Pharmaceutical compositions including insulin and a glucagon / GLP-1 / GIP receptor agonist

Abstract

The present invention relates to a composition comprising insulin and a triple agonist having activity on the glucagon receptor, the GLP-1 receptor and the GIP receptor, and to a combination preparation comprising the composition.

Description

Technical Field

[0001] This invention relates to compositions containing insulin and a triple agonist active against glucagon, GLP-1 and GIP receptors, as well as complex formulations containing the same composition. Background Technology

[0002] Insulin is a blood glucose-regulating hormone secreted by the pancreas. It transports excess glucose from the blood into cells, providing them with energy and maintaining blood glucose levels within a normal range. However, in diabetic patients, insulin cannot function properly due to insulin deficiency, insulin resistance, and loss of beta-cell function. As a result, diabetic patients cannot utilize blood glucose as an energy source and exhibit hyperglycemia, leading to glucose excretion in the urine, which causes various complications. Therefore, diabetic patients with abnormal insulin production (type I) or insulin resistance (type II) require insulin therapy, and insulin administration can regulate blood glucose levels to a normal range.

[0003] In recent years, 25% or more of type 1 diabetes cases and 80% or more of type 2 diabetes cases have had high blood sugar levels and obesity. Insulin alone is known to cause weight gain as a side effect, despite its excellent glycemic control. Therefore, there is a need to develop drugs that can effectively reduce the weight gain side effect of insulin while exhibiting its glycemic-lowering effect.

[0004] The applicant has developed various insulin analogs that retain insulin activity (WO 2014 / 133324A1 and WO 2017 / 039267), and triple agonists active against glucagon, GLP-1, and GIP receptors (WO2017 / 116204 and WO 2017 / 116205). However, these have not yet been used as combination formulations. Summary of the Invention

[0005] Technical issues

[0006] This application demonstrates that, compared with the administration of insulin or triple agonists alone, the combined use of insulin and triple agonists exhibits significantly superior glycemic control and effectively reduces insulin-induced weight gain.

[0007] Technical solution

[0008] One object of the present invention is to provide a composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0009] Specifically, one object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of insulin-related diseases, the composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0010] Another object of the present invention is to provide a pharmaceutical composition for reducing weight gain caused by insulin administration: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0011] Another object of the present invention is to provide a compound formulation for weight loss in patients administering insulin, the compound formulation comprising: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0012] Another object of the present invention is to provide a kit for the prevention or treatment of insulin-related diseases, the kit comprising: (i) insulin; and (ii) isolated peptides active against glucagon, GLP-1 and GIP receptors.

[0013] Another object of the present invention is to provide the use of the composition in the preparation of medicinal materials.

[0014] Another object of the present invention is to provide a method for preventing or treating insulin-related diseases, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0015] Another object of the present invention is to provide a method for reducing weight gain caused by insulin administration, the method comprising administering to a subject a separated peptide that is active against glucagon, GLP-1 and GIP receptors.

[0016] Specifically, another object of the present invention is to provide a method for reducing weight gain caused by insulin administration, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0017] Beneficial effects

[0018] The present invention provides a novel combination therapy comprising a composition or compound formulation of insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof, which not only exhibits preventive or therapeutic effects on insulin-related diseases such as diabetes, but also reduces side effects caused by insulin administration such as weight gain. Attached Figure Description

[0019] Figure 1 and 2The study demonstrated the effects of combined administration of long-acting insulin conjugates and long-acting triple agonist conjugates on glycemic control and body weight changes in type 2 diabetic mice. Detailed Implementation

[0020] One aspect of the present invention relates to a composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0021] In one embodiment, the present invention relates to a pharmaceutical composition for the prevention or treatment of insulin-related diseases, the composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0022] In another embodiment, the present invention relates to a pharmaceutical composition for reducing weight gain caused by insulin administration, the composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0023] In the composition according to any of the foregoing embodiments, insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of insulin is conjugated with insulin.

[0024] In the composition according to any of the foregoing embodiments, the isolated peptide active to glucagon, GLP-1 and GIP receptors is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the isolated peptide is conjugated to the isolated peptide.

[0025] In the composition according to any of the foregoing embodiments, insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of insulin is conjugated with insulin; and

[0026] The isolated peptides, which are active against glucagon, GLP-1 and GIP receptors, are in the form of long-acting conjugates, in which biocompatible substances that can increase the in vivo half-life of the isolated peptides are conjugated to the isolated peptides.

[0027] In any of the compositions according to the foregoing embodiments, the composition is administered to a subject who requires insulin administration.

[0028] In the composition according to any of the foregoing embodiments, insulin-related diseases are selected from the group consisting of insulin resistance, diabetes, hyperglycemia, and obesity.

[0029] In any of the compositions according to the foregoing embodiments, the composition has both a blood glucose-lowering effect and an effect of inhibiting weight gain caused by insulin administration alone.

[0030] In the composition according to any of the foregoing embodiments, the composition comprises: insulin or a long-acting conjugate thereof; and a separate peptide or a long-acting conjugate thereof that is active against glucagon, GLP-1, and GIP receptors.

[0031] The mixture contains insulin or its conjugates and isolated peptides or their long-acting conjugates that are active against glucagon, GLP-1 and GIP receptors in a molar ratio of 1:1 to 100:1, or contains insulin or its conjugates and isolated peptides or their long-acting conjugates that are active against glucagon, GLP-1 and GIP receptors in a molar ratio of 1:1 to 1:100.

[0032] In the composition according to any of the foregoing embodiments, the isolated peptide active to glucagon, GLP-1 and GIP receptors is an analogue of natural glucagon having alterations selected from the group consisting of substitutions, additions, deletions, modifications and combinations thereof of at least one amino acid in the sequence of natural glucagon.

[0033] In the composition according to any of the foregoing embodiments, the amino acid sequence to be added is derived from the amino acid sequence of natural GLP-1, natural GIP, or natural exenatide-4.

[0034] In the composition according to any of the foregoing embodiments, the isolated peptide active against glucagon, GLP-1, and GIP receptors contains an amino acid sequence represented by the following general formula 1:

[0035] 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),

[0036] In general formula 1,

[0037] Xaa1 is histidine (His, H), 4-imidazolium acetyl (CA), or tyrosine (Tyr, Y);

[0038] Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or aminoisobutyric acid (Aib);

[0039] Xaa3 is either glutamic acid (Glu, E) or glutamine (Gln, Q);

[0040] Xaa7 is either threonine (Thr, T) or isoleucine (Ile, I);

[0041] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V).

[0042] Xaa12 is lysine (Lys, K), serine (Ser, S) or isoleucine (Ile, I);

[0043] Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C);

[0044] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

[0045] Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L).

[0046] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

[0047] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C) or lysine (Lys, K).

[0048] Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);

[0049] Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V);

[0050] Xaa20 is lysine (Lys, K), glutamine (Gln, Q) or arginine (Arg, R);

[0051] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C) or aspartic acid (Asp, D);

[0052] Xaa23 is either isoleucine (Ile, I) or valine (Val, V);

[0053] XXaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E).

[0054] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M).

[0055] Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D).

[0056] Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H).

[0057] Xaa30 is cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H), or is absent; and

[0058] 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.

[0059] Where m is Cys, Pro, or Gly-Pro, and n is Cys, Gly, Ser, or His-Gly or does not exist.

[0060] In the composition according to any of the foregoing embodiments, Xaa14 is leucine or methionine; and Xaa15 is cysteine, aspartic acid, or leucine.

[0061] In the composition according to any of the foregoing embodiments, in general formula 1,

[0062] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0063] Xaa7 is threonine.

[0064] Xaa10 is tyrosine, cysteine, or valine;

[0065] Xaa12 is either lysine or isoleucine;

[0066] Xaa13 is tyrosine, alanine, glutamine, or cysteine;

[0067] Xaa14 is leucine, cysteine, or methionine;

[0068] Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid.

[0069] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0070] Xaa18 is alanine, glutamine, arginine, or histidine;

[0071] Xaa19 is alanine, glutamine, valine, or cysteine;

[0072] Xaa20 is lysine, arginine, or glutamine;

[0073] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0074] Xaa23 is either isoleucine or valine;

[0075] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and

[0076] Xaa27 is either leucine or lysine.

[0077] In the composition according to any of the foregoing embodiments, the peptide contains an amino acid sequence represented by the following general formula 2:

[0078] 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),

[0079] In general formula 2,

[0080] Xaa1 is 4-imidazolylacetyl, histidine, or tyrosine;

[0081] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0082] Xaa10 is either tyrosine or cysteine;

[0083] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0084] Xaa14 is leucine, methionine, or tyrosine;

[0085] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0086] Xaa16 is glycine, glutamic acid, or serine;

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

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

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

[0090] Xaa20 is lysine, glutamine, or arginine;

[0091] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0092] Xaa23 is either isoleucine or valine;

[0093] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0094] Xaa28 is lysine, cysteine, asparagine, or aspartic acid.

[0095] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0096] Xaa30 is cysteine, glycine, lysine, or histidine;

[0097] Xaa31 is either proline or cysteine; and

[0098] Xaa40 is either cysteine ​​or absent.

[0099] In the composition according to any of the foregoing embodiments, in general formula 1,

[0100] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0101] Xaa7 is threonine;

[0102] Xaa10 is tyrosine, cysteine, or valine;

[0103] Xaa12 is either lysine or isoleucine;

[0104] Xaa13 is tyrosine, alanine, or cysteine;

[0105] Xaa14 is leucine or methionine;

[0106] Xaa15 is either cysteine ​​or aspartic acid;

[0107] Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

[0108] Xaa18 is alanine, arginine, or histidine;

[0109] Xaa19 is alanine, glutamine, or cysteine;

[0110] Xaa20 is either lysine or glutamine;

[0111] Xaa21 is glutamic acid, cysteine, or aspartic acid;

[0112] Xaa23 is valine;

[0113] Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and

[0114] Xaa27 is either leucine or lysine.

