Glucagon / glpi / gip receptor triple agonist

Abstract

The present invention is entitled Triple Agonists at the Glucagon / GLP-1 / GIP Receptor. The present invention relates to triple agonists that are active at all of the glucagon, GLP-1 and GIP receptors and uses thereof.

Description

[0001] This application is a divisional application. The original application was filed on December 30, 2016, with application number 2016800823523 (PCT / KR2016 / 015554) and invention title "Glucagon / GLP-1 / GIP receptor triple agonist". Technical Field

[0002] This invention relates to a triple agonist that is active against all three receptors: glucagon, GLP-1, and GIP, and its uses. Background Technology

[0003] Obesity and diabetes, including type 2 diabetes, are representative metabolic diseases prevalent in modern society. These diseases are considered health threats worldwide, and the economic costs associated with their incidence are currently increasing rapidly.

[0004] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-releasing peptide (GIP) are representative gastrointestinal hormones and neuronal hormones involved in controlling glucose levels based on food intake. Glucagon is a peptide hormone secreted by the pancreas and is involved in controlling blood glucose levels along with the aforementioned two substances.

[0005] GLP-1 is a hormone secreted by the small intestine in response to food intake. GLP-1 promotes insulin secretion from the pancreas and inhibits glucagon secretion in a glucose-dependent manner, thereby helping to lower blood sugar levels. Additionally, GLP-1 slows digestion in the gastrointestinal tract by acting as a satiety factor and reduces food intake by delaying the emptying of digested food from the gastrointestinal tract. Furthermore, administration of GLP-1 to rats has been reported to inhibit food intake and reduce body weight, and these effects have been confirmed to occur equally in both normal and obese states, thus demonstrating the potential of GLP-1 as a drug for the treatment of obesity.

[0006] Like GLP-1, GIP, one of the gastrointestinal hormones secreted in response to food intake, is a 42-amino acid hormone secreted by intestinal K cells. GIP has been reported to promote insulin secretion in the pancreas in a blood sugar-dependent manner and help lower blood sugar levels, thereby exhibiting effects such as increased GLP-1 activation and anti-inflammatory effects.

[0007] When blood sugar levels drop due to factors such as medication, illness, hormone deficiency, or enzyme deficiency, glucagon is produced in the pancreas. In the liver, glucagon signals the breakdown of glycogen to induce glucose release and raise blood sugar levels back to normal. In addition to increasing blood sugar levels, glucagon also suppresses appetite in animals and humans and activates hormone-sensitive lipases in adipocytes to promote lipolysis and energy expenditure, thus exhibiting an anti-obesity effect.

[0008] Therefore, based on the effects of GLP-1 in controlling blood sugar levels and reducing weight, active research is underway to develop GLP-1 as a therapeutic agent for the treatment of diabetes and obesity. Currently, lizard venom exotropic peptide-4, derived from lizard venom and possessing approximately 50% amino acid homology with GLP-1, is under development as a therapeutic agent for the same type of disease. However, therapeutic agents containing both GLP-1 and lizard venom exotropic peptide-4 have reported side effects such as vomiting and nausea (SyedYY., Drugs, 2015 Jul; 75(10):1141-52).

[0009] In addition, in order to maximize weight loss and as an alternative to the aforementioned GLP-1-based therapeutics, research has focused on dual agonists that are active against both the GLP-1 receptor and the glucagon receptor, and they have shown to be more effective in weight loss due to the activation of the glucagon receptor compared to existing GLP-1 monotherapy (Jonathan W et al., Nat Chem Bio., 2009 Oct(5); 749-757).

[0010] Furthermore, in studies involving triple agonists that are active against all three receptors (GLP-1, GIP, and glucagon), recent efforts have focused on increasing resistance to dipeptidyl peptidase-IV (DPP-IV), which breaks down gastrointestinal hormones to remove their activity, by substituting amino acid sequences, and then increasing the half-life of the triple agonists by adding acyl groups to specific regions (Finan B et al., Nat Med., 2015 Jan; 21(1): 27-36). However, their effects on activating the three different types of receptors are not significant, and the triple agonists do not exhibit various activity ratios.

[0011] Therefore, there is a need for developing novel substances that can highly activate GLP-1, GIP, and glucagon receptors and have the effect of controlling blood sugar levels and reducing weight without causing any side effects such as vomiting and nausea.

[0012] Furthermore, there is a need to develop novel substances with various activity ratios to GLP-1, GIP, and glucagon receptors. For example, there is an increasing need for substances that have a weight-reducing effect but significantly higher glycemic control due to high GLP-1 and GIP activity, yet have relatively low glucagon activity for the purpose of lowering blood sugar; or substances that have high activity to all of GLP-1, GIP, and glucagon, thus exhibiting a significantly high weight-reducing effect. Summary of the Invention

[0013] Technical issues

[0014] The purpose of this invention is to provide isolated peptides that are active against glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor.

[0015] Another object of the present invention is to provide polynucleotides encoding isolated peptides, recombinant expression vectors including polynucleotides, and transformants including polynucleotides or recombinant expression vectors.

[0016] Another object of the present invention is to provide a method for preparing isolated peptides.

[0017] Another object of the present invention is to provide compositions containing isolated peptides.

[0018] Another object of the present invention is to provide a method for treating a target disease, the method comprising administering an isolated peptide or a composition containing an isolated peptide to a subject in need of it.

[0019] Another object of the present invention is to provide the use of isolated peptides or compositions for the preparation of pharmaceuticals.

[0020] Technical solution

[0021] To achieve the above objectives, in one aspect, the present invention provides isolated peptides that are active against glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor.

[0022] In one embodiment, the peptide is an analog of natural glucagon having a variation selected from substitution, addition, deletion, modification, and combinations thereof on at least one amino acid of the natural glucagon sequence.

[0023] In another specific embodiment, the added amino acid sequence is derived from the natural GLP-1 amino acid sequence, the natural GIP amino acid sequence, or the natural venomous lizard exopeptide-4 amino acid sequence.

[0024] In another specific embodiment, the peptide is a separated peptide comprising an amino acid sequence represented by the following general formula 1:

[0025] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa1 7-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO:103)

[0026] In general formula 1,

[0027] Xaa1 can be histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y);

[0028] Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib (aminoisobutyric acid);

[0029] Xaa3 is either glutamic acid (Glu, E) or glutamine (Gln, Q);

[0030] Xaa7 is either threonine (Thr, T) or isoleucine (Ile, I);

[0031] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V).

