1 has the effect of slowing down the digestive action in the gastrointestinal tract by acting as a satiety factor, and reducing the amount of food intake by delaying the time for emptying the digested food in the gastrointestinal tract. In addition, it has been reported that administering GLP-1 to rats has the effects of inhibiting food intake and reducing body weight, and it has been confirmed that these effects occur equally in normal and obese states, thus showing the potential of GLP-1 as a medicament for treating obesity.

[0006] GIP, one of the gastrointestinal hormones secreted by the stimulation of food intake, like GLP-1, is a hormone composed of 42 amino acids secreted by intestinal K cells. It has been reported that GIP performs the function of promoting insulin secretion in the pancreas and helping to reduce blood sugar levels in a blood sugar-dependent manner, thereby showing the effects of increasing GLP-1 activation, anti-inflammatory effects, etc.

[0007] When blood glucose levels drop due to reasons such as drugs, diseases, lack of hormones or enzymes, glucagon is produced in the pancreas. Glucagon signals the breakdown of glycogen in the liver to induce the release of glucose and increase blood glucose levels to normal levels. In addition to the effect of increasing blood glucose levels, glucagon also suppresses the appetite of animals and humans, and activates the hormone-sensitive lipase in adipocytes to promote lipolysis and energy consumption, thus showing an anti-obesity effect.

[0008] Thus, based on the effects of GLP-1 in controlling blood glucose levels and reducing weight, active research is underway to develop GLP-1 as a therapeutic agent for treating diabetes and obesity. Currently, exendin-4, which is prepared from lizard venom and has about 50% amino acid homology with GLP-1, is in the process of being developed as a therapeutic agent for treating the same types of diseases. However, it has been reported that therapeutic agents containing GLP-1 and exendin-4 show side effects such as vomiting and nausea (Syed Y Y., Drugs, 2015 Jul; 75(10): 1141 - 52).

[0009] In addition, to maximize weight loss and as an alternative to the above GLP-1-based therapeutic substances, research has been focused on dual agonists that are active on both GLP-1 and glucagon receptors, and they show more effectiveness in weight loss due to the activation of the glucagon receptor compared to the existing GLP-1 treatment alone (Jonathan W et al., Nat Chem Bio., 2009 Oct(5); 749 - 757).

[0010] In addition, in research related to triple agonists that are simultaneously active on all of GLP-1, GIP, and glucagon receptors, efforts have recently been made to increase resistance to dipeptidyl peptidase-IV (DPP-IV), which breaks down gastrointestinal hormones to remove their activity, by substituting the amino acid sequence, and then to increase the half-life of the triple agonist by adding an acyl group to its specific region (Finan B et al., Nat Med., 2015 Jan; 21(1): 27 - 36). However, their effects of activating three different types of receptors are not significant, and the triple agonists do not show their various active ratios.

[0011] Therefore, there is a need to develop a new substance that can highly activate GLP-1, GIP, and glucagon receptors and has the effects of controlling blood glucose levels and reducing weight without causing any side effects such as vomiting and nausea.

[0012] In addition, there is a need to develop novel substances having various activity ratios against GLP-1, GIP, and glucagon receptors. For example, there is an increasing need to develop substances that have an effect of reducing body weight but have a significantly higher effect of controlling blood glucose levels due to high GLP-1 and GIP activities, and on the other hand have relatively low glucagon activity for hypoglycemic effects; or substances that have high activities against all of GLP-1, GIP, and glucagon and thus have a significantly high effect of reducing body weight.

[0013] In addition, GLP-1, GIP, and glucagon have low stability in the body, and thus they have the drawback that when they are applied to humans for therapeutic use, they must be administered daily or twice daily. Summary of the Invention

[0014] Technical Problem

[0015] An object of the present invention is to provide a conjugate of a peptide having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0016] Another object of the present invention is to provide a polynucleotide encoding the conjugate, a vector comprising the polynucleotide, and a transformant comprising the polynucleotide or the vector.

[0017] Yet another object of the present invention is to provide a composition containing the conjugate.

[0018] Yet another object of the present invention is to provide a method for treating a target disease, the method comprising administering the conjugate or a composition containing the conjugate to a subject in need thereof.

[0019] Yet another object of the present invention is to provide the use of the conjugate or its composition for the preparation of a medicament.

[0020] Technical Solution

[0021] To achieve the above object, in one aspect, the present invention provides a conjugate of a peptide having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0022] In a specific embodiment, the conjugate is represented by the following Chemical Formula 1:

[0023] [Chemical Formula 1]

[0024] X-La-F

[0025] Wherein,

[0026] X is a peptide that is active against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor;

[0027] L is a linker;

[0028] a is 0 or a positive integer, provided that when a is 2 or greater, each L is independent of one another; and

[0029] F is a substance capable of increasing the half-life of X.

[0030] In another specific embodiment, X is an analogue of native glucagon having a variation selected from substitution, addition, deletion, modification, and combinations thereof on at least one amino acid of the native glucagon sequence.

[0031] In yet another specific embodiment, the added amino acid sequence is derived from the native GLP-1 amino acid sequence, the native GIP amino acid sequence, or the native exendin-4 amino acid sequence.

[0032] In yet another specific embodiment, X is a peptide comprising an amino acid sequence represented by the following general formula 1:

[0033] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103)

[0034] Wherein, in General formula 1,

[0035] Xaa1 is histidine (His, H), 4-imidazolylacetyl (CA), or tyrosine (Tyr, Y);

[0036] Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib (amino isobutyric acid);

[0037] Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);

[0038] Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);

[0039] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or valine (Val, V);

[0040] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

[0041] Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine (Cys, C);

[0042] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

[0043] Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L);

[0044] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

[0045] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);

[0046] Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);

[0047] Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), or valine (Val, V);

[0048] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

[0049] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);

[0050] Xaa23 is isoleucine (Ile, I) or valine (Val, V);

[0051] Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E);

[0052] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);

[0053] Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D);

[0054] Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);

[0055] Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or is absent; and

[0056] R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO:106), m-SSGAPPPS-n (SEQ ID NO:107), or m-SSGQPPPS-n (SEQ ID NO:108), or is absent;

[0057] wherein,

[0058] m is -Cys-, -Pro-, or -Gly-Pro-,

[0059] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.

[0060] In yet another specific embodiment,

[0061] In general formula 1,

[0062] Xaa14 is leucine or methionine; and

[0063] Xaa15 is cysteine, aspartic acid, or leucine.

[0064] In yet another specific embodiment, in general formula 1,

[0065] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0066] Xaa7 is threonine;

[0067] Xaa10 is tyrosine, cysteine, or valine;

[0068] Xaa12 is lysine or isoleucine;

[0069] Xaa13 is tyrosine, alanine, glutamine, or cysteine;

[0070] Xaa14 is leucine, cysteine, or methionine;

[0071] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

[0072] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0073] Xaa18 is alanine, glutamine, arginine, or histidine;

[0074] Xaa19 is alanine, glutamine, valine, or cysteine;

[0075] Xaa20 is lysine, arginine, or glutamine;

[0076] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0077] Xaa23 is isoleucine or valine;

[0078] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and Xaa27 is leucine or lysine.

[0079] In yet another specific embodiment, in general formula 1,

[0080] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0081] Xaa7 is threonine;

[0082] Xaa10 is tyrosine, cysteine, or valine;

[0083] Xaa12 is lysine or isoleucine;

[0084] Xaa13 is tyrosine, alanine, or cysteine;

[0085] Xaa14 is leucine or methionine;

[0086] Xaa15 is cysteine or aspartic acid;

[0087] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

[0088] Xaa18 is alanine, arginine, or histidine;

[0089] Xaa19 is alanine, glutamine, or cysteine;

[0090] Xaa20 is lysine or glutamine;

[0091] Xaa21 is glutamate, cysteine, or aspartate;

[0092] Xaa23 is valine;

[0093] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartate; and Xaa27 is leucine or lysine.