[0115] In the composition according to any of the foregoing embodiments, in general formula 2,

[0116] Xaa13 is alanine, tyrosine, or cysteine;

[0117] Xaa15 is either aspartic acid or glutamic acid;

[0118] Xaa17 is glutamine, arginine, cysteine, or lysine;

[0119] Xaa18 is alanine, arginine, or histidine;

[0120] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

[0121] Xaa23 is either isoleucine or valine;

[0122] Xaa24 is cysteine, glutamine, or asparagine;

[0123] Xaa28 is cysteine, asparagine, or aspartic acid;

[0124] Xaa29 is glutamine, cysteine, or histidine; and

[0125] Xaa30 is cysteine, lysine, or histidine.

[0126] In the composition according to any of the foregoing embodiments, in general formula 1,

[0127] Xaa2 is α-methyl-glutamic acid or Aib;

[0128] Xaa7 is threonine;

[0129] Xaa10 is either tyrosine or cysteine;

[0130] Xaa12 is either lysine or isoleucine;

[0131] Xaa13 is tyrosine, alanine, or cysteine;

[0132] Xaa14 is leucine or methionine;

[0133] Xaa15 is either cysteine ​​or aspartic acid;

[0134] Xaa16 is glutamic acid;

[0135] Xaa17 is arginine, isoleucine, cysteine, or lysine;

[0136] Xaa18 is alanine, arginine, or histidine;

[0137] Xaa19 is alanine, glutamine, or cysteine;

[0138] Xaa20 is either lysine or glutamine;

[0139] Xaa21 is either glutamic acid or aspartic acid;

[0140] Xaa23 is valine;

[0141] Xaa24 is glutamine, asparagine, or aspartic acid;

[0142] Xaa27 is leucine; and

[0143] Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0144] In the composition according to any of the foregoing embodiments, in general formula 1,

[0145] Xaa1 is histidine or 4-imidazolyl acetyl;

[0146] Xaa2 is α-methyl-glutamic acid or Aib;

[0147] Xaa3 is glutamine;

[0148] Xaa7 is threonine;

[0149] Xaa10 is tyrosine;

[0150] Xaa12 is isoleucine;

[0151] Xaa13 is either alanine or cysteine;

[0152] Xaa14 is methionine;

[0153] Xaa15 is aspartic acid;

[0154] Xaa16 is glutamic acid;

[0155] Xaa17 is either isoleucine or lysine;

[0156] Xaa18 is either alanine or histidine;

[0157] Xaa19 is either glutamine or cysteine;

[0158] Xaa20 is lysine;

[0159] Xaa21 is aspartic acid;

[0160] Xaa23 is valine;

[0161] Xaa24 is asparagine;

[0162] Xaa27 is leucine;

[0163] Xaa28 is either alanine or asparagine;

[0164] Xaa29 is either glutamine or threonine; and

[0165] Xaa30 is cysteine ​​or lysine, or it may not be present.

[0166] In the composition according to any of the foregoing embodiments, the peptide contains an amino acid sequence represented by the following general formula 3:

[0167] 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),

[0168] In general formula 3,

[0169] Xaa1 is either histidine or tyrosine;

[0170] Xaa2 is α-methyl-glutamic acid or Aib;

[0171] Xaa13 is alanine, tyrosine, or cysteine;

[0172] Xaa17 is arginine, cysteine, or lysine;

[0173] Xaa18 is either alanine or arginine;

[0174] Xaa19 is either alanine or cysteine;

[0175] Xaa21 is either glutamic acid or aspartic acid;

[0176] Xaa24 is either glutamine or asparagine;

[0177] Xaa28 is either cysteine ​​or aspartic acid;

[0178] Xaa29 is cysteine, histidine, or glutamine;

[0179] Xaa30 is either cysteine ​​or histidine;

[0180] Xaa31 is either proline or cysteine; and

[0181] Xaa40 is either cysteine ​​or absent.

[0182] In the composition according to any of the foregoing embodiments, 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 not present.

[0183] In the composition according to any of the foregoing embodiments, the isolated peptide active against glucagon, GLP-1 and GIP receptors contains an amino acid sequence selected from SEQ ID NO:1 to 102.

[0184] In the composition according to any of the foregoing embodiments, the isolated peptide active against glucagon, GLP-1 and GIP receptors contains the amino acid sequence shown in SEQ ID NO:42.

[0185] In the composition according to any of the foregoing embodiments, in the general formula, the amino acids at positions 16 and 20 from the N-terminus form a ring with each other.

[0186] In the composition according to any of the foregoing embodiments, the C-terminus of a separated peptide active against glucagon, GLP-1, and GIP receptors is amidated.

[0187] In the composition according to any of the foregoing embodiments, the insulin is natural insulin, or an insulin analog having alterations selected from the group consisting of substitutions, additions, deletions, modifications, and combinations thereof of at least one amino acid in natural insulin.

[0188] In the composition according to any of the foregoing embodiments, the insulin analog has a change of at least one amino acid selected from the group consisting of: amino acids at positions 1, 2, 3, 5, 8, 10, 12, 16, 23, 24, 25, 26, 27, 28, 29 and 30 of the natural insulin B-chain and amino acids at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19 and 21 of the natural insulin A-chain, said change being a substitution, deletion or combination of another amino acid.

[0189] In the composition according to any of the foregoing embodiments, the insulin analog comprises: an A-chain of SEQ ID NO:119 represented by general formula 4; and a B-chain of SEQ ID NO:120 represented by general formula 5.

[0190] Formula 4

[0191] Xaa1-Xaa2-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 119),

[0192] In general formula 4,

[0193] Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;

[0194] Xaa2 is either alanine or isoleucine;

[0195] Xaa5 is alanine, glutamic acid, glutamine, histidine, or asparagine;

[0196] Xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine;

[0197] Xaa14 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid, or asparagine.

[0198] Xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine.

[0199] Xaa19 is alanine, tyrosine, serine, glutamic acid, histidine, threonine, or asparagine; and

[0200] Xaa21 is asparagine, glycine, histidine, or alanine.

[0201] General Formula 5

[0202] Phe-Val-Asn-Gln-His-Leu-Cys-Xaa8-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Xaa23-Xaa24-Xaa25-Tyr-Xaa27-Xaa28-Lys-Thr (SEQ ID NO:120),

[0203] In general formula 5,

[0204] Xaa8 is either alanine or glycine;

[0205] Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid, or it may not be present.

[0206] Xaa23 is either glycine or alanine;

[0207] Xaa24 is either alanine or phenylalanine;

[0208] Xaa25 is alanine, phenylalanine, aspartic acid, or glutamic acid, or it may not be present.

[0209] Xaa27 is threonine or is absent; and

[0210] Xaa28 is proline, glutamic acid, or aspartic acid, or it may not be present.

[0211] (Excluding peptides containing the A-chain of SEQ ID NO:121 and the B-chain of SEQ ID NO:122).

[0212] In the composition according to any of the foregoing embodiments, the insulin analog has a substitution of at least one amino acid selected from the group consisting of amino acids at positions 8, 23, 24 and 25 of the natural insulin B-chain and amino acids at positions 1, 2 and 19 of the natural insulin A-chain, or a substitution of the amino acid at position 14 of the natural insulin A-chain with glutamic acid or asparagine.

[0213] In any of the compositions according to the foregoing embodiments, the insulin analog contains an amino acid sequence selected from the group consisting of SEQ ID NO: 124, 126, 128, 130, 132, 134, 136, 138 and 140.

[0214] In the composition according to any of the foregoing embodiments, the insulin analog has been modified by: replacing the 16th amino acid in the B-chain of natural insulin with glutamic acid; deleting the 25th amino acid in the B-chain of natural insulin; replacing the 14th amino acid in the A-chain of natural insulin with glutamic acid or alanine; or a combination thereof.

[0215] In the composition according to any of the foregoing embodiments, the insulin analog contains the amino acid sequence of SEQ ID NO: 142 or 144.

[0216] In the composition according to any of the foregoing embodiments, the insulin analog has been modified by replacing the 16th amino acid in the B-chain of natural insulin with glutamic acid, serine, threonine, or aspartic acid; replacing the 25th amino acid in the B-chain of natural insulin with aspartic acid or glutamic acid; replacing the 14th amino acid in the A-chain of natural insulin with histidine, lysine, alanine, or aspartic acid; replacing the 19th amino acid in the A-chain of natural insulin with glutamic acid, serine, or threonine; or combinations thereof.

[0217] In the composition according to any of the foregoing embodiments, the insulin analog contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 and 170.

[0218] In the composition according to any of the foregoing embodiments, the insulin analog is in the form of two polypeptide chains consisting of an A-chain of SEQ ID NO:119 represented by general formula 4 and a B-chain of SEQ ID NO:120 represented by general formula 5.

[0219] In the composition according to any of the foregoing embodiments, the A-chain and the B-chain are linked by disulfide bonds.

[0220] In the composition according to any of the foregoing embodiments, the conjugate is represented by chemical formula 1:

[0221] Chemical Formula 1

[0222] XL a -F,

[0223] in,

[0224] X is an insulin or a separate peptide that is active against glucagon, GLP-1, and GIP receptors;

[0225] L stands for connector;

[0226] a is 0 or a natural number, provided that each L is independent of the others when a is 2 or greater;

[0227] F is a substance that can increase the half-life of X; and

[0228] "-" indicates a covalent or non-covalent bond.

[0229] In the composition according to any of the foregoing embodiments, F is selected from the group consisting of: polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, carbohydrates, heparin, and elastin.

[0230] In the composition according to any of the foregoing embodiments, the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof.

[0231] In the composition according to any of the foregoing embodiments, F is the Fc region of immunoglobulin.

[0232] In the composition according to any of the foregoing embodiments, F is the IgG Fc region.

[0233] In the composition according to any of the foregoing embodiments, the immunoglobulin Fc region is deglycosylated.

[0234] In the composition according to any of the foregoing embodiments, the immunoglobulin Fc region is selected from the group consisting of:

[0235] (a) CH1 domain, CH2 domain, CH3 domain and CH4 domain;

[0236] (b) CH1 and CH2 domains;

[0237] (c) CH1 and CH3 domains;

[0238] (d) CH2 and CH3 domains;

[0239] (e) A combination of at least one of the CH1, CH2, CH3, and CH4 domains with an immunoglobulin hinge region or a portion thereof; and

[0240] (f) Dimers of each domain in the heavy chain constant region and the light chain constant region.