[0032] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

[0033] Xaa13 can be glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C);

[0034] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

[0035] Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L);

[0036] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

[0037] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K).

[0038] Xaa18 can be alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);

[0039] Xaa19 can be alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V);

[0040] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

[0041] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D);

[0042] Xaa23 is either isoleucine (Ile, I) or valine (Val, V);

[0043] Xaa24 can be alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E).

[0044] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);

[0045] Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D).

[0046] Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H).

[0047] Xaa30 may be cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or may be absent; and

[0048] 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;

[0049] in,

[0050] m can be -Cys-, -Pro-, or -Gly-Pro-.

[0051] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or it does not exist.

[0052] In another specific implementation,

[0053] In general formula 1,

[0054] Xaa14 is either leucine or methionine; and

[0055] Xaa15 is cysteine, aspartic acid, or leucine.

[0056] In another specific embodiment, in general formula 1,

[0057] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0058] Xaa7 is threonine;

[0059] Xaa10 is tyrosine, cysteine, or valine;

[0060] Xaa12 is either lysine or isoleucine;

[0061] Xaa13 is tyrosine, alanine, glutamine, or cysteine;

[0062] Xaa14 is leucine, cysteine, or methionine;

[0063] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

[0064] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0065] Xaa18 is alanine, glutamine, arginine, or histidine;

[0066] Xaa19 is alanine, glutamine, valine, or cysteine;

[0067] Xaa20 is lysine, arginine, or glutamine;

[0068] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0069] Xaa23 is either isoleucine or valine;

[0070] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and

[0071] Xaa27 is either leucine or lysine.

[0072] In another specific embodiment, the peptide comprises an amino acid sequence represented by the following general formula 2:

[0073] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xa a17 -

[0074] In general formula 2,

[0075] Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

[0076] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0077] Xaa10 is either tyrosine or cysteine;

[0078] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0079] Xaa14 is leucine, methionine, or tyrosine;

[0080] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0081] Xaa16 is glycine, glutamic acid, or serine;

[0082] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0083] Xaa18 is alanine, glutamine, arginine, or histidine;

[0084] Xaa19 is alanine, glutamine, cysteine, or valine;

[0085] Xaa20 is lysine, glutamine, or arginine;

[0086] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0087] Xaa23 is either isoleucine or valine;

[0088] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0089] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

[0090] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0091] Xaa30 is cysteine, glycine, lysine, or histidine;

[0092] Xaa31 is proline or cysteine; and

[0093] Xaa40 is either cysteine ​​or absent.

[0094] In another specific embodiment, in general formula 1,

[0095] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0096] Xaa7 is threonine;

[0097] Xaa10 is tyrosine, cysteine, or valine;

[0098] Xaa12 is either lysine or isoleucine;

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

[0100] Xaa14 is leucine or methionine;

[0101] Xaa15 is either cysteine ​​or aspartic acid.

[0102] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

[0103] Xaa18 is alanine, arginine, or histidine;

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

[0105] Xaa20 is either lysine or glutamine;

[0106] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0107] Xaa23 is valine;

[0108] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and Xaa27 is leucine or lysine.

[0109] In another specific embodiment, in general formula 2,

[0110] Xaa13 is alanine, tyrosine, or cysteine;

[0111] Xaa15 is either aspartic acid or glutamic acid;

[0112] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0113] Xaa18 is alanine, arginine, or histidine;

[0114] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

[0115] Xaa23 is either isoleucine or valine;

[0116] Xaa24 is cysteine, glutamine, or asparagine;

[0117] Xaa28 is cysteine, asparagine, or aspartic acid;

[0118] Xaa29 is glutamine, cysteine, or histidine; and

[0119] Xaa30 is cysteine, lysine, or histidine.

[0120] In another specific embodiment, in general formula 1,

[0121] Xaa2 is α-methyl-glutamic acid or Aib;

[0122] Xaa7 is threonine;

[0123] Xaa10 is either tyrosine or cysteine;

[0124] Xaa12 is either lysine or isoleucine;

[0125] Xaa13 is tyrosine, alanine, or cysteine;

[0126] Xaa14 is leucine or methionine;

[0127] Xaa15 is either cysteine ​​or aspartic acid.

[0128] Xaa16 is glutamic acid;

[0129] Xaa17 is arginine, isoleucine, cysteine, or lysine;

[0130] Xaa18 is alanine, arginine, or histidine;

[0131] Xaa19 is alanine, glutamine, or cysteine;

[0132] Xaa20 is either lysine or glutamine;

[0133] Xaa21 is either glutamic acid or aspartic acid;

[0134] Xaa23 is valine;

[0135] Xaa24 is glutamine, asparagine, or aspartic acid;

[0136] Xaa27 is leucine; and

[0137] Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0138] In another specific embodiment, in general formula 1,

[0139] Xaa1 is histidine or 4-imidazoacetyl;

[0140] Xaa2 is α-methyl-glutamic acid or Aib;

[0141] Xaa3 is glutamine;

[0142] Xaa7 is threonine;

[0143] Xaa10 is tyrosine;

[0144] Xaa12 is isoleucine;

[0145] Xaa13 is either alanine or cysteine;

[0146] Xaa14 is methionine;

[0147] Xaa15 is aspartic acid;

[0148] Xaa16 is glutamic acid;

[0149] Xaa17 is either isoleucine or lysine;

[0150] Xaa18 is either alanine or histidine;

[0151] Xaa19 is either glutamine or cysteine;

[0152] Xaa20 is lysine;

[0153] Xaa21 is aspartic acid;

[0154] Xaa23 is valine;

[0155] Xaa24 is asparagine;

[0156] Xaa27 is leucine;

[0157] Xaa28 is either alanine or asparagine;

[0158] Xaa29 is either glutamine or threonine; and

[0159] Xaa30 may be cysteine, lysine, or absent.