[0094] In yet another specific embodiment, in General Formula 1,

[0095] Xaa2 is α-methyl-glutamate or Aib;

[0096] Xaa7 is threonine;

[0097] Xaa10 is tyrosine or cysteine;

[0098] Xaa12 is lysine or isoleucine;

[0099] Xaa13 is tyrosine, alanine, or cysteine;

[0100] Xaa14 is leucine or methionine;

[0101] Xaa15 is cysteine or aspartate;

[0102] Xaa16 is glutamate;

[0103] Xaa17 is arginine, isoleucine, cysteine, or lysine; Xaa18 is alanine, arginine, or histidine;

[0104] Xaa19 is alanine, glutamine, or cysteine;

[0105] Xaa20 is lysine or glutamine;

[0106] Xaa21 is glutamate or aspartate;

[0107] Xaa23 is valine;

[0108] Xaa24 is glutamine, asparagine, or aspartate; Xaa27 is leucine; and

[0109] Xaa28 is cysteine, alanine, asparagine, or aspartate. In yet another specific embodiment, in General Formula 1, Xaa1 is histidine or 4-imidazolylacetyl;

[0110] Xaa2 is α-methyl-glutamic acid or Aib;

[0111] Xaa3 is glutamine;

[0112] Xaa7 is threonine;

[0113] Xaa10 is tyrosine;

[0114] Xaa12 is isoleucine;

[0115] Xaa13 is alanine or cysteine;

[0116] Xaa14 is methionine;

[0117] Xaa15 is aspartic acid;

[0118] Xaa16 is glutamic acid;

[0119] Xaa17 is isoleucine or lysine;

[0120] Xaa18 is alanine or histidine;

[0121] Xaa19 is glutamine or cysteine;

[0122] Xaa20 is lysine;

[0123] Xaa21 is aspartic acid;

[0124] Xaa23 is valine;

[0125] Xaa24 is asparagine;

[0126] Xaa27 is leucine;

[0127] Xaa28 is alanine or asparagine;

[0128] Xaa29 is glutamine or threonine; and

[0129] Xaa30 is cysteine, or lysine, or is absent.

[0130] In yet another specific embodiment,

[0131] In general formula 1,

[0132] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0133] Xaa3 is glutamine;

[0134] Xaa7 is threonine;

[0135] Xaa10 is tyrosine, cysteine, or valine;

[0136] Xaa12 is lysine;

[0137] Xaa13 is tyrosine;

[0138] Xaa14 is leucine;

[0139] Xaa15 is aspartic acid;

[0140] Xaa16 is glycine, glutamic acid, or serine;

[0141] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0142] Xaa18 is alanine, arginine, or histidine;

[0143] Xaa19 is alanine or glutamine;

[0144] Xaa20 is lysine or glutamine;

[0145] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0146] Xaa23 is valine;

[0147] Xaa24 is alanine, glutamine, or cysteine;

[0148] Xaa27 is leucine or lysine; and

[0149] Xaa29 is glycine, glutamine, threonine, or histidine.

[0150] In yet another specific embodiment, X is a peptide comprising an amino acid sequence represented by the following general formula 2:

[0151] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General formula 2, SEQ ID NO: 104)

[0152] In general formula 2,

[0153] Xaa1 is 4-imidazolylacetyl, histidine, or tyrosine;

[0154] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0155] Xaa10 is tyrosine or cysteine;

[0156] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0157] Xaa14 is leucine, methionine, or tyrosine;

[0158] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0159] Xaa16 is glycine, glutamic acid, or serine;

[0160] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0161] Xaa18 is alanine, glutamine, arginine, or histidine;

[0162] Xaa19 is alanine, glutamine, cysteine, or valine;

[0163] Xaa20 is lysine, glutamine, or arginine;

[0164] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0165] Xaa23 is isoleucine or valine;

[0166] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0167] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

[0168] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0169] Xaa30 is cysteine, glycine, lysine, or histidine;

[0170] Xaa31 is proline or cysteine; and

[0171] Xaa40 is cysteine, or is absent.

[0172] In yet another specific embodiment, in General Formula 2,

[0173] Xaa13 is alanine, tyrosine, or cysteine;

[0174] Xaa15 is aspartic acid or glutamic acid;

[0175] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0176] Xaa18 is alanine, arginine, or histidine;

[0177] Xaa21 is cysteine, glutamate, glutamine, or aspartic acid;

[0178] Xaa23 is isoleucine or valine;

[0179] Xaa24 is cysteine, glutamine, or asparagine;

[0180] Xaa28 is cysteine, asparagine, or aspartic acid;

[0181] Xaa29 is glutamine, cysteine, or histidine; and

[0182] Xaa30 is cysteine, lysine, or histidine.

[0183] In yet another specific embodiment, X is a peptide comprising an amino acid sequence represented by the following general formula 3:

[0184] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General formula 3, SEQ ID NO: 105).

[0185] In General formula 3,

[0186] Xaa1 is histidine or tyrosine;

[0187] Xaa2 is α-methyl-glutamic acid or Aib;

[0188] Xaa13 is alanine, tyrosine, or cysteine;

[0189] Xaa17 is arginine, cysteine, or lysine;

[0190] Xaa18 is alanine or arginine;

[0191] Xaa19 is alanine or cysteine;

[0192] Xaa21 is glutamate or aspartic acid;

[0193] Xaa24 is glutamine or asparagine;

[0194] Xaa28 is cysteine or aspartic acid;

[0195] Xaa29 is cysteine, histidine, or glutamine;

[0196] Xaa30 is cysteine or histidine;

[0197] Xaa31 is proline or cysteine; and

[0198] Xaa40 is cysteine or is absent.

[0199] In yet another specific embodiment, R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or is absent.

[0200] In yet another specific embodiment, in Formulas 1 to 3, the 16th amino acid and the 20th amino acid from the N-terminus together form a loop.

[0201] In yet another specific embodiment, X is a peptide comprising an amino acid sequence selected from SEQ ID NOS: 1 to 102.

[0202] In yet another specific embodiment, F is selected from polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissues, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

[0203] In yet another specific embodiment, F is a polymer selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextrans, polyvinyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0204] In yet another specific embodiment, F is the Fc region of an immunoglobulin.

[0205] In yet another specific embodiment, F is the IgG Fc region.

[0206] In yet another specific embodiment, L is selected from peptides, fatty acids, saccharides, polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0207] In yet another specific embodiment, L is a polymer selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof.

[0208] In yet another specific embodiment, L is polyethylene glycol.

[0209] Yet another aspect of the present invention provides a polynucleotide encoding the conjugate, a vector comprising the polynucleotide, and a transformant comprising the polynucleotide or the vector.

[0210] Yet another aspect of the present invention provides a composition comprising the conjugate.

[0211] In one specific embodiment, the composition is a pharmaceutical composition.

[0212] In another specific embodiment, the composition is for preventing or treating metabolic syndrome.

[0213] In yet another specific embodiment, metabolic syndrome may include glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary heart disease.

[0214] Yet another aspect of the present invention provides a method for treating a target disease, the method comprising administering to a subject in need thereof the conjugate or the composition comprising the conjugate.

[0215] In one specific embodiment, the disease is metabolic syndrome.

[0216] Yet another aspect of the present invention provides the use of the conjugate or the composition comprising the conjugate in the preparation of a medicament.

[0217] In one specific embodiment, the medicament is for preventing or treating metabolic syndrome.

[0218] Advantages of the Invention

[0219] The conjugate of the triple agonist according to the present invention has activity against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor and can thus be applied to the treatment of metabolic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figure 1 The histogram shows the measurement results of the body weight change in an obese animal model (mouse) at two-day intervals during the 28-day administration of the long-acting conjugate of the triple agonist to the mouse every two days by a high-fat diet (p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA relative to the vehicle);

[0221] Figure 2 The histogram shows the measurement results of the mesenteric fat mass in an obese animal model (mouse) at two-day intervals during the 28-day administration of the long-acting conjugate of the triple agonist to the mouse every two days by a high-fat diet (p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA relative to the vehicle). DETAILED DESCRIPTION

[0222] Hereinafter, the present invention will be described in more detail.

[0223] Meanwhile, each of the descriptions and exemplary embodiments disclosed herein can be applied to other descriptions and exemplary embodiments. That is, all combinations of the various elements disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the specific disclosure provided below.

[0224] Throughout the specification of the present invention, not only the conventional single-letter and three-letter codes of naturally occurring amino acids are used, but also those three-letter codes generally allowed for other amino acids such as α-aminoisobutyric acid (Aib), Sar (N-methylglycine), and α-methyl-glutamic acid.

[0225] In addition, the amino acids mentioned herein are abbreviated as follows according to the nomenclature rules of IUPAC-IUB:

[0226] Alanine (Ala, A) Arginine (Arg, R)

[0227] Asparagine (Asn, N) Aspartic acid (Asp, D)

[0228] Cysteine (Cys, C) Glutamic acid (Glu, E)

[0229] Glutamine (Gln, Q) Glycine (Gly, G)

[0230] Histidine (His, H) Isoleucine (Ile, I)

[0231] Leucine (Leu, L) Lysine (Lys, K)

[0232] Methionine (Met, M) Phenylalanine (Phe, F)

[0233] Proline (Pro, P) Serine (Ser, S)

[0234] Threonine (Thr, T) Tryptophan (Trp, W)

[0235] Tyrosine (Tyr, Y) Valine (Val, V)

[0236] One aspect of the present invention provides conjugates of peptides that are active against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0237] In the present invention, the conjugate of a peptide that is active against the glucagon receptor, the GLP-1 receptor, and the GIP receptor can be in a form in which a biocompatible substance is conjugated to the peptide to increase the in vivo half-life of the peptide. In the present invention, the biocompatible substance can be used interchangeably with a carrier.