[0241] In the composition according to any of the foregoing embodiments, the immunoglobulin Fc region has the following defects: loss of a site capable of forming a disulfide bond, loss of some amino acids at the N-terminus of the native Fc, addition of a methionine residue at the N-terminus of the native Fc, loss of a complement binding site, or loss of an antibody-dependent cell-mediated cytotoxicity (ADCC) site.

[0242] In the composition according to any of the foregoing embodiments, the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

[0243] In the composition according to any of the foregoing embodiments, the immunoglobulin Fc region is a mixture of domains having different sources of immunoglobulins derived from a group consisting of IgG, IgA, IgD, IgE and IgM.

[0244] In the composition according to any of the foregoing embodiments, L is selected from the group consisting of peptides, fatty acids, sugars, polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0245] In the composition according to any of the foregoing embodiments, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0246] In the composition according to any of the foregoing embodiments, L is polyethylene glycol.

[0247] One aspect of the present invention relates to a compound formulation for weight loss in patients administering insulin, the compound formulation comprising: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0248] One aspect of the present invention relates to a kit for the prevention or treatment of insulin-related diseases, the kit comprising: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0249] One aspect of the present invention relates to the use of the composition in the preparation of medicinal materials.

[0250] One aspect of the present invention relates to a method for preventing or treating insulin-related diseases, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1, and GIP receptors.

[0251] One aspect of the present invention relates to a method for reducing weight gain caused by insulin administration, the method comprising administering to a subject a isolated peptide that is active against glucagon, GLP-1 and GIP receptors.

[0252] One aspect of the invention relates to a method for reducing weight gain caused by insulin administration, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0253] Methods for implementing the present invention

[0254] The present invention will now be described in detail.

[0255] Each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited to the specific descriptions below.

[0256] Furthermore, those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be included in this invention.

[0257] Throughout this specification, standard single-letter and three-letter codes for naturally occurring amino acids are used, as well as other amino acids typically permitted to have three-letter codes, such as α-aminoisobutyric acid (Aib), N-methylglycine (Sar), and α-methylglutamic acid. Amino acids referred to in abbreviations herein are described according to IUPAC-IUB rules:

[0258] Alanine (Ala, A), Arginine (Arg, R)

[0259] Asparagine (Asn, N), Aspartic acid (Asp, D)

[0260] Cysteine ​​(Cys, C) and Glutamic acid (Glu, E)

[0261] Glutamine (Gln, Q), glycine (Gly, G)

[0262] Histidine (His, H) and Isoleucine (Ile, I)

[0263] Leucine (Leu, L) and Lysine (Lys, K)

[0264] Methionine (Met, M) and Phenylalanine (Phe, F)

[0265] Proline (Pro, P) and serine (Ser, S)

[0266] Threonine (Thr, T) and tryptophan (Trp, W)

[0267] Tyrosine (Tyr, Y) and valine (Val, V)

[0268] One aspect of the present invention is to provide a composition comprising: (i) insulin; and (ii) a isolated peptide (also known as a “triple agonist”) active against glucagon, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0269] In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of insulin-related diseases, the composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0270] In another embodiment, the present invention provides a pharmaceutical composition for reducing weight loss caused by insulin administration, the composition comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0271] Specifically, the composition may contain:

[0272] (a) Insulin; and isolated peptides active against glucagon, GLP-1 and GIP receptors;

[0273] (b) A long-acting conjugate of insulin, wherein insulin is conjugated with a biocompatible substance that can increase the in vivo half-life of insulin; and a separated peptide that is active against glucagon, GLP-1 and GIP receptors.

[0274] (c) Insulin; and long-acting conjugates of triple agonists, wherein a isolated peptide active against glucagon, GLP-1, and GIP receptors is conjugated with a biocompatible substance that increases the in vivo half-life of the isolated peptide; or

[0275] (d) Long-acting conjugates of insulin; and long-acting conjugates of triple agonists.

[0276] The pharmaceutical composition of the present invention for the prevention or treatment of insulin-related diseases comprises: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein the composition can reduce the side effects (e.g., weight gain) caused by insulin administration while exhibiting the efficacy of insulin, and has a preventive or therapeutic effect on insulin-related diseases caused by insulin deficiency, insufficiency or dysfunction.

[0277] Exemplary embodiments of the present invention have demonstrated a hypoglycemic effect and an inhibitory effect on weight gain in db / db mice administered a composition containing insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof, indicating that the pharmaceutical composition according to the present invention can alleviate the side effects of insulin while maintaining the efficacy of insulin.

[0278] As used herein, the term "insulin-related disease" refers to a disease caused by an inherent abnormality in insulin's glycemic control function, such as insulin deficiency, insufficiency, or dysfunction, and any disease for which prevention or treatment is desired through insulin administration is included within the scope of this invention. Specifically, insulin-related diseases can be selected from the group consisting of insulin resistance disorders, diabetes, hyperglycemia, and obesity.

[0279] Insulin therapy for insulin-related diseases can achieve the desired therapeutic effect, but it can cause unexpected side effects, such as weight gain, leading to other types of illness and patient suffering. The pharmaceutical composition according to the invention, by combining insulin and a triple agonist, can demonstrate therapeutic efficacy against insulin-related diseases while suppressing weight gain as a side effect of insulin.

[0280] The compositions of the present invention may contain insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof, the ratio of which can exhibit therapeutic effects on insulin-related diseases and reduce side effects, especially weight gain.

[0281] Specifically, the composition may contain both insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof, wherein the composition may contain insulin and a triple agonist in a molar ratio of 1:1 to 100:1 or 1:1 to 1:100, but is not limited thereto.

[0282] In one embodiment of the invention, the composition can be provided for therapeutic use by combining insulin or a conjugate thereof with a substance or a conjugate thereof that is active against glucagon, GLP-1, and GIP receptors. The term "insulin" is as described above.

[0283] In this document, the term "composition" may be used interchangeably with "assembly," which contains insulin or a conjugate thereof and substances active against glucagon, GLP-1, and GIP receptors. The composition may be provided in kit form.

[0284] As used herein, "combination" has the purpose of administering insulin or its conjugates in combination with a substance or its conjugates that is active against glucagon, GLP-1, and GIP receptors, and the term may be understood synonymously with "used in combination." The composition may be administered in the following forms:

[0285] a) in the form of a mixture, wherein (i) insulin or a conjugate thereof and (ii) a substance or a conjugate thereof active against glucagon, GLP-1 and GIP receptors; or

[0286] b) in the form of isolating (i) insulin or its conjugates and (ii) substances or their conjugates that are active against glucagon, GLP-1 and GIP receptors, but not limited thereto.

[0287] When the composition is in the form of isolated insulin or its conjugates and substances or their conjugates that are active against glucagon, GLP-1 and GIP receptors, the insulin or its conjugates and substances or their conjugates that are active against glucagon, GLP-1 and GIP receptors can be prepared as separate formulations and administered simultaneously, separately, sequentially or in reverse order.

[0288] In this invention, combination administration should be understood not only as simultaneous administration, but also as a form of administration in which insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors act together on the subject, such that each acts at a level equal to or greater than its original function. Therefore, the term "combination" should be understood to mean simultaneous, single, sequential, or reverse administration. When administration is sequential, reverse, or single, the order of administration is not particularly limited, but delaying the administration of the second component should not negate the beneficial effects of the combination.

[0289] In this invention, the term "composition" refers to the composition itself, which contains insulin or a conjugate thereof and a substance or a conjugate thereof that is active against glucagon, GLP-1 and GIP receptors, or it may be a composition containing the composition and having therapeutic use, but is not limited thereto. Examples of this invention may be compositions having preventive or therapeutic use against insulin-related diseases, but are not limited thereto.

[0290] The compositions according to the invention are for combined administration of: insulin or its conjugates; and substances or their conjugates active against glucagon, GLP-1, and GIP receptors, wherein insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors can be prepared as a single formulation or prepared separately. Specifically, insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors can be administered simultaneously, separately, sequentially, or in reverse order, but are not limited thereto.

[0291] In this invention, the term "set" may contain combinations or compositions according to the invention for the combined administration of insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors. Specifically, a set according to the invention may contain: a formulation prepared from insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors; or a single formulation prepared from insulin or its conjugates and substances or their conjugates active against glucagon, GLP-1, and GIP receptors, and the set may further contain substances necessary for the combined administration of these two substances, but is not limited thereto.

[0292] Insulin and substances active against glucagon, GLP-1, and GIP receptors include insulin and various substances, such as compounds or peptides, that have significant levels of activity against glucagon, GLP-1, and GIP receptors.

[0293] In this invention, "peptides active against glucagon, GLP-1, and GIP receptors" can be used interchangeably with triple agonists. The full text of International Patent Publications WO2017 / 116204 and WO 2017 / 116205, relating to triple agonists and their long-acting conjugates, is incorporated herein by reference.

[0294] Examples of triple agonists include a variety of substances, such as various peptides, that have significant levels of activity against glucagon, GLP-1, and GIP receptors.

[0295] For substances that have significant levels of activity against glucagon, GLP-1, and GIP receptors, although there are no particular limitations, the in vitro activity against one or more, specifically two or more, and more specifically all three of the natural ligands (natural glucagon, natural GLP-1, and natural GIP) is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more, with respect to the corresponding receptors.

[0296] Methods for measuring the in vitro activity of these triple agonists can be found in Test Example 1 of this paper, but are not specifically limited thereto.

[0297] At the same time, the triple agonist is characterized by having one, two or more, specifically three, of the following activities i) to iii), specifically having significant levels of activity:

[0298] i) Activation of GLP-1 receptor; ii) Activation of glucagon receptor; and iii) Activation of GIP receptor.

[0299] Receptor activation may include, for example, exhibiting 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and 100% or more of in vitro activity against the receptor compared to the natural form. However, activation is not limited to this.

[0300] Compared to any one of natural GLP-1, natural glucagon, and natural GIP, triple agonists may have an increased in vivo half-life, but are not particularly limited thereto.