[0160] In another specific implementation,

[0161] In general formula 1,

[0162] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0163] Xaa3 is glutamine;

[0164] Xaa7 is threonine;

[0165] Xaa10 is tyrosine, cysteine, or valine;

[0166] Xaa12 is lysine;

[0167] Xaa13 is tyrosine;

[0168] Xaa14 is leucine;

[0169] Xaa15 is aspartic acid;

[0170] Xaa16 is glycine, glutamic acid, or serine;

[0171] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0172] Xaa18 is alanine, arginine, or histidine;

[0173] Xaa19 is either alanine or glutamine;

[0174] Xaa20 is either lysine or glutamine;

[0175] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0176] Xaa23 is valine;

[0177] Xaa24 is alanine, glutamine, or cysteine;

[0178] Xaa27 is either leucine or lysine; and

[0179] Xaa29 is glycine, glutamine, threonine, or histidine.

[0180] In another specific embodiment, the peptide is a peptide comprising an amino acid sequence represented by the following general formula 3:

[0181] 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).

[0182] In general formula 3,

[0183] Xaa1 is histidine or tyrosine;

[0184] Xaa2 is α-methyl-glutamic acid or Aib;

[0185] Xaa13 is alanine, tyrosine, or cysteine;

[0186] Xaa17 is arginine, cysteine, or lysine;

[0187] Xaa18 is either alanine or arginine;

[0188] Xaa19 is either alanine or cysteine;

[0189] Xaa21 is either glutamic acid or aspartic acid;

[0190] Xaa24 is either glutamine or asparagine;

[0191] Xaa28 is either cysteine ​​or aspartic acid;

[0192] Xaa29 is cysteine, histidine, or glutamine;

[0193] Xaa30 is either cysteine ​​or histidine;

[0194] Xaa31 is proline or cysteine; and

[0195] Xaa40 is either cysteine ​​or absent.

[0196] In another specific embodiment, R1 is cysteine, 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 not present.

[0197] In another specific embodiment, in general formulas 1 to 3, the 16th and 20th amino acids from the N-terminus together form a ring.

[0198] In another specific embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOS:1 to 102.

[0199] In another specific embodiment, the peptide has at least one of the following activities i) to iii):

[0200] i) Activation of GLP-1 receptor;

[0201] ii) Activation of glucagon receptors; and

[0202] iii) Activation of GIP receptors.

[0203] In another specific embodiment, the peptide has an increased in vivo half-life compared to any one of natural GLP-1, natural glucagon, and natural GIP.

[0204] In another specific embodiment, the C-terminus of the peptide is amidated.

[0205] Another aspect of the invention provides a polynucleotide encoding a separated peptide, a recombinant expression vector comprising the polynucleotide, and a transformant comprising the polynucleotide or the recombinant expression vector.

[0206] Another aspect of the present invention provides a method for preparing isolated peptides.

[0207] Another aspect of the invention provides compositions comprising isolated peptides.

[0208] In one specific embodiment, the composition is a pharmaceutical composition.

[0209] In another specific embodiment, the composition is used for the prevention or treatment of metabolic syndrome.

[0210] In another specific embodiment, metabolic syndrome includes glucose intolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary heart disease.

[0211] Another object of the present invention is to provide a method for treating a target disease, the method comprising administering to a subject in need of the isolated peptide or a composition containing the isolated peptide.

[0212] Another object of the present invention is to provide the use of isolated peptides or compositions in pharmaceutical preparation.

[0213] Beneficial effects of the invention

[0214] The peptides according to the invention have activity against glucagon receptors, glucagon-like peptide-1 (GLP-1) receptors and glucose-dependent insulinotropic peptide (GIP) receptors, and are therefore applicable to the treatment of metabolic syndrome. Detailed Implementation

[0215] The invention will be described in more detail below.

[0216] Furthermore, 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 fall within the scope of this invention. Moreover, the scope of this invention should not be limited by the specific disclosures provided below.

[0217] Throughout this invention, not only are the conventional single-letter and three-letter codes for naturally occurring amino acids used, but also those three-letter codes that are generally permitted for other amino acids, such as α-aminoisobutyric acid (Aib), Sar (N-methylglycine), and α-methyl-glutamic acid.

[0218] In addition, the amino acids mentioned in this article are abbreviated as follows according to the IUPAC-IUB naming rules:

[0219]

[0220] One aspect of the present invention provides isolated peptides that are active against glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic peptide (GIP) receptor.

[0221] In this invention, isolated peptides active against glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor, and glucose-dependent insulinotropic peptide (GIP) receptor can be used interchangeably with triple agonists.

[0222] Triple agonists can include a variety of substances (e.g., various peptides) that have significant levels of activity against glucagon, GLP-1, and GIP receptors.

[0223] Triple agonists exhibiting significant levels of activity against glucagon, GLP-1, and GIP receptors compared to their natural ligands (natural glucagon, natural GLP-1, and natural GIP) 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, but are not particularly limited thereto, against one or more, specifically two or more, or more than all three, receptors of glucagon, GLP-1, and GIP.

[0224] The method for measuring the in vitro activity of the triple agonist can be referred to in Example 2 of the present invention, but is not particularly limited thereto.

[0225] Meanwhile, the triple agonist is characterized by having one or more of the following activities (i) to (iii), specifically its significant activities (one or more):

[0226] i) Activation of GLP-1 receptor; ii) Activation of glucagon receptor; and iii) Activation of GIP receptor.

[0227] In particular, receptor activation may include, for example, cases where the in vitro activity relative to the natural ligand of the corresponding receptor 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, but the activation is not limited thereto.

[0228] In addition, the triple agonist can be an agonist with an increased in vivo half-life relative to any one of natural GLP-1, natural glucagon and natural GIP, but is not particularly limited thereto.

[0229] The aforementioned glucagon analogues may be non-natural analogues, but are not particularly limited thereto.

[0230] Specifically, the isolated peptides can be analogs of natural glucagon, but are not particularly limited to this.

[0231] The natural glucagon analogues according to the present invention may include peptides that differ from natural glucagon in at least one amino acid sequence; peptides modified by modification of the natural glucagon sequence; and mimics of natural glucagon.

[0232] Meanwhile, natural glucagon may have the following amino acid sequence, but is not particularly limited to this:

[0233] 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)

[0234] Specifically, the isolated peptide may be an analogue of natural glucagon having variations selected from substitutions, additions, deletions, modifications, and combinations thereof at at least one amino acid in the natural glucagon sequence, but is not particularly limited thereto.

[0235] In addition, amino acid substitution can include replacing amino acids with other amino acids and replacing both with non-natural compounds.