[0238] In the present invention, the conjugate of the peptide can exhibit an increased duration of potency compared to the peptide not conjugated to a carrier, and this conjugate is referred to herein as a "long-acting conjugate".

[0239] At the same time, such a conjugate can be non-naturally occurring.

[0240] In a specific embodiment of the present invention, the conjugate is a conjugate represented by the following Chemical Formula 1:

[0241] [Chemical Formula 1]

[0242] X-La-F

[0243] Wherein,

[0244] X is a peptide that is active against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor;

[0245] L is a linker;

[0246] a is 0 or a positive integer, provided that when a is 2 or greater, each L is independent of one another; and

[0247] F is a substance capable of increasing the half-life of X.

[0248] In the present invention, the "peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor" may correspond to a structure (constituent part, constitution) that forms a part of the above conjugate. Specifically, the peptide corresponds to X in Chemical Formula 1 above.

[0249] In the present invention, the peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor can be used interchangeably with the triple agonist.

[0250] The peptide may include various substances (e.g., various peptides) having a significant level of activity against the glucagon, GLP-1, and GIP receptors.

[0251] Compared with the natural ligands of the corresponding receptors (natural glucagon, natural GLP-1, and natural GIP), the triple agonist having a significant level of activity against the glucagon, GLP-1, and GIP receptors may exhibit in vitro activity of 0.1% or higher, 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, and 100% or higher against one or more, specifically two or more, more specifically all three of the glucagon, GLP-1, and GIP receptors, but is not particularly limited thereto.

[0252] The method for measuring the in vitro activity of the triple agonist can refer to Experimental Example 1 of the present invention, but is not particularly limited thereto.

[0253] Meanwhile, the triple agonist is characterized by having one or more, two or more, and specifically all three of the following activities i) to iii), specifically its significant activity (one or more):

[0254] i) Activation of the GLP-1 receptor; ii) Activation of the glucagon receptor; and iii) Activation of the GIP receptor.

[0255] In particular, activation of the receptor can include, for example, those cases where the in vitro activity of the peptide is 0.1% or higher, 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, and 100% or higher compared to the native ligand of the corresponding receptor, but the activation is not limited thereto.

[0256] In addition, the triple agonist can be an agonist having an increased in vivo half-life relative to any one of native GLP-1, native glucagon, and native GIP, but is not particularly limited thereto.

[0257] The above glucagon analogs can be non-naturally occurring analogs, but are not particularly limited thereto.

[0258] Specifically, the isolated peptide can be an analog of native glucagon, but is not particularly limited thereto.

[0259] The native glucagon analog according to the present invention can include a peptide having at least one difference in the amino acid sequence compared to native glucagon; a peptide modified by modification of the native glucagon sequence; and a mimetic of native glucagon.

[0260] Meanwhile, native glucagon can have the following amino acid sequence, but is not particularly limited thereto:

[0261] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO:118)

[0262] Specifically, the isolated peptide can be an analog of native glucagon having a variant with a substitution, addition, deletion, modification, and combinations thereof of at least one amino acid selected from the native glucagon sequence, but is not particularly limited thereto.

[0263] In addition, the substitution of amino acids can include both substitution with amino acids and substitution with non-natural compounds.

[0264] In addition, the addition can be carried out at the N-terminus and / or C-terminus of the peptide. Meanwhile, the length of the added amino acids is not particularly limited, but 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, and 11 or more amino acids can be added. And in a broad sense, the addition can include the addition of polypeptides, but is not particularly limited thereto.

[0265] More specifically, the glucagon analog can be one in which 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 amino acids selected from the amino acids at positions 1, 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the natural glucagon amino acid sequence are replaced by other amino acids, and additionally, can be one in which 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, or 11 or more amino acids are independently or additionally added to its C-terminus, but is not particularly limited thereto.

[0266] Even more specifically, the glucagon analog can be one in which 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 amino acids selected from the amino acids at positions 1, 2, 3, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the natural glucagon amino acid sequence are replaced by other amino acids, and additionally, can be one in which 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, or 11 or more amino acids are independently or additionally added to its C-terminus, but is not particularly limited thereto.

[0267] Even more specifically, the glucagon analogs can be those in which one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen amino acids selected from the amino acids at positions 1, 2, 3, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 28, and 29 in the native glucagon amino acid sequence are replaced by other amino acids, and additionally, can be those in which one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more amino acids are independently or additionally added to its C-terminus, but are not particularly limited thereto.

[0268] Even more specifically, the glucagon analogs can be those in which one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or fourteen amino acids selected from the amino acids at positions 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the native glucagon amino acid sequence are replaced by other amino acids, and additionally, can be those in which one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more amino acids are independently or additionally added to its C-terminus, but are not particularly limited thereto.

[0269] The amino acids to be introduced into the above native glucagon can be selected from tyrosine, α-methyl-glutamic acid, Aib, methionine, glutamic acid, histidine, lysine, leucine, isoleucine, glutamine, valine, glycine, alanine, cysteine, serine, alanine, aspartic acid, and arginine, but are not particularly limited thereto.

[0270] For example, the amino acid sequence(s) to be added can be at least one amino acid sequence derived from the native GLP-1, native GIP, or native exendin-4 amino acid sequence.

[0271] Glucagon analogs or triple agonists may include intramolecular bridges (e.g., covalent crosslinks, non-covalent crosslinks), and specifically, in the form including a ring, e.g., in the form of a ring formed between the 16th amino acid and the 20th amino acid in the glucagon analog or triple agonist, but not particularly limited thereto.

[0272] Non-limiting examples of the ring may include a lactam bridge (or lactam ring).

[0273] In addition, glucagon analogs or triple agonists include all those modified to include a ring, or include amino acids capable of forming a ring at the target position.

[0274] For example, the glucagon analog or triple agonist may be an analog or triple agonist in which the amino acid pair at the 16th and 20th positions are respectively replaced by glutamate or lysine capable of forming a ring, but the glucagon analog or triple agonist is not limited thereto.

[0275] The ring may be formed between amino acid side chains within the glucagon analog or triple agonist; for example, they may be in the form of a lactam ring between the side chain of lysine and the side chain of glutamate, but the ring is not particularly limited thereto.

[0276] Examples of glucagon analogs prepared by a combination of these methods may include peptides whose amino acid sequences differ from the amino acid sequence of native glucagon in at least one amino acid, and in which the α-carbon in its N-terminus is removed, while being active against glucagon receptor, GLP-1 receptor, GIP receptor, etc., but not limited thereto, and analogs of native glucagon applicable to the present invention can be prepared by combining various methods for the preparation of analogs.

[0277] In addition, regarding the triple agonist of the present invention, in order to increase the in vivo half-life of the triple agonist, some amino acids may be replaced by other amino acids or non-natural compounds to avoid being recognized by peptidases, but the triple agonist is not particularly limited thereto.

[0278] Specifically, the peptide may be a peptide in which the in vivo half-life is increased by avoiding recognition by peptidases by replacing the second amino acid sequence in the amino acid sequence of the triple agonist, but any substitution or modification of amino acids to avoid recognition by in vivo peptidases is not limited.

[0279] In addition, such modifications for preparing analogs of native glucagon may include all modifications using L- or D-amino acids and / or non-natural amino acids; and / or modifications of the native sequence, for example, modifications of side chain functional groups, intramolecular covalent bonding (e.g., ring formation between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation, etc.

[0280] In addition, the modifications may also include all those in which one or more amino acids are added to the amino and / or carboxyl terminus of native glucagon.

[0281] During substitution or addition of amino acids, 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 may include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. Peptides comprising such amino acids and typical peptide sequences can be synthesized and purchased from commercial suppliers, e.g., American Peptide Company, Bachem (USA), or Anygen (Korea).

[0282] Amino acid derivatives can be obtained in the same manner, and as one such example, 4-imidazole acetic acid (4-imidazolylacetic acid) can be used.

[0283] In addition, the peptides according to the present invention can be in variant forms, in which the amino and / or carboxyl terminus, etc. of the peptide are chemically modified or protected by organic groups, or amino acids can be added to the termini of the peptide for its protection from in vivo proteases while increasing its stability.

[0284] In particular, in the case of chemically synthesized peptides, their N- and C-termini are charged, and thus the N- and C-termini of the peptide can be acetylated and / or amidated, but the peptide is not particularly limited thereto.