[0301] Although there are no particular restrictions, these peptides can be peptides that do not exist naturally.

[0302] Specifically, triple agonists can be analogs of natural glucagon, but are not particularly limited to this.

[0303] The natural glucagon analogues according to the present invention may comprise a peptide having at least one difference in amino acid sequence compared to natural glucagon, a peptide obtained by modifying the sequence of natural glucagon, and a mimic of natural glucagon.

[0304] Meanwhile, although there are no particular restrictions, natural glucagon can have the following amino acid sequence:

[0305] 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)

[0306] Specifically, the triple agonist can be a natural glucagon analogue having alterations to a group consisting of substitutions, additions, deletions, modifications, and combinations thereof selected from at least one amino acid in the natural glucagon sequence, but is not particularly limited thereto.

[0307] The substitution of amino acids can include replacing them with amino acids and replacing them with non-natural compounds.

[0308] The addition can be made at the N-terminus and / or C-terminus of the triple agonist. There is no particular limitation on the length of the amino acid to be added, 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, but is not particularly limited to, the addition of peptides.

[0309] The amino acids to be introduced into natural glucagon can be selected from the group consisting of 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 specifically limited to this group.

[0310] For example, the amino acid sequence to be added can be at least one amino acid sequence derived from the amino acid sequence of natural GLP-1, natural GIP, or natural exenatide-4.

[0311] Such triple agonists may contain intramolecular bridges (e.g., covalent or non-covalent bridges), and specifically, triple agonists may be in the form of rings, for example, in the form of a ring formed between the amino acids at positions 16 and 20 of the triple agonist, but are not particularly limited thereto.

[0312] Non-limiting examples of rings may include lactam bridges (or lactam rings).

[0313] Furthermore, examples of triple agonists include all triple agonists that are modified to contain amino acids capable of forming a ring at the target site in order to include a ring.

[0314] For example, in a triple agonist, the pair of amino acids at positions 16 and 25 can be replaced by glutamic acid or lysine, which can form a ring, but are not limited thereto.

[0315] The ring can be formed between the amino acid side chains within a triple agonist, for example, in the form of a lactam ring formed between a lysine side chain and a glutamic acid side chain, but is not particularly limited thereto.

[0316] Examples of triple agonists prepared by combining these methods include, but are not limited to, peptides that differ from natural glucagon in at least one amino acid sequence, wherein the α-carbon of the N-terminal amino acid residue has been removed, and which are active against glucagon, GLP-1 and GIP receptors, etc., and triple agonists to be applicable to the present invention can be prepared by a combination of various methods for preparing analogues.

[0317] Although there are no particular limitations, in 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 agonist-degrading enzymes.

[0318] Specifically, the triple agonist of the present invention may be a peptide having an increased in vivo half-life by replacing the second amino acid sequence in the amino acid sequence of the triple agonist to avoid recognition by degrading enzymes, but includes, but is not limited to, any amino acid substitutions or modifications for avoiding recognition by in vivo degrading enzymes.

[0319] Such modifications used to prepare natural glucagon analogs include: changes to use L- or D-type amino acids and / or non-natural amino acids; and / or all changes made by modifying the sequence of the natural form, such as altering side chain functional groups, intramolecular covalent bonds such as ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc.

[0320] In addition, such changes also include the addition of one or more amino acids at the amino and / or carboxyl termini of natural glucagon.

[0321] Examples of amino acids to be replaced or added can include not only the 20 amino acids commonly found in human proteins, but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptide sequences containing these amino acids and typical peptides can be synthesized by commercial suppliers and purchased from commercial suppliers such as American Peptide Company and Bachem in the United States, or Antigen in South Korea.

[0322] Amino acid derivatives can also be obtained in a similar manner, and for example, 4-imidazolidineacetic acid can be used.

[0323] The peptides of the present invention that are active against glucagon, GLP-1 and GIP receptors can be in the form that the N-terminus and / or C-terminus are chemically modified or protected by organic groups, or amino acids are added to the end of the peptide, etc., to prevent the influence of intrareceptor protein cleavage enzymes and increase stability.

[0324] In particular, chemically synthesized peptides have charged N and C ends, and therefore the N end can be acetylated and / or the C end can be amidated to eliminate these charges, but these are not particularly limited thereto. Furthermore, forms in which the C end remains unchanged, i.e., forms with a carboxyl group, are also included.

[0325] The peptides active against glucagon, GLP-1, and GIP receptors according to the present invention, i.e., triple agonists, comprise all of the peptide itself, its salts (e.g., pharmaceutically acceptable salts of the peptide), or their solvates. The peptides active against glucagon, GLP-1, and GIP receptors may be in the form of any pharmaceutically acceptable salt.

[0326] There are no particular restrictions on the type of salt. However, salt is preferably in a form that is safe and effective for subjects, such as mammals, but is not particularly limited thereto.

[0327] The term "pharmaceutical acceptable" refers to a substance that, within the scope of pharmaceutical decisions, can be used effectively for its intended purpose without causing excessive toxicity, irritation, allergic reactions, etc.

[0328] As used herein, "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Suitable examples of acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Examples of salts derived from suitable bases include: alkali metals, such as sodium and potassium; alkaline earth metals, such as magnesium; ammonium; etc.

[0329] 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.

[0330] Clearly, all of the above descriptions apply to insulin, which will be described later.

[0331] In one specific aspect, peptides active against glucagon, GLP-1, and GIP receptors may contain an amino acid sequence represented by the following general formula 1:

[0332] 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),

[0333] In general formula 1,

[0334] Xaa1 is histidine, 4-imidazolylacetyl or tyrosine;

[0335] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0336] Xaa3 is glutamic acid or glutamine;

[0337] Xaa7 is either threonine or isoleucine;

[0338] Xaa10 is leucine, tyrosine, lysine, cysteine, or valine;

[0339] Xaa12 is lysine, serine, or isoleucine;

[0340] Xaa13 is glutamine, tyrosine, alanine, or cysteine;

[0341] Xaa14 is leucine, methionine, or tyrosine;

[0342] Xaa15 is cysteine, aspartic acid, glutamic acid, or leucine.

[0343] Xaa16 is glycine, glutamic acid, or serine;

[0344] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0345] Xaa18 is alanine, glutamine, arginine, or histidine;

[0346] Xaa19 is alanine, glutamine, cysteine, or valine;

[0347] Xaa20 is lysine, glutamine, or arginine;

[0348] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0349] Xaa23 is either isoleucine or valine;

[0350] XXaa24 is alanine, glutamine, cysteine, asparagine, aspartic acid, or glutamic acid;

[0351] Xaa27 is valine, leucine, lysine, or methionine;

[0352] Xaa28 is cysteine, lysine, alanine, asparagine, or aspartic acid;

[0353] Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine;

[0354] Xaa30 is cysteine, glycine, lysine, or histidine, or may not be present; and

[0355] R1 is cysteine, GKKNDWKHNIT (SEQ ID NO:106), m-SSGAPPPS-n (SEQ ID NO:107), or m-SSGQPPPS-n (SEQ ID NO:108), or is absent.

[0356] Where m is Cys, Pro, or Gly-Pro, and

[0357] n is Cys, Gly, Ser, or His-Gly, or does not exist.

[0358] Examples of triple agonists may include triple agonists containing an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 102, or may consist (substantially) a triple agonist consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 102, but are not limited thereto.

[0359] Although described herein as “a peptide consisting of a specific SEQ ID NO,” the addition of nonsense sequences, naturally occurring mutations, and their silenced mutations upstream or downstream of the amino acid sequence of the corresponding sequence number is not excluded, provided that there is an equivalent or corresponding activity to the peptide consisting of the amino acid sequence of the corresponding sequence number, and peptides with such sequence additions or mutations are obviously within the scope of this invention.

[0360] The above description can be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0361] Specifically, in general formula 1 above, Xaa14 can be leucine or methionine; and Xaa15 can be cysteine, aspartic acid, or leucine. In this case, the other variables besides Xaa14 and Xaa15 described in general formula 1 can have combinations of the above-mentioned amino acids.

[0362] Examples of these peptides may be, but are not limited to, peptides containing or (substantially) consisting of amino acid sequences selected from the group consisting of SEQ ID NO: 1 to 12, 14 to 17 and 21 to 102.

[0363] These peptides can significantly activate at least one of the glucagon, GLP-1, and GIP receptors, but are not particularly limited thereto. Specifically, the peptides can significantly activate the GLP-1 receptor, or can further significantly activate the glucagon and / or GIP receptors, but are not particularly limited thereto.

[0364] More specifically,

[0365] In general formula 1,

[0366] Xaa2 can be glycine, α-methyl-glutamic acid, or Aib;

[0367] Xaa7 can be threonine.

[0368] Xaa10 can be tyrosine, cysteine, or valine;

[0369] Xaa12 can be either lysine or isoleucine;

[0370] Xaa13 can be tyrosine, alanine, glutamine, or cysteine;

[0371] Xaa14 can be leucine, cysteine, or methionine;

[0372] Xaa15 can be cysteine, leucine, glutamic acid, or aspartic acid;

[0373] Xaa17 can be glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;

[0374] Xaa18 can be alanine, glutamine, arginine, or histidine;

[0375] Xaa19 can be alanine, glutamine, valine, or cysteine;

[0376] Xaa20 can be lysine, arginine, or glutamine;

[0377] Xaa21 can be glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

[0378] Xaa23 can be isoleucine or valine;

[0379] Xaa24 can be cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and

[0380] Xaa27 can be either leucine or lysine, but the variable is not specifically limited to these.

[0381] In addition to the variables described in this section, the remaining variables of General Formula 1 may have the above-described combinations of amino acids.

[0382] More specifically,

[0383] In general formula 1,

[0384] Xaa2 can be glycine, α-methyl-glutamic acid, or Aib;

[0385] Xaa7 can be threonine.