[0236] Additionally, addition can be made at the N-terminus and / or C-terminus of the peptide. There is no particular limitation on the length of the added amino acids, but 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, and eleven or more amino acids can be added. In a broader sense, addition can include, but is not particularly limited to, the addition of polypeptides.

[0237] More specifically, glucagon analogues may be 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, or thirteen or more amino acids selected from positions 1, 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 of the amino acid sequence of natural glucagon. Those in which one or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 amino acids are replaced by other amino acids, and additionally, 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 their C-terminus, but are not particularly limited thereto.

[0238] More specifically, glucagon analogues may 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, seventeen or more, eighteen or more, nineteen or more, tenteen or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen or more amino acids are substituted with other amino acids, and additionally, may 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 their C-terminus, but are not particularly limited thereto.

[0239] More specifically, glucagon analogues may 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 acid sequence of natural glucagon are substituted with other amino acids, and additionally, may 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 their C-terminus, but are not particularly limited thereto.

[0240] More specifically, glucagon analogues may be 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 positions 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28 and 29 of the amino acid sequence of natural glucagon are substituted with other amino acids, and additionally, may 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 their C-terminus, but are not particularly limited thereto.

[0241] The amino acids to be introduced from the above-mentioned natural 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 to these.

[0242] For example, the amino acid sequence (one or more) to be added may be at least one amino acid sequence derived from natural GLP-1, natural GIP, or natural venomous lizard exopeptide-4 amino acid sequence.

[0243] Glucagon analogs or triple agonists may include intramolecular bridges (e.g., covalently cross-linked or non-covalently cross-linked), and specifically, in the form of rings, for example, in the form of a ring formed between the 16th and 20th amino acids of the glucagon analog or triple agonist, but are not particularly limited thereto.

[0244] Non-limiting examples of rings may include lactam bridges (or lactam rings).

[0245] In addition, glucagon analogs or triple agonists include all of those modified to include a ring, or to include amino acids capable of forming a ring at the target position.

[0246] For example, glucagon analogs or triple agonists may be analogs or triple agonists in which the amino acid pairs at positions 16 and 20 are replaced by cyclic glutamic acid or lysine, respectively, but glucagon analogs or triple agonists are not limited thereto.

[0247] The ring can be formed between the amino acid side chains within glucagon analogs or triple agonists; for example, they can be in the form of a lactam ring between the side chains of lysine and glutamic acid, but the ring is not particularly limited thereto.

[0248] Examples of glucagon analogs prepared by combination of these methods may include peptides whose amino acid sequence differs from that of natural glucagon in at least one amino acid, and wherein the α-carbon at its N-terminus is removed, while being active for glucagon receptors, GLP-1 receptors, and GIP receptors, etc., but are not limited thereto, and analogs of natural glucagon suitable for the present invention may be prepared by combination of various methods used for the preparation of analogs.

[0249] 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 recognition by peptidases, but the triple agonist is not particularly limited to this.

[0250] Specifically, a peptide can be one in which the half-life in vivo is increased by replacing the second amino acid sequence in the amino acid sequence of the triple agonist to avoid recognition by peptidases, but there are no restrictions on any substitution or modification of amino acids to avoid recognition by peptidases in vivo.

[0251] In addition, such modifications used to prepare analogs of natural glucagon may include all modifications using L- or D-type amino acids and / or non-natural amino acids; and / or modifications of the natural sequence, such as modifications of side chain functional groups, intramolecular covalent bonding (e.g., ring formation between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation, etc.

[0252] Additionally, modifications may include all of those in which one or more amino acids are added to the amino and / or carboxyl termini of natural glucagon.

[0253] During the 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 containing these amino acids and typical peptide sequences can be synthesized and purchased from commercial suppliers such as American Peptide Company, Bachem (USA), or Anygen (Korea).

[0254] Amino acid derivatives can be obtained in the same manner, and as an example, 4-imidazolic acid (4-imidazoacetic acid) can be used.

[0255] In addition, the peptides according to the invention may be in variant forms, wherein the amino and / or carboxyl termini of the peptide are chemically modified or protected by organic groups, or amino acids may be added to the termini for their protection against in vivo proteases while increasing their stability.

[0256] In particular, in the case of chemically synthesized peptides, their N- and C-termini are charged, and therefore the N- and C-termini of the peptides can be acetylated and / or amidated, but peptides are not particularly limited to this.

[0257] Additionally, the peptides according to the invention may include all of those in the form of the peptide itself, its salts (e.g., pharmaceutically acceptable salts), or its solvates. Furthermore, the peptide may be in any pharmaceutically acceptable form.

[0258] There are no particular restrictions on the types of salt. However, salts are preferably safe and effective for the target organism (e.g., mammals), but are not particularly limited thereto.

[0259] The term "pharmaceutical acceptable" refers to a substance that can be used effectively for its intended purpose within the limits determined by pharmacology and medicine, without causing excessive toxicity, irritation, allergic reactions, etc.

[0260] 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 acids may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Examples of salts derived from suitable bases may include alkali metals such as sodium and potassium; alkaline earth metals such as magnesium; ammonium, etc.

[0261] As used herein, the term "solvent" refers to a complex formed between a peptide or its salt according to the invention and a solvent molecule.

[0262] In one specific embodiment, the peptide may be a separate peptide comprising an amino acid sequence represented by the following general formula 1.

[0263] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa1 7-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO:103)

[0264] In the above general formula 1,

[0265] Xaa1 can be histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y);

[0266] Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib (aminoisobutyric acid);

[0267] Xaa3 is either glutamic acid (Glu, E) or glutamine (Gln, Q);

[0268] Xaa7 is either threonine (Thr, T) or isoleucine (Ile, I);

[0269] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V).

[0270] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

[0271] Xaa13 can be glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C);

[0272] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

[0273] Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L);

[0274] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

[0275] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K).

[0276] Xaa18 can be alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);

[0277] Xaa19 can be alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V);

[0278] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

[0279] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D);

[0280] Xaa23 is either isoleucine (Ile, I) or valine (Val, V);

[0281] Xaa24 can be alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E).

[0282] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);

[0283] Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D).

[0284] Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H).

[0285] Xaa30 may be cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or may be absent.

[0286] 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;

[0287] in,

[0288] m can be -Cys-, -Pro-, or -Gly-Pro-;

[0289] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or does not exist.