[0285] In addition, the peptides according to the present invention may include all those in the form of the peptide itself, its salts (e.g., its pharmaceutically acceptable salts), or its solvates. In addition, the peptide can be in any pharmaceutically acceptable form.

[0286] The types of salts are not particularly limited. However, the salts are preferably salts that are safe and effective for a subject (e.g., a mammal), but are not particularly limited thereto.

[0287] The term "pharmaceutically acceptable" refers to a substance that can be effectively used for an intended use within the scope determined by pharmaco-medical decisions without causing excessive toxicity, irritation, allergic reactions, etc.

[0288] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable salts can 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, benzenesulfonic acid, etc. Examples of salts derived from suitable bases can include alkali metals such as sodium, potassium, etc.; alkaline earth metals such as magnesium; ammonium, etc.

[0289] As used herein, the term "solvate" refers to a complex formed between a peptide or its salt according to the present invention and a solvent molecule.

[0290] In a specific embodiment, X can be a peptide comprising an amino acid sequence represented by the following general formula 1.

[0291] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103)

[0292] In the above general formula 1,

[0293] Xaa1 is histidine (His, H), 4-imidazolylacetyl (CA), or tyrosine (Tyr, Y);

[0294] Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib;

[0295] Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);

[0296] Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);

[0297] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or valine (Val, V);

[0298] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

[0299] Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine (Cys, C);

[0300] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

[0301] Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L);

[0302] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

[0303] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);

[0304] Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);

[0305] Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), or valine (Val, V);

[0306] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

[0307] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);

[0308] Xaa23 is isoleucine (Ile, I) or valine (Val, V);

[0309] Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E);

[0310] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);

[0311] Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D);

[0312] Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);

[0313] Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or is absent;

[0314] R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO:106), m-SSGAPPPS-n (SEQ ID NO:107), or m-SSGQPPPS-n (SEQ ID NO:108), or is absent;

[0315] wherein,

[0316] m is -Cys-, -Pro-, or -Gly-Pro-;

[0317] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.

[0318] For example, the triple agonist can be an agonist comprising an amino acid sequence selected from SEQ ID NOS: 1 to 102; and an agonist consisting of (substantially) an amino acid sequence selected from SEQ ID NOS: 1 to 102, but not limited thereto.

[0319] In addition, although described in the present invention as "a peptide consisting of a specific SEQ ID NO", as long as the peptide has the same or corresponding activity as the peptide consisting of the amino acid sequence of the corresponding SEQ ID NO, it does not exclude mutations that occur by the addition of nonsense sequences upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO, or mutations that occur naturally, or its silent mutations, and even when sequence additions or mutations are present, it clearly falls within the scope of the present invention.

[0320] The above can be applied to other specific embodiments or aspects of the present invention, but not limited thereto.

[0321] Specifically, in the above general formula 1, Xaa14 can be leucine or methionine, and Xaa15 can be cysteine, aspartic acid, or leucine.

[0322] Examples of the peptide may include a peptide comprising or (substantially) consisting of an amino acid sequence selected from SEQ ID NOS: 1 to 12, 14 to 17, and 21 to 102, but are not particularly limited thereto.

[0323] The peptide may significantly activate at least one of the glucagon receptor, GLP-1 receptor, and GIP receptor, but is not particularly limited thereto. Specifically, the peptide may be a peptide that significantly activates the GLP-1 receptor, or additionally significantly activates the glucagon receptor and / or GIP receptor, but is not particularly limited thereto.

[0324] Even more specifically, the peptide may be:

[0325] In the above general formula 1,

[0326] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0327] Xaa7 is threonine;

[0328] Xaa10 is tyrosine, cysteine, or valine;

[0329] Xaa12 is lysine or isoleucine;

[0330] Xaa13 is tyrosine, alanine, glutamine, or cysteine;

[0331] Xaa14 is leucine, cysteine, or methionine;

[0332] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

[0333] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0334] Xaa18 is alanine, glutamine, arginine, or histidine;

[0335] Xaa19 is alanine, glutamine, valine, or cysteine;

[0336] Xaa20 is lysine, arginine, or glutamine;

[0337] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0338] Xaa23 is isoleucine or valine;

[0339] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and

[0340] Xaa27 is leucine or lysine, but is not particularly limited thereto.

[0341] Even more specifically,

[0342] In the above general formula 1,

[0343] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0344] Xaa7 is threonine;

[0345] Xaa10 is tyrosine, cysteine, or valine;

[0346] Xaa12 is lysine or isoleucine;

[0347] Xaa13 is tyrosine, alanine, or cysteine;

[0348] Xaa14 is leucine or methionine;

[0349] Xaa15 is cysteine or aspartic acid;

[0350] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

[0351] Xaa18 is alanine, arginine, or histidine;

[0352] Xaa19 is alanine, glutamine, or cysteine;

[0353] Xaa20 is lysine or glutamine;

[0354] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0355] Xaa23 is valine;

[0356] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and

[0357] Xaa27 is leucine or lysine, but is not particularly limited thereto.

[0358] Even more specifically,

[0359] In the above general formula 1,

[0360] Xaa2 is α-methyl-glutamic acid or Aib;

[0361] Xaa7 is threonine;

[0362] Xaa10 is tyrosine or cysteine;

[0363] Xaa12 is lysine or isoleucine;

[0364] Xaa13 is tyrosine, alanine, or cysteine;

[0365] Xaa14 is leucine or methionine;

[0366] Xaa15 is cysteine or aspartic acid;

[0367] Xaa16 is glutamic acid;

[0368] Xaa17 is arginine, isoleucine, cysteine, or lysine;

[0369] Xaa18 is alanine, arginine, or histidine;

[0370] Xaa19 is alanine, glutamine, or cysteine;

[0371] Xaa20 is lysine or glutamine;

[0372] Xaa21 is glutamic acid or aspartic acid;

[0373] Xaa23 is valine;

[0374] Xaa24 is glutamine, asparagine, or aspartic acid;

[0375] Xaa27 is leucine; and

[0376] Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0377] Specifically,

[0378] In the above general formula 1,

[0379] Xaa1 is histidine or 4-imidazolylacetyl;

[0380] Xaa2 is α-methyl-glutamic acid or Aib;

[0381] Xaa3 is glutamine;

[0382] Xaa7 is threonine;

[0383] Xaa10 is tyrosine;

[0384] Xaa12 is isoleucine;

[0385] Xaa13 is alanine or cysteine;

[0386] Xaa14 is methionine;

[0387] Xaa15 is aspartic acid;

[0388] Xaa16 is glutamic acid;

[0389] Xaa17 is isoleucine or lysine;

[0390] Xaa18 is alanine or histidine;

[0391] Xaa19 is glutamine or cysteine;

[0392] Xaa20 is lysine;

[0393] Xaa21 is aspartic acid;

[0394] Xaa23 is valine;

[0395] Xaa24 is asparagine;

[0396] Xaa27 is leucine;

[0397] Xaa28 is alanine or asparagine;

[0398] Xaa29 is glutamine or threonine; and

[0399] Xaa30 is cysteine or lysine, or is absent.

[0400] More specifically,

[0401] In the above general formula 1,

[0402] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0403] Xaa3 is glutamine;

[0404] Xaa7 is threonine;

[0405] Xaa10 is tyrosine, cysteine, or valine;

[0406] Xaa12 is lysine;

[0407] Xaa13 is tyrosine;

[0408] Xaa14 is leucine;

[0409] Xaa15 is aspartic acid;

[0410] Xaa16 is glycine, glutamic acid, or serine;

[0411] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0412] Xaa18 is alanine, arginine, or histidine;

[0413] Xaa19 is alanine or glutamine;

[0414] Xaa20 is lysine or glutamine;

[0415] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0416] Xaa23 is valine;

[0417] Xaa24 is alanine, glutamine, or cysteine;

[0418] Xaa27 is leucine or lysine; and

[0419] Xaa29 is glycine, glutamine, threonine, or histidine;

[0420] However, it is not particularly limited thereto.

[0421] These peptides may correspond to the case where the peptide has a significant activation level for both the GLP-1 receptor and the glucagon receptor, or has a higher activation level for the GIP receptor; the case where the peptide has a significant activation level for all of the GLP-1 receptor, the glucagon receptor, and the GIP receptor; or the case where the peptide has a significant activation level for both the GLP-1 receptor and the GIP receptor and has a higher activation level for the glucagon receptor; however, it is not particularly limited thereto.

[0422] When the peptide has a significant activation level for both the GLP-1 receptor and the GIP receptor and also has a higher activation level for the glucagon receptor, it can provide a peptide with a more improved ability to control blood glucose levels and a weight loss effect. However, when the peptide has a significant activation level for all of the GLP-1 receptor, the glucagon receptor, and the GIP receptor, there is an advantage of maximizing the weight loss effect, but the effect is not particularly limited thereto.