[0386] Xaa10 can be tyrosine, cysteine, or valine;

[0387] Xaa12 can be either lysine or isoleucine;

[0388] Xaa13 can be tyrosine, alanine, or cysteine;

[0389] Xaa14 can be leucine or methionine;

[0390] Xaa15 can be either cysteine ​​or aspartic acid;

[0391] Xaa17 can be glutamine, arginine, isoleucine, cysteine, or lysine;

[0392] Xaa18 can be alanine, arginine, or histidine;

[0393] Xaa19 can be alanine, glutamine, or cysteine;

[0394] Xaa20 can be lysine or glutamine;

[0395] Xaa21 can be glutamic acid, cysteine, or aspartic acid;

[0396] Xaa23 can be valine;

[0397] Xaa24 can be alanine, glutamine, cysteine, asparagine, or aspartic acid; and

[0398] Xaa27 can be either leucine or lysine, but the variable is not specifically limited to these.

[0399] In addition to the variables described in this section, the remaining variables of General Formula 1 may have the above-described combinations of amino acids.

[0400] More specifically,

[0401] In general formula 1,

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

[0403] Xaa7 is threonine.

[0404] Xaa10 is either tyrosine or cysteine;

[0405] Xaa12 is either lysine or isoleucine;

[0406] Xaa13 is tyrosine, alanine, or cysteine;

[0407] Xaa14 is leucine or methionine;

[0408] Xaa15 is either cysteine ​​or aspartic acid;

[0409] Xaa16 is glutamic acid;

[0410] Xaa17 is arginine, isoleucine, cysteine, or lysine;

[0411] Xaa18 is alanine, arginine, or histidine;

[0412] Xaa19 is alanine, glutamine, or cysteine;

[0413] Xaa20 is either lysine or glutamine;

[0414] Xaa21 is either glutamic acid or aspartic acid;

[0415] Xaa23 is valine;

[0416] Xaa24 is glutamine, asparagine, or aspartic acid;

[0417] Xaa27 is leucine; and

[0418] Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0419] In addition to the variables described in this section, the remaining variables of General Formula 1 may have the above-described combinations of amino acids.

[0420] Specifically,

[0421] In general formula 1,

[0422] Xaa1 can be histidine or 4-imidazolyl acetyl group.

[0423] Xaa2 can be α-methyl-glutamic acid or Aib;

[0424] Xaa3 can be glutamine;

[0425] Xaa7 can be threonine.

[0426] Xaa10 can be tyrosine;

[0427] Xaa12 can be isoleucine;

[0428] Xaa13 can be either alanine or cysteine;

[0429] Xaa14 can be methionine;

[0430] Xaa15 can be aspartic acid;

[0431] Xaa16 can be glutamic acid;

[0432] Xaa17 can be either isoleucine or lysine;

[0433] Xaa18 can be either alanine or histidine;

[0434] Xaa19 can be glutamine or cysteine;

[0435] Xaa20 can be lysine;

[0436] Xaa21 can be aspartic acid;

[0437] Xaa23 can be valine;

[0438] Xaa24 can be asparagine;

[0439] Xaa27 can be leucine;

[0440] Xaa28 can be alanine or asparagine;

[0441] Xaa29 can be glutamine or threonine; and

[0442] Xaa30 can be cysteine ​​or lysine, or it may not be present.

[0443] In addition to the variables described in this section, the remaining variables of General Formula 1 may have the above-described combinations of amino acids.

[0444] More specifically,

[0445] In general formula 1,

[0446] Xaa2 can be glycine, α-methyl-glutamic acid, or Aib;

[0447] Xaa3 can be glutamine;

[0448] Xaa7 can be threonine.

[0449] Xaa10 can be tyrosine, cysteine, or valine;

[0450] Xaa12 can be lysine;

[0451] Xaa13 can be tyrosine;

[0452] Xaa14 can be leucine;

[0453] Xaa15 can be aspartic acid;

[0454] Xaa16 can be glycine, glutamic acid, or serine;

[0455] Xaa17 can be glutamine, arginine, cysteine, or lysine;

[0456] Xaa18 can be alanine, arginine, or histidine;

[0457] Xaa19 can be either alanine or glutamine;

[0458] Xaa20 can be lysine or glutamine;

[0459] Xaa21 can be glutamic acid, cysteine, or aspartic acid;

[0460] Xaa23 can be valine;

[0461] Xaa24 can be alanine, glutamine, or cysteine;

[0462] Xaa27 can be leucine or lysine; and

[0463] Xaa29 can be glycine, glutamine, threonine, or histidine, but the variable is not specifically limited to these.

[0464] In addition to the variables described in this section, the remaining variables of General Formula 1 may have the above-described combinations of amino acids.

[0465] These peptides that are active against glucagon, GLP-1, and GIP receptors can correspond to: peptides with significantly higher activity against GLP-1 and glucagon receptors than against GIP receptors; peptides with significantly higher activity against GLP-1, glucagon, and GIP receptors; and peptides with significantly higher activity against GLP-1 and GIP receptors than against glucagon receptors, but peptides are not limited to these categories.

[0466] Peptides exhibiting significantly higher activity against GLP-1 and glucagon receptors than against GIP receptors can provide greater weight loss and glycemic control, with peptides showing significant activity against all three receptors (GLP-1, glucagon, and GIP) providing the greatest weight loss. However, peptides are not limited to these categories.

[0467] Examples of these peptides may include, but are not particularly limited to, peptides containing or (substantially) consisting of amino acid sequences selected from the group consisting of SEQ ID NO: 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.

[0468] In one specific aspect, peptides active against glucagon, GLP-1, and GIP receptors may contain an amino acid sequence represented by the following general formula 2:

[0469] 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),

[0470] In general formula 2,

[0471] Xaa1 is 4-imidazolylacetyl, histidine, or tyrosine;

[0472] Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

[0473] Xaa10 is either tyrosine or cysteine;

[0474] Xaa13 is alanine, glutamine, tyrosine, or cysteine;

[0475] Xaa14 is leucine, methionine, or tyrosine;

[0476] Xaa15 is aspartic acid, glutamic acid, or leucine;

[0477] Xaa16 is glycine, glutamic acid, or serine;

[0478] Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;

[0479] Xaa18 is alanine, glutamine, arginine, or histidine;

[0480] Xaa19 is alanine, glutamine, cysteine, or valine;

[0481] Xaa20 is lysine, glutamine, or arginine;

[0482] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

[0483] Xaa23 is either isoleucine or valine;

[0484] Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

[0485] Xaa28 is lysine, cysteine, asparagine, or aspartic acid.

[0486] Xaa29 is glycine, glutamine, cysteine, or histidine;

[0487] Xaa30 is cysteine, glycine, lysine, or histidine;

[0488] Xaa31 is either proline or cysteine; and

[0489] Xaa40 is either cysteine ​​or absent.

[0490] More specifically, in general formula 2,

[0491] Xaa13 can be alanine, tyrosine, or cysteine;

[0492] Xaa15 can be either aspartic acid or glutamic acid;

[0493] Xaa17 can be glutamine, arginine, cysteine, or lysine;

[0494] Xaa18 can be alanine, arginine, or histidine;

[0495] Xaa21 can be cysteine, glutamic acid, glutamine, or aspartic acid;

[0496] Xaa23 can be isoleucine or valine;

[0497] Xaa24 can be cysteine, glutamine, or asparagine;

[0498] Xaa28 can be cysteine, asparagine, or aspartic acid.

[0499] Xaa29 can be glutamine, cysteine, or histidine; and

[0500] Xaa30 can be cysteine, lysine, or histidine.

[0501] In addition to the variables described in this section, the remaining variables in general formula 2 may have the above-described combinations of amino acids.

[0502] Examples of peptides active against glucagon, GLP-1, and GIP receptors may be peptides containing or (substantially) composed of amino acid sequences selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77 and 95 to 102, and more specifically SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77 and 96 to 102, but are not particularly limited thereto.

[0503] In one specific aspect, peptides active against glucagon, GLP-1, and GIP receptors may contain an amino acid sequence represented by the following general formula 3:

[0504] 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),

[0505] In general formula 3,

[0506] Xaa1 is either histidine or tyrosine;

[0507] Xaa2 is α-methyl-glutamic acid or Aib;

[0508] Xaa13 is alanine, tyrosine, or cysteine;

[0509] Xaa17 is arginine, cysteine, or lysine;

[0510] Xaa18 is either alanine or arginine;

[0511] Xaa19 is either alanine or cysteine;

[0512] Xaa21 is either glutamic acid or aspartic acid;

[0513] Xaa24 is either glutamine or asparagine;

[0514] Xaa28 is either cysteine ​​or aspartic acid;

[0515] Xaa29 is cysteine, histidine, or glutamine;

[0516] Xaa30 is either cysteine ​​or histidine;

[0517] Xaa31 is either proline or cysteine; and

[0518] Xaa40 is either cysteine ​​or absent.

[0519] Examples of peptides active against glucagon, GLP-1, and GIP receptors may be peptides containing or (essentially) composed of amino acid sequences selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 64 to 71, 75 to 77, and 96 to 102, but are not particularly limited thereto.

[0520] In 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 may be absent, but is not particularly limited thereto.

[0521] Furthermore, the peptides of the present invention can be synthesized according to their length using methods known in the art, such as automated peptide synthesizers, and can be produced using genetic engineering techniques.

[0522] Specifically, the peptides of the present invention can be prepared using standard synthetic methods, recombinant expression systems, or any other methods known in the art. Therefore, the peptides according to the present invention can be synthesized by a variety of methods, including, for example, the following:

[0523] (a) Methods for synthesizing peptides by solid-phase or liquid-phase methods or by assembling them stepwise or from fragments, and for isolating and purifying the final peptide products;

[0524] (b) A method for expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from a host cell culture;

[0525] (c) A method for in vitro cell-free expression of a nucleic acid construct encoding a peptide and recovery of the expression product therefrom; or

[0526] A method for obtaining peptide fragments through any combination of methods (a), (b), and (c), obtaining peptides by linking fragments, and then recovering peptides.

[0527] As used herein, the term "insulin" refers to a hormone secreted by pancreatic β-cells and generally controls blood glucose levels by promoting intracellular glucose uptake and inhibiting lipolysis. Proinsulin, which lacks glucose-regulating function, is processed into insulin, which does possess glucose-regulating capabilities. Insulin consists of two polypeptide chains, the A-chain and the B-chain, containing 21 and 30 amino acids respectively, interconnected by two disulfide bridges. Insulin can be human insulin. The A-chain and B-chain of natural human insulin contain the amino acid sequences shown in SEQ ID NO:121 and 122, respectively.