[0290] For example, a triple agonist may be an agonist comprising an amino acid sequence selected from SEQ ID NOS:1 to 102; and an agonist consisting (substantially) of an amino acid sequence selected from SEQ ID NOS:1 to 102, but is not limited thereto.

[0291] Furthermore, although described in this invention as a “peptide composed of a specific SEQ ID NO”, as long as the peptide has the same or corresponding activity as the peptide composed of the amino acid sequence of the corresponding SEQ ID NO, it does not exclude mutations that may occur by the addition of meaningless sequences upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO, or naturally occurring mutations, or their silent mutations, and it is clearly within the scope of this invention even when sequence addition or mutation is present.

[0292] The above description can be applied to other specific embodiments or aspects of the present invention, but is not limited thereto.

[0293] Specifically, in the above general formula, Xaa14 can be leucine or methionine, and Xaa15 can be cysteine, aspartic acid, or leucine.

[0294] Examples of peptides may include, but are not particularly limited to, peptides comprising or (substantially) composed of amino acid sequences selected from SEQ ID NOS:1 to 12, 14 to 17 and 21 to 102.

[0295] The peptide can significantly activate at least one of the glucagon receptor, GLP-1 receptor, and GIP receptor, but is not particularly limited thereto. Specifically, the peptide can 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.

[0296] Even more specifically, peptides can be:

[0297] In the above general formula 1,

[0298] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0299] Xaa7 is threonine;

[0300] Xaa10 is tyrosine, cysteine, or valine;

[0301] Xaa12 is either lysine or isoleucine;

[0302] Xaa13 is tyrosine, alanine, glutamine, or cysteine;

[0303] Xaa14 is leucine, cysteine, or methionine;

[0304] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

[0305] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0306] Xaa18 is alanine, glutamine, arginine, or histidine;

[0307] Xaa19 is alanine, glutamine, valine, or cysteine;

[0308] Xaa20 is lysine, arginine, or glutamine;

[0309] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0310] Xaa23 is either isoleucine or valine;

[0311] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and

[0312] Xaa27 can be leucine or lysine, but is not specifically limited to these.

[0313] Even more specifically,

[0314] In the above general formula 1,

[0315] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0316] Xaa7 is threonine;

[0317] Xaa10 is tyrosine, cysteine, or valine;

[0318] Xaa12 is either lysine or isoleucine;

[0319] Xaa13 is tyrosine, alanine, or cysteine;

[0320] Xaa14 is leucine or methionine;

[0321] Xaa15 is either cysteine ​​or aspartic acid.

[0322] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

[0323] Xaa18 is alanine, arginine, or histidine;

[0324] Xaa19 is alanine, glutamine, or cysteine;

[0325] Xaa20 is either lysine or glutamine;

[0326] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0327] Xaa23 is valine;

[0328] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and Xaa27 is leucine or lysine, but not particularly limited thereto.

[0329] Even more specifically,

[0330] In the above general formula 1,

[0331] Xaa2 is α-methyl-glutamic acid or Aib;

[0332] Xaa7 is threonine;

[0333] Xaa10 is either tyrosine or cysteine;

[0334] Xaa12 is either lysine or isoleucine;

[0335] Xaa13 is tyrosine, alanine, or cysteine;

[0336] Xaa14 is leucine or methionine;

[0337] Xaa15 is either cysteine ​​or aspartic acid.

[0338] Xaa16 is glutamic acid;

[0339] Xaa17 is arginine, isoleucine, cysteine, or lysine;

[0340] Xaa18 is alanine, arginine, or histidine;

[0341] Xaa19 is alanine, glutamine, or cysteine;

[0342] Xaa20 is either lysine or glutamine;

[0343] Xaa21 is either glutamic acid or aspartic acid;

[0344] Xaa23 is valine;

[0345] Xaa24 is glutamine, asparagine, or aspartic acid;

[0346] Xaa27 is leucine; and

[0347] Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0348] Specifically,

[0349] In the above general formula 1,

[0350] Xaa1 is histidine or 4-imidazoacetyl;

[0351] Xaa2 is α-methyl-glutamic acid or Aib;

[0352] Xaa3 is glutamine;

[0353] Xaa7 is threonine;

[0354] Xaa10 is tyrosine;

[0355] Xaa12 is isoleucine;

[0356] Xaa13 is either alanine or cysteine;

[0357] Xaa14 is methionine;

[0358] Xaa15 is aspartic acid;

[0359] Xaa16 is glutamic acid;

[0360] Xaa17 is either isoleucine or lysine;

[0361] Xaa18 is either alanine or histidine;

[0362] Xaa19 is either glutamine or cysteine;

[0363] Xaa20 is lysine;

[0364] Xaa21 is aspartic acid;

[0365] Xaa23 is valine;

[0366] Xaa24 is asparagine;

[0367] Xaa27 is leucine;

[0368] Xaa28 is either alanine or asparagine;

[0369] Xaa29 is either glutamine or threonine; and

[0370] Xaa30 may be cysteine ​​or lysine, or it may not be present.

[0371] More specifically,

[0372] In the above general formula 1,

[0373] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0374] Xaa3 is glutamine;

[0375] Xaa7 is threonine;

[0376] Xaa10 is tyrosine, cysteine, or valine;

[0377] Xaa12 is lysine;

[0378] Xaa13 is tyrosine;

[0379] Xaa14 is leucine;

[0380] Xaa15 is aspartic acid;

[0381] Xaa16 is glycine, glutamic acid, or serine;

[0382] Xaa17 is glutamine, arginine, cysteine, or lysine; Xaa18 is alanine, arginine, or histidine.

[0383] Xaa19 is either alanine or glutamine;

[0384] Xaa20 is either lysine or glutamine;

[0385] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0386] Xaa23 is valine;

[0387] Xaa24 is alanine, glutamine, or cysteine;

[0388] Xaa27 is either leucine or lysine; and

[0389] Xaa28 is glycine, glutamine, threonine, or histidine;

[0390] However, this is not a specific limitation.

[0391] These peptides may be equivalent to the following: a peptide exhibiting significant activation levels for both GLP-1 receptors and glucagon receptors, or a higher activation level compared to the GIP receptor; a peptide exhibiting significant activation levels for all three receptors (GLP-1, glucagon, and GIP); or a peptide exhibiting significant activation levels for both GLP-1 and GIP receptors, and a higher activation level compared to the glucagon receptor; but are not particularly limited thereto.