[0423] Examples of the peptide may include peptides comprising an amino acid sequence selected from SEQ ID NOS: 8, 9, 21 to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88, 89, 91 to 93, and 95 to 102; or peptides consisting of (substantially) thereof, but it is not particularly limited thereto.

[0424] In one specific embodiment, the peptide may include an amino acid sequence represented by the following general formula 2.

[0425] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General formula 2, SEQ ID NO: 104)

[0426] In the above general formula 2,

[0427] Xaa1 is 4-imidazolylacetyl, histidine, or tyrosine;

[0428] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0429] Xaa10 is tyrosine or cysteine;

[0430] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0431] Xaa14 is leucine, methionine, or tyrosine;

[0432] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0433] Xaa16 is glycine, glutamic acid, or serine;

[0434] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0435] Xaa18 is alanine, glutamine, arginine, or histidine;

[0436] Xaa19 is alanine, glutamine, cysteine, or valine;

[0437] Xaa20 is lysine, glutamine, or arginine;

[0438] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0439] Xaa23 is isoleucine or valine;

[0440] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0441] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

[0442] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0443] Xaa30 is cysteine, glycine, lysine, or histidine;

[0444] Xaa31 is proline or cysteine; and

[0445] Xaa40 is cysteine or absent.

[0446] More specifically, in the above general formula 2,

[0447] Xaa13 is alanine, tyrosine, or cysteine;

[0448] Xaa15 is aspartic acid or glutamic acid;

[0449] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0450] Xaa18 is alanine, arginine, or histidine;

[0451] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

[0452] Xaa23 is isoleucine or valine;

[0453] Xaa24 is cysteine, glutamine, or asparagine;

[0454] Xaa28 is cysteine, asparagine, or aspartic acid;

[0455] Xaa29 is glutamine, cysteine, or histidine; and

[0456] Xaa30 is cysteine, lysine, or histidine.

[0457] Examples of the peptide may include peptides comprising an amino acid sequence selected from SEQ ID NOS: 21, 22, 42, 43, 50, 64 to 77, and 95 to 102; more specifically, peptides comprising an amino acid sequence selected from SEQ ID NOS: 21, 22, 42, 43, 50, 64 to 77, and 96 to 102; or peptides consisting of (substantially) the same, but not particularly limited thereto.

[0458] In one specific embodiment, the peptide may include an amino acid sequence represented by the following general formula 3.

[0459] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General formula 3, SEQ ID NO: 105)

[0460] In the above general formula 3,

[0461] Xaa1 is histidine or tyrosine;

[0462] Xaa2 is α-methyl-glutamic acid or Aib;

[0463] Xaa13 is alanine, tyrosine or cysteine;

[0464] Xaa17 is arginine, cysteine, or lysine;

[0465] Xaa18 is alanine or arginine;

[0466] Xaa19 is alanine or cysteine;

[0467] Xaa21 is glutamic acid or aspartic acid;

[0468] Xaa24 is glutamine or asparagine,

[0469] Xaa28 is cysteine or aspartic acid;

[0470] Xaa29 is cysteine, histidine, or glutamine;

[0471] Xaa30 is cysteine or histidine;

[0472] Xaa31 is proline or cysteine; and

[0473] Xaa40 is cysteine or is absent.

[0474] Examples of the peptide may include peptides comprising an amino acid sequence selected from SEQ ID NOS: 21, 22, 42, 43, 50, 64 to 71, 75 to 77, and 96 to 102; or peptides consisting of (substantially) the same, but are not particularly limited thereto.

[0475] In addition, in the above general formula (1), R1 may be cysteine, GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or may be absent, but is not particularly limited thereto.

[0476] In addition, the peptides of the present invention can be synthesized by methods well known in the art (e.g., by a peptide synthesizer) according to their lengths, and can be produced by genetic engineering techniques.

[0477] Specifically, the peptides of the present invention can be prepared by standard synthesis methods, recombinant expression systems, or any other methods known in the art. Therefore, the peptides of the present invention can be synthesized by many methods, including, for example, the methods described below:

[0478] (a) A method of synthesizing a peptide stepwise or by fragment assembly by a solid-phase or liquid-phase method, and then separating and purifying the final peptide product; or

[0479] (b) A method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0480] (c) A method of performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product therefrom; or

[0481] A method of obtaining peptide fragments by any combination of methods (a), (b), and (c), obtaining a peptide by ligating the peptide fragments, and then recovering the peptide.

[0482] In the above conjugate, F is a substance that can increase the half-life of X - i.e., a peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor - and its structure corresponds to the part constituting the conjugate of the present invention.

[0483] F and X can be bound to each other by a covalent chemical bond or a non-covalent chemical bond; or F and X can be bound to each other by L through a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0484] The substance that can increase the half-life of X can be a biocompatible substance, for example, a substance selected from polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, connective tissues in vivo, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0485] Elastin can be human tropoelastin as a water-soluble precursor, and can be a polymer of a certain sequence or some repeating units of tropoelastin, for example, including all elastin-like polypeptides, but is not particularly limited thereto.

[0486] Examples of the polymer can be a polymer selected from polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, but is not particularly limited thereto.

[0487] Polyethylene glycol includes all forms of homopolymers of ethylene glycol, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG), but is not particularly limited thereto.

[0488] In addition, the biocompatible substance can include polyamino acids, such as polylysine, polyaspartic acid, and polyglutamic acid, but is not limited thereto.

[0489] In addition, the fatty acid can be a fatty acid that has a binding affinity for albumin in vivo, but is not particularly limited thereto.

[0490] In a more specific embodiment, the FcRn-binding substance can be the immunoglobulin Fc region, and more specifically, can be the IgG Fc region, but is not particularly limited thereto.

[0491] At least one amino acid side chain in the peptide of the present invention can be attached to the biocompatible substance to increase solubility and / or half-life in vivo, and / or increase its bioavailability. These modifications can reduce the clearance of therapeutic proteins and peptides.

[0492] The biocompatible polymer can be water-soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharmaceutically acceptable.

[0493] F can be directly connected to X (i.e., a is 0 in Chemical Formula 1) or can be connected through a linker (L).

[0494] Specifically, L can be a peptide linker or a non-peptide linker, but is not limited thereto.

[0495] When L is a peptide linker, it may include one or more amino acids, for example, 1 to 1000 amino acids, but is not particularly limited thereto. In the present invention, various known peptide linkers can be used for the connection between F and X (for example, including [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, etc., where x is a natural number of at least 1), but the peptide linker is not limited thereto.

[0496] In the present invention, the "non-peptide linker" includes a biocompatible polymer to which at least two repeating units are attached. The repeating units are connected to each other by any covalent bond instead of a peptide bond. The non-peptide linker can be a structure that becomes part of the conjugate of the present invention and corresponds to L in Chemical Formula 1 above.

[0497] In La of Chemical Formula 1, a can be 1 or greater, and when a is 2 or greater, each L can be independent of each other.

[0498] As used herein, the term "non-peptide linker" can be used interchangeably with "non-peptide polymer".

[0499] In addition, in one specific embodiment, the conjugate can be a conjugate in which F and X are covalently connected to each other through a non-peptide linker having two reactive end groups respectively connected to X (specifically, a peptide drug) and F (specifically, an immunoglobulin Fc region).

[0500] Specifically, the non-peptide linker can be a non-peptide linker selected from fatty acids, saccharides, polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0501] Although not particularly limited, the non-peptide linker can be a non-peptide linker selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ether, biodegradable polymers such as polylactic acid (PLA) and poly(lactic acid-glycolic acid) (PLGA), lipid polymers, chitin, hyaluronic acid, oligonucleotide, and combinations thereof. In a more specific embodiment, the non-peptide polymer can be polyethylene glycol, but is not limited thereto. In addition, derivatives of the above substances known in the art and derivatives that can be easily manufactured at the technical level in the art belong to the scope of the present invention.

[0502] The non-peptide linker used in the present invention can be any polymer that is resistant to proteases in vivo, without limitation. The molecular weight of the non-peptide polymer can be in the range of 1 kDa to 100 kDa, and specifically, in the range of 1 kDa to 20 kDa, but is not limited thereto. In addition, the non-peptide linker of the present invention connected to the polypeptide corresponding to F can include not only a single type of polymer but also a combination of different types of polymers.

[0503] In a specific embodiment, the two ends of the non-peptide linker can be connected to the amino or thiol group of F (such as the immunoglobulin Fc region), or the amino or thiol group of X, respectively.