[0528] A-Chain:

[0529] Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 121)

[0530] B-Chain:

[0531] Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (SEQ ID NO: 122)

[0532] As used herein, the term "proinsulin" refers to the precursor molecule of insulin. Proinsulin may contain the insulin A-chain and B-chain, as well as the C-peptide between them. Proinsulin may be human proinsulin.

[0533] In this invention, insulin may be natural insulin, or an analogue, derivative or fragment thereof, having changes selected from the group consisting of: substitution, addition, deletion, modification or combination thereof of at least one amino acid in natural insulin.

[0534] As used in this article, the term "insulin analogue" refers to non-natural insulin that is different from natural insulin.

[0535] Examples of insulin analogs include analogs obtained by altering some amino acids of natural insulin through the addition, deletion, or substitution. For example, an insulin analog may be an insulin analog in which at least one amino acid selected from the group consisting of amino acids at positions 1, 2, 3, 5, 8, 10, 12, 16, 23, 24, 25, 26, 27, 28, 29, and 30 of the natural amino acid B-chain and amino acids at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19, and 21 of the natural amino acid A-chain is substituted or deleted by another amino acid, but is not limited thereto. Insulin analogs of the present invention can be referenced to the disclosures of Korean Patent Publication Nos. 10-2014-0106452 or 10-2017-0026284 (or WO 2014 / 133324 A1 or WO2017 / 039267), the entire descriptions of which are incorporated herein by reference.

[0536] The insulin analogues to be used in this invention may be in the form of a single polypeptide chain or two polypeptide chains, more preferably two polypeptide chains, but are not limited thereto. The two polypeptide chains may consist of two polypeptides—a polypeptide corresponding to the A-chain of natural insulin and a polypeptide corresponding to the B-chain of natural insulin. When comparing either of the two polypeptide chains with the A-chain or B-chain of natural insulin in terms of sequence identity, the expression corresponding to the A-chain or B-chain of natural insulin may refer to having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity, but is not particularly limited thereto, and this can be readily understood by those skilled in the art by comparing the sequences constituting the two polypeptide chains with the sequences of the A-chain or B-chain of natural insulin.

[0537] As used herein, the term "homology" is intended to indicate the degree of similarity to the amino acid sequence of a wild-type protein or the nucleotide sequence encoding it, and the term includes sequences that have sequence identity with the amino acid sequence or nucleotide sequence of the present invention at least the aforementioned percentage level. Homology can be determined by visually comparing two given sequences, or it can be determined using bioinformatics algorithms, thereby analyzing the degree of homology by permuting the compared subject sequences. Homology between two given amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA). The automated permutation algorithms in the above modules include the sequence permutation algorithms of Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman. Other useful algorithms for sequence permutation and homology determination are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

[0538] Information about the insulin sequence used in this invention and the nucleotide sequence encoding it can be obtained from known databases, such as NCBI.

[0539] In one specific exemplary embodiment, the insulin analog of the present invention comprises: an A-chain represented by SEQ ID NO:119 of general formula 4; and a B-chain represented by SEQ ID NO:120 of general formula 5.

[0540] Formula 4

[0541] Xaa1-Xaa2-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 119),

[0542] In general formula 4,

[0543] Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;

[0544] Xaa2 is either alanine or isoleucine;

[0545] Xaa5 is alanine, glutamic acid, glutamine, histidine, or asparagine;

[0546] Xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine;

[0547] Xaa14 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid, or asparagine.

[0548] Xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine.

[0549] Xaa19 is alanine, tyrosine, serine, glutamic acid, histidine, threonine, or asparagine; and

[0550] Xaa21 is asparagine, glycine, histidine, or alanine.

[0551] General Formula 5

[0552] Phe-Val-Asn-Gln-His-Leu-Cys-Xaa8-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Xaa23-Xaa24-Xaa25-Tyr-Xaa27-Xaa28-Lys-Thr (SEQ ID NO: 120)

[0553] In general formula 5,

[0554] Xaa8 is either alanine or glycine;

[0555] Xaa16 is tyrosine, glutamic acid, serine, threonine, or aspartic acid, or it may not be present.

[0556] Xaa23 is either glycine or alanine;

[0557] Xaa24 is either alanine or phenylalanine;

[0558] Xaa25 is alanine, phenylalanine, aspartic acid, or glutamic acid, or it may not be present.

[0559] Xaa27 is threonine or is absent; and

[0560] Xaa28 is proline, glutamic acid, or aspartic acid, or it may not be present.

[0561] (Excluding peptides containing the A-chain of SEQ ID NO:121 and the B-chain of SEQ ID NO:122).

[0562] More specifically, insulin analogs may have a substitution of at least one amino acid from the group consisting of amino acids selected from positions 8, 23, 24, and 25 of the natural insulin B-chain and amino acids selected from positions 1, 2, and 19 of the natural insulin A-chain, and / or a substitution of amino acid at position 14 of the natural insulin A-chain with glutamic acid or asparagine. In particular, insulin analogs may contain or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 124, 126, 128, 130, 132, 134, 136, 138, and 140, but insulin analogs are not limited thereto.

[0563] Alternatively, insulin analogs may have a substitution of amino acid at position 16 of the B-chain of natural insulin with glutamic acid, and / or a deletion of amino acid at position 25 of the B-chain of natural insulin and / or a substitution of amino acid at position 14 of the A-chain of natural insulin with glutamic acid or alanine, and in particular, insulin analogs may contain or consist of the amino acid sequence of SEQ ID NO: 142 or 144, but insulin analogs are not limited thereto.

[0564] Alternatively, insulin analogs may have a substitution of the amino acid at position 16 of the natural insulin B-chain with glutamic acid, serine, threonine, or aspartic acid, and / or a substitution of the amino acid at position 25 of the natural insulin B-chain with aspartic acid or glutamic acid, and / or a substitution of the amino acid at position 14 of the natural insulin A-chain with histidine, lysine, alanine, or aspartic acid, and / or a substitution of the amino acid at position 19 of the natural insulin A-chain with glutamic acid, serine, or threonine. In particular, insulin analogs may contain or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170, but insulin analogs are not limited thereto.

[0565] Meanwhile, the insulin analogue of the present invention can be a short-chain insulin form without removing the C-peptide, containing the A-chain represented by SEQ ID NO:119 of general formula 4 and the B-chain represented by SEQ ID NO:120 of general formula 5, and has insulin activity and function.

[0566] Furthermore, the insulin analogs of the present invention can be prepared by removing the C-peptide from proinsulin containing a C-peptide between the A-chain and the B-chain. The removal of the C-peptide can be carried out by methods known in the art, and specifically, insulin analogs can be prepared by treatment with trypsin and carboxypeptidase B, but are not limited thereto.

[0567] Specifically, the insulin analog of the present invention may be composed of an A-chain of SEQ ID NO:119 represented by general formula 4 and a B-chain of SEQ ID NO:120 represented by general formula 5, and more specifically, the insulin analog may be in the form of two polypeptide chains, wherein the A-chain and the B-chain are connected to each other by two disulfide bridges, but is not limited thereto.

[0568] Obviously, insulin analogs shown by specific serial numbers and prepared in the form of two polypeptide chains by removing the C-peptide from insulin analogs in the form of proinsulin are also included within the scope of this invention.

[0569] Specifically, insulin analogs may have the following changes in the sequence of natural insulin, especially natural human insulin, and may be selected from analogs 1 to 24.

[0570] Analog 1 <![CDATA[A 1 G->A]]> Analog 2 <![CDATA[A 2 I->A]]> Analog 3 <![CDATA[A 19 Y->A]]> Analog 4 <![CDATA[B 8 G->A]]> Analog 5 <![CDATA[B 23 G->A]]> Analog 6 <![CDATA[B 24 F->A]]> Analog 7 <![CDATA[B 25 F->A]]> Analog 8 <![CDATA[A 14 Y->E]]> Analog 9 <![CDATA[A 14 Y->N]]> Analog 10 <![CDATA[A 14 Tyr->Glu+B 25 Missing]]> Analog 11 <![CDATA[A 14 Tyr->Ala+B 16 Tyr->Glu.B 25 Missing]]> Analog 12 <![CDATA[A 14 Y->H]]> Analog 13 <![CDATA[A 14 Y->K]]> Analog 14 <![CDATA[A 19 Y->E]]> Analog 15 <![CDATA[A 19 Y->S]]> Analog 16 <![CDATA[A 19 Y->T]]> Analog 17 <![CDATA[B 16 Y->E]]> Analog 18 <![CDATA[B 16 Y->S]]> Analog 19 <![CDATA[B 16 Y->T]]> Analog 20 <![CDATA[A 14 Y->A]]> Analog 21 <![CDATA[A 14 Y->D]]> Analog 22 <![CDATA[B 16 Y->D]]> Analog 23 <![CDATA[B 25 F->D]]> Analog 24 <![CDATA[B 25 F->E]]>

[0571] As used herein, the term "insulin derivative" includes peptides that differ from natural insulin in at least one amino acid sequence, peptides prepared by modifying the sequence of natural insulin, and natural insulin mimics that can regulate blood glucose control in the body like natural insulin. Such derivatives of natural insulin may have blood glucose control functions in the body.

[0572] Specifically, insulin derivatives can be obtained by any method or combination of altering certain amino acids in natural insulin through substitution, addition, deletion, or modification.

[0573] Specifically, derivatives of natural insulin may exhibit at least 80% amino acid sequence homology with each of the A-chain and B-chain of natural insulin and / or may be, but are not limited to, forms in which a group of one amino acid residue of insulin undergoes chemical substitution (e.g., α-methylation or α-hydroxylation), deletion (e.g., deamination), or modification (e.g., N-methylation).

[0574] The natural insulin derivative to be used in this invention can be prepared by a combination of various methods for preparing derivatives.