[0392] When a peptide has a significant activation level on both GLP-1 receptor and GIP receptor, and a higher activation level than on glucagon receptor, it can provide a peptide with a greater ability to control blood sugar levels and reduce weight. However, when a peptide has a significant activation level on all three receptors (GLP-1, glucagon, and GIP), it has the advantage of maximizing the weight reduction effect, but this effect is not particularly limited to this.

[0393] Examples of peptides 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 composed (substantially) of, but not particularly limited thereto.

[0394] In one specific embodiment, the peptide may include an amino acid sequence represented by the following general formula 2.

[0395] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-X aa21-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)

[0396] In the above general formula 2,

[0397] Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

[0398] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0399] Xaa10 is either tyrosine or cysteine;

[0400] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0401] Xaa14 is leucine, methionine, or tyrosine;

[0402] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0403] Xaa16 is glycine, glutamic acid, or serine;

[0404] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0405] Xaa18 is alanine, glutamine, arginine, or histidine;

[0406] Xaa19 is alanine, glutamine, cysteine, or valine;

[0407] Xaa20 is lysine, glutamine, or arginine;

[0408] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0409] Xaa23 is either isoleucine or valine;

[0410] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0411] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

[0412] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0413] Xaa30 is cysteine, glycine, lysine, or histidine;

[0414] Xaa31 is proline or cysteine; and

[0415] Xaa40 is either cysteine ​​or absent.

[0416] More specifically, in general formula 2 above,

[0417] Xaa13 is alanine, tyrosine, or cysteine;

[0418] Xaa15 is either aspartic acid or glutamic acid;

[0419] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0420] Xaa18 is alanine, arginine, or histidine;

[0421] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

[0422] Xaa23 is either isoleucine or valine;

[0423] Xaa24 is cysteine, glutamine, or asparagine;

[0424] Xaa28 is cysteine, asparagine, or aspartic acid;

[0425] Xaa29 is glutamine, cysteine, or histidine; and

[0426] Xaa30 is cysteine, lysine, or histidine.

[0427] Examples of peptides 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 composed (substantially) of, but not particularly limited thereto.

[0428] In one specific embodiment, the peptide may include an amino acid sequence represented by the following general formula 3.

[0429] 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),

[0430] In the above general formula 3,

[0431] Xaa1 is histidine or tyrosine;

[0432] Xaa2 is α-methyl-glutamic acid or Aib;

[0433] Xaa13 is alanine, tyrosine, or cysteine;

[0434] Xaa17 is arginine, cysteine, or lysine;

[0435] Xaa18 is either alanine or arginine;

[0436] Xaa19 is either alanine or cysteine;

[0437] Xaa21 is either glutamic acid or aspartic acid;

[0438] Xaa24 is either glutamine or asparagine.

[0439] Xaa28 is either cysteine ​​or aspartic acid;

[0440] Xaa29 is cysteine, histidine, or glutamine;

[0441] Xaa30 is either cysteine ​​or histidine;

[0442] Xaa31 is proline or cysteine; and

[0443] Xaa40 is either cysteine ​​or absent.

[0444] Examples of peptides 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 composed (substantially) of, but not particularly limited thereto.

[0445] Additionally, in the above general formula 1, R1 can 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 it may not exist, but is not particularly limited thereto.

[0446] Another aspect of the present invention provides a polynucleotide encoding a separated peptide, a recombinant expression vector containing the polynucleotide, and a transformant containing the polynucleotide or the recombinant expression vector.

[0447] Peptides are explained in the same way as above.

[0448] In addition, the isolated polynucleotide encoding the peptide, within the scope of this invention, includes polynucleotide sequences having 75% or higher, specifically 85% or higher, more specifically 90% or higher, or even more specifically 95% or higher sequence identity with the corresponding sequence.

[0449] As used herein, the term "homology" indicates sequence similarity to a wild-type amino acid sequence or wild-type nucleic acid sequence, and homology comparisons can be performed visually or using commercially available comparison programs. Using commercially available computer programs, homology between two or more sequences can be expressed as a percentage (%), and homology (%) between adjacent sequences can be calculated.

[0450] As used herein, the term “recombinant vector” refers to a DNA construct in which a polynucleotide encoding a target peptide (e.g., peptide) is operatively linked to a suitable regulatory sequence to enable the expression of the target peptide (e.g., peptide) in a host cell.

[0451] Regulatory sequences include a promoter that initiates transcription, any operator gene sequence used to regulate transcription, a sequence encoding a suitable mRNA ribosome-binding domain, and a termination sequence used to regulate transcription and translation. After transformation into a suitable host cell, the recombinant vector can replicate or function regardless of how the host genome is processed, or it can integrate into the host genome itself.

[0452] The recombinant vectors used in this invention are not particularly limited, as long as the vector can replicate in host cells and can be constructed using any vector known in the art. Examples of commonly used vectors include natural or recombinant plasmids, granules, viruses, and bacteriophages. There are no particular limitations on the vectors used in this invention, but any expression vector known in the art can be used.

[0453] The recombinant vector is used for the transformation of host cells to generate the peptides 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 used for the recombinant production of the peptides of the present invention.

[0454] As used herein, the term "transformation" refers to the process of introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell, thereby enabling the expression of the protein encoded by the polynucleotide within the host cell. For the transformed polynucleotide, it is acceptable whether it is inserted into and located on or outside the host cell's chromosome, as long as it can be expressed in the host cell; both scenarios are included.

[0455] In addition, polynucleotides include DNA and RNA encoding target proteins. Polynucleotides can be introduced in any form, as long as they can be introduced into and expressed in a host cell. For example, polynucleotides can be introduced into host cells in the form of an expression cassette, a gene construct containing all the necessary elements for self-expression. Expression cassettes typically contain a promoter operatively linked to the polynucleotide, a transcription termination signal, a ribosome-binding domain, and a translation termination signal. Expression cassettes can be in the form of self-replicating expression vectors. Alternatively, polynucleotides can be introduced into host cells as is and operatively linked to sequences necessary for their expression in the host cell, but are not limited to this.

[0456] Additionally, as used herein, the term "operably linked" refers to a functional link between a promoter sequence and the aforementioned gene sequence, the promoter sequence initiating and mediating the transcription of a polynucleotide encoding the target peptide of the present invention.

[0457] Suitable hosts for use in this invention are not particularly limited, as long as they can express the polynucleotides of this 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.