[0504] Specifically, the non-peptide polymer may include reactive groups that can be connected to F (such as, the immunoglobulin Fc region) and X at its two ends, respectively, and specifically, reactive groups that can be connected to the amino group at the N-terminus of X or lysine, or the thiol group of cysteine, or the amino group at the N-terminus of F (such as, the immunoglobulin Fc region) or lysine, or the thiol group of cysteine, but the reactive groups are not limited thereto.

[0505] In addition, the reactive groups of the non-peptide polymer that can be connected to the immunoglobulin Fc region and X can be selected from aldehyde groups, maleimide groups, and succinimide derivatives, but are not limited thereto.

[0506] In the above, examples of the aldehyde group may include propionaldehyde group or butyraldehyde group, but are not limited thereto.

[0507] In the above, as succinimide derivatives, succinimidyl valerate, succinimidyl methyl butyrate, succinimidyl methyl propionate, succinimidyl butyrate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate can be used, but the succinimide derivatives are not limited thereto.

[0508] The non-peptide linker can be connected to X and F through these reactive groups, but the reactive groups are not particularly limited thereto.

[0509] In addition, the final product generated by reductive alkylation through an aldehyde bond is more stable than that connected through an amide bond. The aldehyde reactive group selectively reacts with the N-terminus under low pH conditions, while it can form a covalent bond with lysine residues at high pH (such as, pH 9.0).

[0510] The reactive groups at the two ends of the non-peptide linker can be the same as or different from each other. For example, a maleimide reactive group can be provided at one end, while an aldehyde group, propionaldehyde group, or butyraldehyde group can be provided at the other end. However, as long as F (specifically the immunoglobulin Fc region) can be connected to X, the reactive groups are not particularly limited thereto.

[0511] For example, the non-peptide linker can have a maleimide group as a reactive group at one end, while having an aldehyde group, propionaldehyde group, or butyraldehyde group, etc. at the other end.

[0512] When polyethylene glycol having reactive hydroxyl groups at both ends is used as a non-peptide polymer, the hydroxyl groups can be activated into various reactive groups through known chemical reactions, or polyethylene glycol with commercially available modified reactive groups can be used to prepare the long-acting protein conjugates of the present invention.

[0513] In a specific embodiment, the non-peptide polymer can be a non-peptide polymer that can be linked to the cysteine residue of X, and more specifically, to the -SH group of cysteine, but is not limited thereto.

[0514] When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X through a thioether bond, and the aldehyde group can be linked to F, specifically to the -NH2 of immunoglobulin Fc, through reductive alkylation, but is not limited thereto, and the above is only one embodiment.

[0515] In addition, in the above conjugate, the reactive group of the non-peptide polymer can be linked to the -NH2 located at the N-terminus of immunoglobulin Fc, but this is only one embodiment.

[0516] In the present invention, the "immunoglobulin Fc region" refers to a region that does not include the variable regions of the heavy and light chains of immunoglobulin and includes the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). The immunoglobulin Fc region can be a structure that becomes part of the protein conjugate of the present invention.

[0517] The immunoglobulin Fc region may include the hinge region in the heavy chain constant region, but is not limited thereto. In addition, the immunoglobulin Fc region of the present invention can be an extended Fc region that does not include the variable regions of the heavy and light chains of immunoglobulin and includes part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), as long as the immunoglobulin Fc region has an effect that is substantially the same as or improved compared to the natural type. In addition, the immunoglobulin Fc region of the present invention can be a region in which a relatively long portion corresponding to CH2 and / or CH3 is removed.

[0518] For example, the immunoglobulin Fc region of the present invention can be 1) the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain; 2) the CH1 domain and the CH2 domain; 3) the CH1 domain and the CH3 domain; 4) the CH2 domain and the CH3 domain; 5) a combination between one or two or more domains among the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and the immunoglobulin hinge region (or a part of the hinge region); and 6) a dimer between each domain of the heavy chain constant region and the light chain constant region, but is not limited thereto.

[0519] In addition, in a specific embodiment, the immunoglobulin Fc region can be in a dimeric form, and one molecule of X can be covalently linked to one Fc region in the dimeric form, wherein the immunoglobulin Fc and X can be linked to each other through a non-peptide polymer. Meanwhile, it is also possible that two molecules of X are symmetrically linked to the Fc region in the dimeric form. However, the linkage is not limited thereto.

[0520] In addition, the immunoglobulin Fc region of the present invention includes not only the natural amino acid sequence but also sequence derivatives thereof. Amino acid sequence derivatives refer to amino acid sequences that have differences at at least one amino acid residue due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0521] For example, it is known that amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 in the conjugation of immunoglobulin Fc can be used as suitable modification sites.

[0522] In addition, various other derivatives are possible, including derivatives with deletions of regions capable of forming disulfide bonds, or deletions of some amino acid residues at the N-terminus of natural Fc or addition of a methionine residue at the N-terminus of natural Fc. Further, in order to remove effector functions, deletions can occur in complement binding sites such as the C1q binding site and the antibody-dependent cell cytotoxicity (ADCC) site. Techniques for preparing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97 / 34631, WO 96 / 32478, etc.

[0523] Amino acid exchanges in proteins and peptides that do not completely alter the activity are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most frequently occurring exchanges are 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 both directions. Depending on the situation, the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0524] The above Fc derivatives exhibit the same biological activities as the Fc region of the present invention, and they may have improved structural stability against heat, pH, etc.

[0525] Furthermore, the immunoglobulin Fc region can be obtained in its native form isolated from the body of a human or an animal such as a cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., or it can be a recombinant immunoglobulin Fc region or its derivative obtained from transformed animal cells or microorganisms. Herein, the Fc region can be obtained from native immunoglobulins by isolating whole immunoglobulins from the body of a living human or animal and treating the isolated immunoglobulins with a protease. When whole immunoglobulins are treated with papain, the immunoglobulins are cleaved into Fab and Fc regions, whereas when whole immunoglobulins are treated with pepsin, the immunoglobulins are cleaved into pF’c and F(ab)2 fragments. Fc or pF’c can be isolated using size exclusion chromatography, etc. In a more specific embodiment, the immunoglobulin Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism expressing a human-derived Fc region.

[0526] In addition, the immunoglobulin Fc region can be in the form of native glycans, glycans increased or decreased compared to the native type, or a deglycosylated form. The increase, decrease, or removal of immunoglobulin Fc glycans can be achieved by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. The immunoglobulin Fc region obtained by removing glycans from the Fc region shows a significantly reduced binding affinity to C1q, as well as a decrease or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and thus it does not induce unnecessary immune responses in the body. In this regard, the immunoglobulin Fc region in the deglycosylated or non-glycosylated (aglycosylated) immunoglobulin Fc region may be a more suitable form for fulfilling the original purpose of the present invention as a drug carrier.

[0527] As used herein, the term “deglycosylation” refers to the enzymatic removal of sugar moieties from the Fc region, and the term “aglycosylation” refers to an unglycosylated Fc region produced in prokaryotes, more specifically, Escherichia coli (E. coli.).

[0528] Meanwhile, the immunoglobulin Fc region can be derived from a human or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs. In a more specific embodiment, it is derived from a human.

[0529] In addition, the Fc region of an immunoglobulin (Ig) can be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In more specific embodiments, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, and in even more specific embodiments, it is derived from IgG, which is known to enhance the half-life of ligand-binding proteins. In even more specific embodiments still, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiments, the IgG4 Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0530] Specifically, as used herein, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is linked to a single-chain polypeptide of a different origin to form a dimer or multimer. That is, the dimer or multimer can be formed from two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0531] In addition, the above conjugates can have enhanced effects and long-acting properties compared to native GLP-1, GIP, or glucagon, or compared to X where F is unmodified, and in addition to those described above, these conjugates include those encapsulated by biodegradable nanoparticles.

[0532] Another aspect of the present invention provides a polynucleotide encoding the conjugate, a vector containing the polynucleotide, and a transformant containing the polynucleotide or the vector containing the polynucleotide.

[0533] The conjugate is the same as that explained above.

[0534] The polynucleotide can be a polynucleotide encoding the conjugate in the form of a fusion protein.

[0535] In addition, an isolated polynucleotide encoding the conjugate includes, within the scope of the present invention, a polynucleotide sequence having 75% or higher, specifically 85% or higher, more specifically 90% or higher, and even more specifically 95% or higher sequence identity with the corresponding sequence.

[0536] As used herein, the term "homology" indicates sequence similarity to a wild-type amino acid sequence or a wild-type nucleic acid sequence, and homology comparisons can be made visually or using commercially available comparison programs. Using commercially available computer programs, the homology between two or more sequences can be expressed as a percentage (%), and the homology (%) between adjacent sequences can be calculated.