[0575] Such alterations used to prepare insulin derivatives include: alterations using L- or D-type amino acids and / or non-natural amino acids; and / or alterations through modification of the natural form sequence or post-translational modifications (e.g., methylation, acylation, ubiquitination, intermolecular covalent bonds, etc.).

[0576] In addition, such changes also include the addition of one or more amino acids at the amino and / or carboxyl termini of insulin.

[0577] The amino acids to be replaced or added can be not only the 20 common amino acids in human proteins, but also atypical amino acids or amino acids that do not exist naturally. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptide sequences containing these amino acids and typical peptides can be synthesized by commercial suppliers or purchased from commercial suppliers, such as American Peptide Company and Bachem in the United States, or Antigen in South Korea, but there are no particular restrictions.

[0578] As used herein, the term "fragment of natural insulin, insulin analog, or insulin derivative" refers to a form in which one or more amino acids have been removed from the amino or carboxyl terminus of natural insulin, insulin analog, or natural insulin derivative. These fragments may retain glycemic control function in the body.

[0579] Furthermore, the insulin analogs of the present invention can be prepared by using, alone or in combination, appropriate methods for preparing derivatives and fragments of natural insulin.

[0580] Specifically, the insulin analogs according to the present invention comprise changes to specific amino acid residues in the A-chain and B-chain of the natural insulin described above, and specifically, the insulin analogs may have changes to specific amino acid residues in the A-chain of the natural insulin and / or changes to specific amino acid residues in the B-chain of the natural insulin.

[0581] The pharmaceutical compositions according to the invention may contain, as one of the following substances as insulin, which are: (a) natural insulin, (b) insulin analogs, (c) insulin derivatives, (d) fragments thereof, or (e) combinations thereof.

[0582] In one specific embodiment, the insulin or isolated peptide of the present invention, which is active against glucagon, GLP-1 and GIP receptors, may be in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the insulin or isolated peptide is conjugated to the insulin or isolated peptide.

[0583] As used herein, the term insulin or a “long-acting conjugate” or “conjugate” of a separate peptide active against glucagon, GLP-1, and GIP receptors has a structure in which a biocompatible substance is conjugated to insulin or a separate peptide active against glucagon, GLP-1, and GIP receptors, and may exhibit an increased duration of efficacy compared to insulin or a separate peptide active against glucagon, GLP-1, and GIP receptors without the conjugated biocompatible substance.

[0584] Biocompatible substances in long-acting conjugates may be covalently linked to insulin or isolated peptides active against glucagon, GLP-1, and GIP receptors, but are not particularly limited thereto.

[0585] In this invention, insulin as an element of the conjugate can be insulin having a natural form sequence, or it can be an analogue, derivative or fragment of insulin having a change in at least one amino acid in the natural form sequence by substitution, addition, deletion, modification or a combination thereof. However, any form of insulin can be used as an element of the conjugate of this invention without limitation, as long as it has the blood glucose lowering / raising effect of natural insulin.

[0586] As used herein, the term "biocompatible substance" refers to a substance that can be linked to a physiologically active substance (e.g., isolated peptides active against glucagon, GLP-2, and GIP receptors, insulin, etc.) to increase the duration of efficacy of the physiologically active substance compared to a physiologically active substance without a biocompatible substance portion or carrier. Biocompatible substances can be covalently linked to physiologically active substances, but are not particularly limited thereto.

[0587] Specifically, the conjugate is represented by chemical formula 1:

[0588] Chemical Formula 1

[0589] XL a -F,

[0590] in,

[0591] X is insulin, or a separate peptide that is active against glucagon, GLP-1, and GIP receptors (i.e., a triple agonist).

[0592] L stands for connector;

[0593] a is 0 or a natural number, and each L is independent of the others when a is 2 or greater; and

[0594] F is a substance that can increase the half-life of X.

[0595] The compositions of the present invention may contain (a) insulin and a triple agonist, (b) a long-acting conjugate of insulin and a triple agonist, (c) a long-acting conjugate of insulin and a triple agonist, or (d) a long-acting conjugate of insulin and a long-acting conjugate of a triple agonist, wherein the long-acting form of insulin or the triple agonist exhibits excellent glycemic control on the basis of increased duration in vivo and can alleviate the side effects of insulin.

[0596] In the conjugate, F is X, which is a substance capable of increasing the half-life of insulin or a triple agonist, and corresponds to an element constituting a part of the conjugate of the present invention.

[0597] F and X can be connected to each other by covalent or non-covalent chemical bonds, or F and X can be connected to each other via L by covalent, non-covalent, or a combination thereof.

[0598] Substances that can increase the half-life of X can be biocompatible substances and can be selected from, for example, the group consisting of 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, connective tissue in vivo, nucleotides, fibronectin, transferrin, carbohydrates, heparin and elastin, but are not limited thereto.

[0599] Elastin can be human elastin, which is a water-soluble precursor and can be a polymer of part of its sequence or some of its repeating units, and examples of it include all elastin-like polypeptides, but are not particularly limited thereto.

[0600] Examples of polymers are selected from, but are not particularly limited to, the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0601] Polyethylene glycol corresponds to, but is not limited to, all forms of the term including ethylene glycol homopolymers, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG).

[0602] Examples of biocompatible substances may include, but are not limited to, polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid.

[0603] Fatty acids can bind to albumin in the body, but are not limited to this.

[0604] In a more specific embodiment, the FcRn binding substance may be the Fc region of an immunoglobulin, and more specifically the IgG Fc region, but is not particularly limited thereto.

[0605] One or more amino acid side chains within the peptides of the present invention can be conjugated to these biocompatible substances to increase in vivo solubility and / or half-life, and / or increase their bioavailability. Such modifications may also reduce the clearance of therapeutic proteins and peptides.

[0606] The aforementioned biocompatible substances may be water-soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharmaceutically acceptable.

[0607] F and X can be directly connected to each other (i.e., a is 0 in chemical formula 1) or they can be connected through a connector (L).

[0608] In this invention, the "immunoglobulin Fc region" refers to the region containing heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of immunoglobulin. The immunoglobulin Fc region can be an element constituting the conjugate portion of this invention.

[0609] The immunoglobulin Fc region may include, but is not limited to, the hinge region within the heavy chain constant region. The immunoglobulin Fc region of the present invention may be an elongated Fc region comprising part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CT1), excluding the heavy and light chain variable regions of the immunoglobulin, provided that the immunoglobulin Fc region has substantially the same or improved effects compared to the native form. Alternatively, the immunoglobulin Fc region of the present invention may be a region corresponding to a relatively long amino acid sequence deletion at CH2 and / or CH3.

[0610] For example, the immunoglobulin Fc region of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of one or more of the CH1, CH2, CH3, and CH4 domains with an immunoglobulin hinge region (or a portion of the hinge region); and 6) a dimer between each domain of the heavy chain constant region and the light chain constant region. However, the immunoglobulin Fc region of the present invention is not limited thereto.

[0611] In one specific embodiment, the immunoglobulin Fc region can be in dimer form, and one X molecule can be covalently linked to a dimer Fc region, wherein immunoglobulin Fc and X can be linked to each other via a non-peptide polymer. Alternatively, two X molecules can be symmetrically linked to a dimer Fc region. Immunoglobulin Fc and X can be linked to each other via non-peptide linkers. However, the immunoglobulin Fc region of the present invention is not limited thereto.

[0612] Furthermore, the Fc region of the immunoglobulin of the present invention contains not only the amino acid sequence in its natural form, but also its derivatives. An amino acid sequence derivative refers to an amino acid sequence that differs from the natural amino acid sequence through the deletion, insertion, non-conservative or conserved substitution of at least one amino acid residue, or a combination thereof.

[0613] For example, for immunoglobulin Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to play an important role in conjugation, can be used as suitable alteration sites.

[0614] For example, various derivatives can be made by removing sites capable of forming disulfide bonds, deleting certain amino acid residues at the N-terminus of the natural Fc, or adding methionine residues at the N-terminus of the natural Fc. Furthermore, to eliminate effector function, complement-binding sites, such as the C1q binding site, can be removed, as can antibody-dependent cell-mediated cytotoxicity (ADCC) sites. Techniques for preparing such sequence derivatives of the Fc region of immunoglobulins are disclosed in international patent publications WO 97 / 34631 and WO 96 / 32478, among others.

[0615] Amino acid exchanges between proteins and peptides that do not completely alter the activity of the protein or peptide are known in the art (H. Neurath and RL Hill, *The Proteins*, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, changes can be made through phosphorylation, sulfation, acrylylation, glycosylation, methylation, farnesylation, acetylation, and amination.

[0616] The aforementioned Fc derivatives exhibit bioactivity comparable to the Fc region of the present invention and can improve the structural stability of the Fc region to heat, pH, etc.

[0617] Furthermore, the Fc region can be obtained from the natural form isolated from living humans or animals such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or it can be a recombinant form obtained from transformed animal cells or microorganisms or their derivatives. In this case, the Fc region can be obtained from the natural form by isolating the whole immunoglobulin from a living human or animal and then treating the isolated immunoglobulin with a protease. The isolated immunoglobulin is cleaved into Fab and Fc by papain treatment and into pF'c and F(ab)2 by pepsin treatment. These can be separated into Fc or pF'c by size exclusion chromatography, etc. In a more specific embodiment, the immunoglobulin Fc region is a recombinant immunoglobulin Fc region, wherein the human Fc region is obtained from microorganisms.

[0618] Furthermore, the immunoglobulin Fc region can be in the form of native glycans, in a form with increased glycans compared to the native form, in a form with decreased glycans compared to the native form, or in a deglycosylated form. The increase, decrease, or removal of immunoglobulin Fc glycans can be achieved using conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods utilizing microorganisms. Immunoglobulin Fc regions with glycans removed from the Fc region exhibit a significantly deteriorated binding affinity to complement C1q, as well as reduced or eliminated antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and therefore do not induce unwanted immune responses in vivo. In this regard, deglycosylated or glycosylated immunoglobulin Fc regions may be a more suitable form to achieve the initial objective of this invention as a drug carrier.