[0458] Another aspect of the present invention provides a method for preparing isolated peptides.

[0459] Peptides are explained in the same way as above.

[0460] Furthermore, the peptides of the present invention can be synthesized according to their length by methods well known in the art (e.g., by an automated peptide synthesizer) and can be produced by genetic engineering techniques.

[0461] Specifically, the peptides of the present invention can be prepared by standard synthetic 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, those described below:

[0462] (a) A method for synthesizing peptides stepwise or by fragment assembly using solid-phase or liquid-phase methods, followed by separation and purification of the final peptide product; or

[0463] (b) A method for expressing a nucleic acid construct (construct) encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0464] (c) A method for in vitro cell-free expression of a nucleic acid construct encoding a peptide, and for recovering the expression product therefrom; or

[0465] A method for obtaining peptide fragments by any combination of methods (a), (b), and (c), obtaining peptides by linking peptide fragments, and then recovering the peptides.

[0466] In a more specific instance, the desired peptide can be generated through genetic manipulation, which includes preparing a fusion gene encoding a fusion protein comprising a fusion partner and a peptide, converting the result into a host cell, expressing the fusion protein, and cleaving the peptide from the fusion protein using a protease or compound and then isolating it. For this purpose, for example, a DNA sequence encoding an amino acid sequence that can be cleaved by a protease such as factor Xa or enterokinase, CNBr, or a compound such as hydroxylamine can be inserted between the fusion partner and the polynucleotide encoding the peptide.

[0467] Another aspect of the present invention provides a composition containing isolated peptides.

[0468] Peptides are explained in the same way as above.

[0469] Specifically, the composition may be a pharmaceutical composition, and more specifically, a pharmaceutical composition for the prevention or treatment of metabolic syndrome.

[0470] As used herein, the term “prevention” refers to all actions that inhibit or delay metabolic syndrome by administering the aforementioned peptides or compositions, while the term “treatment” refers to all actions that improve or beneficially alter the symptoms of metabolic syndrome by administering the aforementioned peptides or compositions.

[0471] As used herein, the term “application” means the introduction of a particular substance into a target by a suitable method, and the route of application of the composition can be any conventional route that enables the delivery of the composition to the target, such as intraperitoneal application, intravenous application, intramuscular application, subcutaneous application, intradermal application, oral application, local application, intranasal application, intrapulmonary application, rectal application, etc., but is not limited thereto.

[0472] As used herein, the term "metabolic syndrome" refers to the symptoms, alone or in combination, of various diseases arising from chronic metabolic disorders. In particular, examples of diseases that fall under the category of metabolic syndrome may include, but are not limited to, impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis attributable to dyslipidemia, atherosclerosis, arteriosclerosis, and coronary artery disease.

[0473] As used in this article, the term "obesity" refers to a medical condition characterized by excessive accumulation of body fat, and individuals are generally defined as obese when their body mass index (BMI; weight (kg) divided by height squared (m)) is 25 or higher. Obesity is most commonly caused by an energy imbalance resulting from over a long period of time, where food intake is excessive relative to energy expenditure. As a metabolic disease affecting the entire body, obesity increases the likelihood of developing diabetes and hyperlipidemia, increases the risk of sexual dysfunction, arthritis, and cardiovascular disease, and in some cases is associated with cancer development.

[0474] The pharmaceutical compositions of the present invention may further contain pharmaceutically acceptable carriers, excipients, or diluents. Pharmaceutically acceptable carriers, excipients, or diluents may be non-naturally occurring.

[0475] As used herein, the term "pharmaceutically acceptable" means having sufficient quantity to produce a therapeutic effect without causing side effects, and can be readily determined by a person skilled in the art based on factors known in the medical field, such as the type of disease, age, weight, health status, sex, patient's drug sensitivity, route of administration, method of administration, frequency of administration, duration of treatment, and combination or simultaneous administration of one or more drugs.

[0476] The pharmaceutical compositions of the present invention containing the peptides of the present invention may further contain pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may include, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc.; for injections, buffers, preserving agents, analgesics, solubilizers, isotonic agents, stabilizers, etc., which may be used in combination; and for topical administration, alkalis, excipients, lubricants, preservatives, etc.

[0477] The formulations of the compositions according to the invention can be prepared in a variety of ways by combining them with the pharmaceutically acceptable carriers described above. For example, for oral administration, the compositions can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, rice paper wafers, etc. For injections, the compositions can be formulated into single-dose ampoules or multi-dose containers. The compositions can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0478] 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. Furthermore, the composition may further contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc.

[0479] In addition, the pharmaceutical compositions of the present invention can be prepared from any formulation type selected from tablets, pills, powders, granules, capsules, suspensions, oral liquid drugs, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations and suppositories.

[0480] In addition, the composition can be formulated into a unit dosage form suitable for the patient’s body, and preferably into a formulation useful for peptide drugs according to typical methods in the pharmaceutical field, for administration via oral or parenteral routes, such as through the skin, vein, muscle, artery, intramedullary, intrathecal, intracardiac, lung, percutaneous, subcutaneous, intraperitoneal, intranasal, gastric, local, sublingual, vaginal, or rectum, but not limited thereto.

[0481] In addition, peptides can be used by mixing with various pharmaceutically acceptable carriers such as saline or organic solvents. To increase stability or absorption, 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 in pharmaceutical products.

[0482] The dosage and frequency of administration of the pharmaceutical compositions of the present invention are determined together with various factors, such as the type of active ingredient (one or more), the route of administration, the patient's age, sex and weight, and the severity of the disease.

[0483] The total effective dose of the compositions of the present invention can be administered to the patient in a single dose or in multiple doses over a long period of time according to a fractionated treatment protocol. In the pharmaceutical compositions of the present invention, the content of one or more active ingredients can be varied according to the severity of the disease. Specifically, the total daily dose of the peptides of the present invention can be from about 0.0001 mg to 500 mg per 1 kg of patient body weight. However, in addition to the route of administration and treatment frequency of the pharmaceutical compositions, various factors including the patient's age, weight, health status, sex, disease severity, diet, and excretion rate are considered in determining the effective dose of the peptides. In this respect, those skilled in the art can readily determine the effective dose suitable for the specific use of the pharmaceutical compositions of the present invention. The pharmaceutical compositions according to the present invention are not particularly limited in formulation and route of administration or method, as long as they exhibit the effects of the present invention.