[0537] As used herein, the term "recombinant vector" refers to a DNA construct in which a polynucleotide encoding a target protein (e.g., a conjugate) is operably linked to suitable regulatory sequences to enable expression of the target protein, conjugate, in a host cell.

[0538] Regulatory sequences include a promoter capable of initiating transcription, any operator gene sequences for regulating transcription, a sequence encoding a suitable mRNA ribosome binding domain, and a sequence for regulating termination of transcription and translation. After transformation into a suitable host cell, the recombinant vector can replicate or function regardless of the host genome, or can integrate into the host genome itself.

[0539] The recombinant vector for use in the present invention is not particularly limited as long as the vector can replicate in a host cell, and it can be constructed using any vector known in the art. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and phages. The vector for use in the present invention is not particularly limited, but any expression vector known in the art can be used.

[0540] The recombinant vector is used for transformation of a host cell to produce the conjugate of the present invention. Additionally, these transformed cells, as part of the present invention, can be used to amplify nucleic acid fragments and vectors, or they can be cultured cells or cell lines for the recombinant production of the conjugate of the present invention.

[0541] As used herein, the term "transformation" refers to the process of introducing a recombinant vector comprising a polynucleotide encoding a target protein into a host cell, thereby enabling expression of the protein encoded by the polynucleotide in the host cell. For the transformed polynucleotide, it does not matter whether it is inserted into the chromosome of the host cell and located thereon or is extrachromosomal, as long as it can be expressed in the host cell, and both cases are included.

[0542] Additionally, polynucleotides include DNA and RNA encoding a target protein. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct comprising all the necessary elements required for self-expression. The expression cassette typically may contain a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding domain, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide can be introduced into the host cell as it is and operably linked to sequences necessary for its expression in the host cell, but is not limited thereto.

[0543] In addition, as used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and the aforementioned gene sequence, where the promoter sequence initiates and mediates the transcription of a polynucleotide encoding a target protein (i.e., the conjugate of the present invention).

[0544] The suitable host for the present invention is not particularly limited as long as it can express the polynucleotide of the present invention. Examples of suitable hosts may include bacteria belonging to the genus Escherichia, such as Escherichia coli; bacteria belonging to the genus Bacillus, such as Bacillus subtilis; bacteria belonging to the genus Pseudomonas, such as Pseudomonas putida; yeasts, such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; insect cells, such as Spodoptera frugiperda (Sf9), and animal cells, such as CHO, COS, and BSC.

[0545] Yet another aspect of the present invention provides a composition containing the conjugate.

[0546] The conjugate is the same as that explained above.

[0547] Specifically, the composition may be a pharmaceutical composition, and more specifically, a pharmaceutical composition for preventing or treating metabolic syndrome.

[0548] As used herein, the term "prevention" refers to all actions of inhibiting or delaying metabolic syndrome by administering the aforementioned conjugate or a composition containing the conjugate, while the term "treatment" refers to all actions of improving or favorably altering the symptoms of metabolic syndrome by administering the aforementioned conjugate or a composition containing the conjugate.

[0549] As used herein, the term "administration" refers to introducing a specific substance into an object by a suitable method, and the administration route of the composition can be any conventional route that enables the composition to be delivered to the target in the body, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, etc.

[0550] As used herein, the term "metabolic syndrome" refers to symptoms in which various diseases occurring due to chronic metabolic disorders occur alone or in combination. In particular, examples of diseases belonging to metabolic syndrome may include glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis attributable to dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease, but are not limited thereto.

[0551] As used herein, the term "obesity" refers to a medical condition with an excessive accumulation of body fat, and a person is generally defined as obese when their body mass index (BMI; the value of weight (kg) divided by the square of height (m)) is 25 or higher. Obesity is most often caused by an energy imbalance resulting from excessive food intake over a long period of time compared to energy expenditure. As a metabolic disease affecting the whole body, obesity increases the likelihood of developing diabetes and hyperlipidemia, increases the risk of the incidence of sexual dysfunction, arthritis, and cardiovascular diseases, and is associated with cancer development in some cases.

[0552] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutically acceptable carrier, excipient, or diluent may be non-naturally occurring.

[0553] As used herein, the term "pharmaceutically acceptable" refers to having properties with a quantity sufficient to present a therapeutic effect and not causing side effects, and can be easily determined by those skilled in the art based on factors well-known in the medical field, such as the type of disease, age, weight, health status, gender, drug sensitivity of the patient, route of administration, method of administration, frequency of administration, duration of treatment, drugs (one or more) administered in combination or simultaneously, and the like.

[0554] The pharmaceutical composition of the present invention containing the peptide of the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc.; for injectables, buffers, preserving agents, analgesics, solubilizers, isotonic agents, stabilizers, etc., which may be used in combination; and for topical administration, alkalis, excipients, lubricants, preserving agents, etc.

[0555] The formulation type of the composition according to the present invention can be variously prepared by combining with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injectables, the composition can be formulated into unit-dose ampoules or multi-dose containers. The composition can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0556] Meanwhile, examples of suitable carriers, excipients and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. Additionally, the composition may further contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc.

[0557] Furthermore, the pharmaceutical composition of the present invention can be prepared in any formulation type selected from tablets, pills, powders, granules, capsules, suspensions, liquid drugs for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, freeze-dried preparations and suppositories.

[0558] Additionally, the composition can be formulated into a unit dosage form suitable for the patient's body and specifically formulated into a preparation useful for protein drugs according to typical methods in the pharmaceutical field, so as to be administered by oral or parenteral routes, such as through the skin, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal administration, but not limited thereto.

[0559] Furthermore, the conjugate can be used by mixing with various pharmaceutically acceptable carriers approved as pharmaceutical drugs such as physiological saline or organic solvents. Carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers can be used as pharmaceutical drugs in order to increase stability or absorbability.

[0560] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined together with various factors according to the type of the active ingredient(s), various factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

[0561] The total effective dose of the composition of the present invention can be administered to a patient as a single dose, or can be administered in multiple doses over a long period according to a fractionated treatment protocol. In the pharmaceutical composition of the present invention, the content of the active ingredient(s) can vary according to the severity of the disease. Specifically, the total daily dose of the conjugate of the present invention can be about 0.0001 mg to 500 mg per 1 kg of patient body weight. However, in addition to the administration route and treatment frequency of the pharmaceutical composition, various factors including the patient's age, weight, health status, gender, disease severity, diet, and excretion rate are considered to determine the effective dose of the conjugate. In this regard, those skilled in the art can easily determine the effective dose suitable for the specific use of the pharmaceutical composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, administration route, and method as long as it exhibits the effects of the present invention.

[0562] The pharmaceutical composition of the present invention shows an excellent duration and potency of in vivo efficacy, and thus can significantly reduce the number and frequency of administrations of the pharmaceutical preparation of the present invention.

[0563] Another aspect of the present invention provides a method for treating a target disease, which comprises administering a conjugate or a composition containing the conjugate to a subject in need thereof. The target disease can be metabolic syndrome.

[0564] The conjugate or the composition containing the conjugate is the same as that explained above.

[0565] The target disease can be metabolic syndrome.

[0566] As used herein, the term "subject" refers to a subject suspected of having metabolic syndrome, and a subject suspected of having metabolic syndrome refers to a mammal including humans, rats, livestock, etc., which has metabolic syndrome or is at risk of developing metabolic syndrome, but includes without limitation any subject that can be treated with the conjugate of the present invention or a composition containing the conjugate.

[0567] The method of the present invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising the conjugate. The total daily dose of the composition may be determined within the scope of the appropriate medical judgment of a physician, and the composition may be administered once daily or in divided doses several times a day. However, for the purposes of the present invention, for any particular patient, the specific therapeutically effective dose of the composition is preferably applied differently according to various factors, including the type and degree of response to be achieved, the specific composition including whether other preparations are used intermittently or therewith, the age, weight, health status, sex and diet of the patient, the time and route of administration, the excretion rate of the composition, the duration of treatment, other drugs used in combination with or simultaneously with the specific composition, and similar factors well known in the medical field.

[0568] Yet another aspect of the present invention provides the use of the conjugate or a composition comprising the conjugate in the preparation of a medicament.

[0569] The conjugate or the composition comprising the conjugate is the same as that explained above.

[0570] The medicament may be used for preventing or treating metabolic syndrome.

[0571] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples.

[0572] Example 1: Preparation of a triple agonist

[0573] Triple agonists that are all active against GLP-1, GIP and glucagon receptors were prepared and their amino acid sequences are shown in Table 1 below.