[0484] The pharmaceutical compositions of the present invention exhibit excellent duration and potency of in vivo efficacy, and thus can significantly reduce the number of administrations and frequency of the pharmaceutical formulations of the present invention.

[0485] Another aspect of the invention provides a method for treating a target disease, comprising administering an isolated peptide or a composition containing the isolated peptide to a subject in need. The target disease may be metabolic syndrome.

[0486] Isolated peptides or compositions containing them, metabolic syndrome, and their treatment are the same as explained above.

[0487] As used herein, the term "subject" means a subject suspected of having metabolic syndrome, and a subject suspected of having metabolic syndrome means a mammal including humans, rats, livestock, etc., that has metabolic syndrome or is at risk of developing metabolic syndrome, but without limitation includes any subject that can be treated with the above-described peptides of the present invention or compositions containing them.

[0488] The treatment methods of the present invention may include administering a pharmaceutically effective amount of a peptide-containing pharmaceutical composition. The total daily dose of the composition may be determined within the appropriate medical judgment of a physician, and the composition may be administered once daily or in divided doses several times daily. However, for the purposes of the present invention, the specific therapeutically effective dose of the composition for any particular patient is preferably applied differently based on various factors, including the type and extent of the desired response, the specific composition including whether other preparations are used intermittently or concurrently, the patient's age, weight, health status, sex and diet, the time and route of administration, the secretion rate of the composition, the duration of treatment, other drugs used in combination with or concurrently with the specific composition, and similar factors known in the medical field.

[0489] Another aspect of the invention provides the use of isolated peptides or peptide-containing compositions in the preparation of pharmaceuticals. The pharmaceuticals can be used to prevent or treat metabolic syndrome.

[0490] The isolated peptides or compositions and metabolic syndromes are the same as explained above.

[0491] The invention will be described in more detail below with reference to the following embodiments. However, these embodiments are for illustrative purposes only, and the invention is not intended to be limited to these embodiments.

[0492] Example 1: Preparation of a triple agonist

[0493] Triple agonists showing activity against all GLP-1, GIP, and glucagon receptors were prepared, and their amino acid sequences are shown in Table 1 below.

[0494] [Table 1]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502] In the sequences described in Table 1, the amino acid represented by X indicates aminoisobutyric acid (Aib) as a non-natural amino acid, and the underlined amino acid indicates a ring formed between the underlined amino acids. Additionally, in Table 1, CA represents 4-imidazoacetyl and Y represents tyrosine.

[0503] Example 2: Measurement of the in vitro activity of the triple agonist

[0504] The activity of the triple agonist prepared in Example 1 was measured by measuring the in vitro cell activity using cell lines in which the GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor were respectively transformed.

[0505] Each of the aforementioned cell lines is a cell line in which the genes for the human GLP-1 receptor, human GCG receptor, and human GIP receptor have been transformed into Chinese hamster ovaries (CHOs) and expressed, and is therefore suitable for measuring the activities of GLP-1, GCG, and GIP. Thus, the activities of each fraction were measured using the separately transformed cell lines.

[0506] To measure the GLP-1 activity of the triple agonist prepared in Example 1, human GLP-1 was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonist prepared in Example 1 was serially diluted 4-fold from 400 nM to 0.00038 nM.

[0507] The culture medium was removed from CHO cells expressing the human GLP-1 receptor, and 5 μL of each of the serially diluted substances was added to the CHO cells. Then, 5 μL of a buffer solution containing cAMP antibody was added, and the cells were incubated at room temperature for 15 minutes. Next, 10 μL of an assay mixture containing cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were calculated and compared with each other. The relative valence compared with human GLP-1 is shown in Table 2 below.

[0508] To measure the GCG activity of the triple agonist prepared in Example 1, human GCG was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonist prepared in Example 1 was serially diluted 4-fold from 400 nM to 0.00038 nM.

[0509] The culture medium was removed from CHO cells expressing the human GCG receptor, and 5 μL of each of the serially diluted substances was added to the CHO cells. Then, 5 μL of a buffer solution containing cAMP antibody was added, and the cells were incubated at room temperature for 15 minutes. Next, 10 μL of an assay mixture containing cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were calculated and compared with each other. The relative valence compared with human GCG is shown in Table 2 below.

[0510] To measure the GIP activity of the triple agonist prepared in Example 1, human GIP was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple agonist prepared in Example 1 was serially diluted 4-fold from 400 nM to 0.00038 nM.

[0511] The culture medium was removed from CHO cells expressing the human GIP receptor, and 5 μL of each of the serially diluted substances was added to the CHO cells. Then, 5 μL of a buffer solution containing cAMP antibody was added, and the cells were incubated at room temperature for 15 minutes. Next, 10 μL of an assay mixture containing cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After the reaction, the cell lysate was applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were calculated and compared with each other. The relative valence compared with human GIP is shown in Table 2 below.

[0512] [Table 2]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518] The novel glucagon analogue prepared above has the function of a triple agonist that can activate all GLP-1 receptors, GIP receptors and glucagon receptors, and therefore glucagon analogues can be used as therapeutic substances for treating patients with metabolic syndrome including diabetes and obesity.

[0519] From the foregoing, those skilled in the art will understand that the present invention can be implemented in other specific forms without modifying the technical concept or essential features of the 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 invention. Rather, the invention is intended to cover not only the exemplary embodiments but also various substitutions, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

1. An isolated peptide active against glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor, and glucose-dependent insulinotropic peptide (GIP) receptor, wherein the amino acid sequence of said peptide is as shown in SEQ ID NO: 32 or 37, and The peptide described herein possesses the following activities i) to iii): i) Activation of GLP-1 receptor; ii) Activation of glucagon receptors; and iii) Activation of GIP receptors.

2. The isolated peptide of claim 1, wherein the C-terminus of the peptide is amidated.

3. A polynucleotide encoding the peptide of claim 1.

4. A pharmaceutical composition for the prevention or treatment of metabolic syndrome, comprising a peptide according to any one of claims 1 to 2.

5. Use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of metabolic syndrome, said pharmaceutical composition comprising a peptide according to any one of claims 1 to 2, wherein said metabolic syndrome includes glucose intolerance, dyslipidemia, obesity, diabetes, or hypertension.

6. The use according to claim 5, wherein the metabolic syndrome includes hypercholesterolemia.

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