[0574] [Table 1]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581] In the sequences described in Table 1, the amino acid represented by X represents amino isobutyric acid (Aib) as a non-natural amino acid, and the underlined amino acids represent the formation of a loop between the underlined amino acids. Additionally, in Table 1, CA represents 4-imidazolylacetyl and Y represents tyrosine.

[0582] Example 2: Preparation of a long-acting conjugate of a triple agonist

[0583] For the pegylation of the cysteine residues of the triple agonists (SEQ ID NOS: 21, 22, 42, 43, 50, 77, and 96) of Example 1 using PEG (10 kDa) having maleimide groups and aldehyde groups at both ends (i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan)), the triple agonist was reacted with maleimide-PEG-aldehyde at a molar ratio of 1:1 to 3, at a protein concentration of 1 mg / mL to 5 mg / mL, at low temperature, for 0.5 to 3 hours. In particular, the reaction was carried out in an environment where 20% to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction product was applied to SP Sepharose HP (GE healthcare, USA) to purify the triple agonist, which was mono-pegylated at its cysteine residues.

[0584] Then, the purified mono-pegylated triple agonist and immunoglobulin Fc were reacted at a molar ratio of 1:1 to 5, at a protein concentration of 10 mg / mL to 50 mg / mL, at 4 °C to 8 °C, for 12 hours to 18 hours. The reaction was carried out in an environment where 10 mM to 50 mM sodium cyanoborohydride (NaCNBH3) (i.e., reducing agent), and 10% to 30% isopropanol were added to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was completed, the reaction product was applied to a butyl Sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA) to purify the conjugate comprising the triple agonist and immunoglobulin Fc.

[0585] After preparation, the purity analyzed by reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography was shown to be 95% or higher.

[0586] In particular, the conjugate in which the triple agonist of SEQ ID NO: 21 and immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 21 and immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 21", and they can be used interchangeably in the present invention.

[0587] In particular, the conjugate in which the triple agonist of SEQ ID NO: 22 and immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 22 and immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 22", and they can be used interchangeably in the present invention.

[0588] Specifically, the conjugate in which the triple agonist of SEQ ID NO: 42 and the immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 42 and the immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 42", and they can be used interchangeably in the present invention.

[0589] Specifically, the conjugate in which the triple agonist of SEQ ID NO: 43 and the immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 43 and the immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 43", and they can be used interchangeably in the present invention.

[0590] Specifically, the conjugate in which the triple agonist of SEQ ID NO: 50 and the immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 50 and the immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 50", and they can be used interchangeably in the present invention.

[0591] Specifically, the conjugate in which the triple agonist of SEQ ID NO: 77 and the immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 77 and the immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 77", and they can be used interchangeably in the present invention.

[0592] Specifically, the conjugate in which the triple agonist of SEQ ID NO: 96 and the immunoglobulin Fc are linked by PEG is named "conjugate comprising the triple agonist of SEQ ID NO: 96 and the immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 96", and they can be used interchangeably in the present invention.

[0593] Experimental Example 1: Measurement of the in vitro activity of a triple agonist and its long-acting conjugate

[0594] The activities of the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were measured by a method of measuring cell activity in vitro using cell lines in which the GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor were respectively transfected.

[0595] Each of the above cell lines is a cell line in which the genes of the human GLP-1 receptor, human GCG receptor, and human GIP receptor are respectively transfected into Chinese hamster ovary (CHO) and can be expressed therein, and thus is suitable for measuring the activities of GLP-1, GCG, and GIP. Therefore, the activities of each part were measured using the respectively transfected cell lines.

[0596] To measure the GLP-1 activity of the triple agonists and long-acting conjugates prepared in Examples 1 and 2, human GLP-1 was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing the human GLP-1 receptor, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μL. Then, a buffer solution containing a cAMP antibody was added thereto in an amount of 5 μL, and the mixture was incubated at room temperature for 15 minutes. Then, a detection mixture containing a cell lysis buffer was added thereto in an amount of 10 μL to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction was completed, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value, and these values were compared with each other. The relative potencies compared with human GLP-1 are shown in Tables 2 and 3 below.

[0597] To measure the GCG activity of the triple agonists and long-acting conjugates prepared in Examples 1 and 2, human GCG was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing the human GCG receptor, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μL. Then, a buffer solution containing a cAMP antibody was added thereto in an amount of 5 μL, and the mixture was incubated at room temperature for 15 minutes. Then, a detection mixture containing a cell lysis buffer was added thereto in an amount of 10 μL to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction was completed, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value, and these values were compared with each other. The relative potencies compared with human GCG are shown in Tables 2 and 3 below.

[0598] To measure the GIP activity of the triple agonists and long-acting conjugates prepared in Examples 1 and 2, human GIP was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing the human GIP receptor, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μL. Then, a buffer solution containing a cAMP antibody was added thereto in an amount of 5 μL, and the mixture was incubated at room temperature for 15 minutes. Then, a detection mixture containing a cell lysis buffer was added thereto in an amount of 10 μL to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction was completed, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value, and these values were compared with each other. The relative potencies compared with human GIP are shown in Tables 2 and 3 below.

[0599] [Table 2] Relative potency ratios of triple agonists

[0600]

[0601]

[0602]

[0603]

[0604]

[0605] [Table 3] Relative potency ratios of long-acting conjugates of triple agonists

[0606]

[0607] The long-acting conjugate of the triple agonist prepared above has the function of a triple agonist that can activate all GLP-1 receptors, GIP receptors, and glucagon receptors, and thus the long-acting conjugate of the triple agonist can be used as a therapeutic substance for treating patients with metabolic syndrome including diabetes and obesity.

[0608] Experimental Example 2: Measurement of the in vivo activity of a long-acting conjugate of a triple agonist

[0609] In this experiment, high-fat diet-induced obese mice, which are widely used as an animal model for obesity, were used. The body weight of the mice before administration was in the range of about 40 g to about 60 g. The mice were housed in groups during the experiment and had free access to water. Lighting was not provided between 6 AM and 6 PM.

[0610] The experimental groups fed with a high-fat diet included: Group 1, the vehicle (injected once every two days) - control group; Group 2, the long-acting conjugate of SEQ ID NO:42 at 1.44 nmol / kg (injected once every two days); Group 3, the long-acting conjugate of SEQ ID NO:42 at 2.88 nmol / kg (injected once every two days); Group 4, the long-acting conjugate of SEQ ID NO:43 at 1.44 nmol / kg (injected once every two days); Group 5, the long-acting conjugate of SEQ ID NO:43 at 2.88 nmol / kg (injected once every two days); Group 6, the long-acting conjugate of SEQ ID NO:50 at 1.44 nmol / kg (injected once every two days); and Group 7, the long-acting conjugate of SEQ ID NO:50 at 2.88 nmol / kg (injected once every two days). The experiment was terminated on day 28, and the change in body weight of the mice in each group was measured at two-day intervals during the experiment. After the experiment was terminated, the amount of mesenteric fat was measured by autopsy. Statistical analysis was performed to compare between the control group and the experimental groups by one-way ANOVA.

[0611] As a result of the measurement of the change in body weight, as can be confirmed in Figure 1 , compared with before administration, all the groups given the long-acting conjugates of SEQ ID NOS:42, 43, and 50 at high doses showed a 56.9%, 57.0%, and 63.5% reduction in body weight, respectively.

[0612] In addition, as a result of the measurement of the amount of fat in the mesentery, as can be confirmed in Figure 2 , compared with the group given the vehicle, all the groups given the long-acting conjugates of SEQ ID NOS:42, 43, and 50 at high doses showed a significant reduction in body fat.

[0613] From the foregoing, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without modifying the technical concept or essential features of the present invention. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A conjugate represented by the following Chemical Formula 1: [Chemical Formula 1] X-La-F Wherein, X is a peptide active against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor; L is a linker; a is 1, and wherein the linker is polyethylene glycol; and F is an immunoglobulin Fc region in dimeric form, wherein X is a peptide consisting of an amino acid sequence selected from SEQ ID NOS: 27, 32, 36, and 37.

2. The conjugate according to claim 1, wherein X is a peptide consisting of an amino acid sequence selected from SEQ ID NOS: 32 and 37.

3. The conjugate according to claim 1, wherein F is an IgG Fc region.

4. A pharmaceutical composition for preventing or treating metabolic syndrome, comprising the conjugate according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, wherein the metabolic syndrome includes glucose intolerance, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis, or coronary heart disease.

6. The pharmaceutical composition according to claim 4, wherein the metabolic syndrome includes arteriosclerosis attributable to dyslipidemia.

7. The pharmaceutical composition according to claim 4, wherein the metabolic syndrome includes atherosclerosis.

8. The pharmaceutical composition according to claim 4, wherein the metabolic syndrome includes hypercholesterolemia.

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