[0619] As used herein, the term “deglycosylation” refers to the enzymatic removal of glycans from the Fc region, and the term “unglycosylated” refers to the unglycosylated Fc region produced in prokaryotes, more specifically in Escherichia coli.

[0620] Meanwhile, the immunoglobulin Fc region can be derived from humans or other animals, including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, and in a more specific embodiment, the immunoglobulin Fc region is derived from humans.

[0621] Furthermore, the immunoglobulin Fc region can be derived from the Fc region of IgG, IgA, IgD, IgE, IgM, or combinations or mixtures thereof. In a more specific embodiment, the immunoglobulin Fc region is derived from the most abundant IgG or IgM in human blood, and in a more specific embodiment, the immunoglobulin Fc region is derived from IgG known to increase the half-life of ligand-binding proteins. In a more specific embodiment, the immunoglobulin Fc region is the IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0622] As used herein, the term "combination" refers to the linking of polypeptides encoding the Fc region of single-chain immunoglobulins from the same source with single-chain polypeptides from different sources when forming dimers or multimers. That is, dimers or multimers can be prepared from two or more fragments selected from a group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0623] Furthermore, compared with natural insulin or triple agonists, or with X without F modification, the above conjugates may have an increased duration of efficacy, and such conjugates not only include the above forms, but also forms encapsulated in biodegradable nanoparticles.

[0624] In addition, L can be a peptide linker or a non-peptide linker.

[0625] When L is a peptide linker, L may contain one or more amino acids, for example, from 1 to 1000 amino acids, but is not particularly limited thereto. In this invention, various known peptide linkers can be used to connect F and X, and examples may include [GS]. x Connector, [GGGS] x Connector, [GGGGS] x Connectors, etc., where x can be a natural number of at least 1. However, peptide connectors are not limited to the examples above.

[0626] In this invention, a "non-peptide linker" comprises a biocompatible polymer having two or more repeating units linked together. The repeating units are linked together by any covalent bond, not a peptide bond. The non-peptide linker can be an element constituting a part of the conjugate of this invention and corresponds to L in Formula 1. In this invention, the non-peptide linker can be used interchangeably with the non-peptide polymer.

[0627] In this invention, the peptide linker contains a reactive group at its end and can therefore form a conjugate by reacting with another element constituting the conjugate. When a non-peptide linker with reactive functional groups at both ends forms a conjugate by combining with X and F in Formula 1 through its respective reactive groups, the non-peptide linker or non-peptide polymer may be referred to as a non-peptide polymer linker portion or a non-peptide linker portion.

[0628] In L a In this context, a can be 1 or greater, and when a is 2 or greater, each L can be independent.

[0629] In one specific embodiment, in the conjugate, F and X are covalently linked to each other by non-peptide linkers containing reactive groups at both ends capable of linking to F (specifically, the Fc region of an immunoglobulin) and X (specifically, a peptide drug).

[0630] Specifically, non-peptide linkers can be selected from the group consisting of fatty acids, sugars, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0631] Although not particularly limited, the high molecular weight polymers in this invention can be in the range of greater than 0 kDa to about 100 kDa, specifically in the range of about 1 kDa to about 100 kDa, and more specifically in the range of about 1 kDa to about 20 kDa, but are not particularly limited thereto.

[0632] As used herein, the term “about” means a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and therefore includes, but is not limited to, all values ​​in the range that are equal to or similar to those described below the term.

[0633] While not particularly limited, the high molecular weight polymer can be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof, but is not particularly limited thereto. In more specific embodiments, L can be polyethylene glycol, but is not limited thereto. Furthermore, derivatives known in the art and derivatives readily prepared by techniques in the art are also included within the scope of this invention.

[0634] As the non-peptide linker used in this invention, any polymer resistant to in vivo proteases can be used without limitation. The molecular weight of the non-peptide polymer is in the range of about 1 kDa to about 100 kDa, and specifically in the range of about 1 kDa to about 20 kDa, but is not limited thereto. Furthermore, as the non-peptide linker of this invention for linking to a polypeptide corresponding to F, a single polymer and combinations of different types of polymers can be used.

[0635] In one specific embodiment, the two ends of the non-peptide linker can be connected to an amino or thiol group of F, such as the Fc region of immunoglobulin, and an amino or thiol group of X, respectively.

[0636] Specifically, the non-peptide polymer may have reactive groups at both ends that can be linked to F (e.g., the Fc region of immunoglobulin) and X respectively, and specifically, but not limited to, reactive groups that can be linked to the amino group or thiol group of lysine or cysteine ​​located at the N-terminus of X or F (e.g., the Fc region of immunoglobulin).

[0637] Alternatively, the reactive groups of non-peptide polymers capable of linking to F, such as the Fc region of immunoglobulins and X, can be selected from, but are not limited to, the group consisting of aldehyde groups, maleimide groups, and succinimide derivatives.

[0638] Examples of aldehyde groups may include, but are not limited to, propionaldehyde or butyraldehyde.

[0639] Examples of succinimide derivatives may include, but are not limited to, succinimide valerate, succinimide methyl butyrate, succinimide methyl propionate, succinimide butyrate, succinimide propionate, N-hydroxysuccinimide, hydroxysuccinimide, succinimide carboxymethyl ester, or succinimide carbonate.

[0640] Non-peptide linkers can be linked to X and F through these reactive groups, but are not particularly limited thereto.

[0641] Furthermore, the final product obtained by reductive alkylation via aldehyde bonds is significantly more stable than that obtained by amide bond linkage. The reactive aldehyde group reacts selectively with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​such as pH 9.0.

[0642] The reactive groups at both ends of the non-peptide linker can be the same or different from each other, and for example, the non-peptide linker may have a maleimide group at one end and an aldehyde, propionaldehyde, or butyraldehyde group at the other end. However, the reactive groups are not particularly limited to this, as long as F (specifically the immunoglobulin Fc region) and X can be attached to both ends of the non-peptide linker.

[0643] For example, non-peptide linkers may have a maleimide group as a reactive group at one end and an aldehyde group, propionaldehyde group, butyraldehyde group, etc. as reactive groups at the other end.

[0644] When using polyethylene glycol with reactive hydroxyl groups at both ends as a non-peptide polymer, or when using commercially available polyethylene glycol with modified reactive groups, the long-acting protein conjugates of the present invention can be prepared by activating hydroxyl groups into various reactive groups through known chemical reactions.

[0645] In one specific embodiment, the non-peptide polymer may be linked to the cysteine ​​residue of the triple agonist, and more specifically to the -SH group of cysteine, but is not limited thereto.

[0646] For example, non-peptide polymers can be linked to cysteine ​​residues at positions 10, 13, 15, 17, 19, 21, 24, 28, 29, 30, 31, 40, or 41 in a triple agonist, without particular limitation.

[0647] Specifically, the reactive group of the non-peptide polymer can be linked to the -SH group of the cysteine ​​residue, and all the above descriptions apply to the reactive group. When using maleimide-PEG-aldehyde, the maleimide group can be linked to the -SH group of X via a thioether bond, and the aldehyde group can be linked to the -NH2 group of F, specifically immunoglobulin Fc, via reductive amidation, but is not limited thereto, and this case corresponds to an example.

[0648] In one specific embodiment, the non-peptide polymer may be linked to an amino group of insulin or its analogues (more specifically an amino group located at the N-terminus or an amino group located on the lysine side chain), but is not limited thereto, and this case corresponds to an instance.

[0649] In conjugates, the reactive groups of non-peptide polymers can be linked to the -NH2 group located at the N-terminus of the Fc region of immunoglobulins, but this corresponds to one instance.

[0650] As used herein, the term “prevention” refers to all actions that suppress or delay the onset of insulin-related diseases by applying the composition, and the term “treatment” refers to all actions that alleviate or beneficially alter the symptoms of insulin-related diseases by applying the composition.

[0651] As used herein, the term “administration” means the introduction of a predetermined substance into a patient by any suitable method, and the route of administration of a composition may be, but is not limited to, any general route by which the composition can reach the target in the body, and examples of routes of administration may include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0652] The pharmaceutical compositions according to the present invention may contain a pharmaceutically acceptable carrier.

[0653] As used herein, the term "pharmaceutical acceptable" means having a sufficient amount to produce a therapeutic effect without causing side effects, and that the amount can be readily determined by a person skilled in the art based on factors well known in the medical field, including the type of disease, the patient's age, weight, health status and sex, the patient's sensitivity to the drug, the route of administration, the manner of administration, the number of administrations, the duration of treatment, and the combination or simultaneous use of drugs.

[0654] Regarding pharmaceutically acceptable carriers, for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc., can be used; for injectable mixtures, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., can be used; and for topical administration, substrates, excipients, lubricants, preservatives, etc., can be used. Formulations of the compositions according to the present invention can be prepared differently by mixing with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, the composition can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, thin tablets, etc.; and for injections, the composition can be prepared in the form of single-dose ampoules or multi-dose containers. Furthermore, the composition can also be formulated in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0655] Examples of suitable carriers, excipients, and diluents for the formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. Furthermore, the composition may further contain fillers, anticoagulants, lubricants, humectants, flavoring agents, preservatives, etc.

[0656] The pharmaceutical compositions of the present invention may be any formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations, and suppositories.

[0657] Conjugates can be used by mixing with various carriers approved as medicinal materials, such as physiological saline or organic solvents. In order 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; other stabilizers; etc. can be used as medicinal materials.

[0658] The dosage and frequency of the pharmaceutical composition of the present invention are determined based on the type of drug as the active ingredient, as well as various factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

[0659] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or it can be administered in multiple doses over a prolonged period through a tiered treatment regimen. The pharmaceutical compositions of the present invention may contain an active ingredient, the content of which may vary depending on the severity of the disease. Specifically, the total dose of the conjugates of the present invention may be from about 0.0001 mg to 500 mg per kg of patient body weight per day. However, in addition to the route of administration and treatment frequency of the pharmaceutical composition, various factors are considered in determining the effective dose of the conjugate for the patient, such as the patient's age, weight, health status and sex, disease severity, diet and excretion rate, and therefore, taking these into account, those skilled in the art can readily determine the effective dose suitable for a specific use of the pharmaceutical composition of the present invention. The pharmaceutical compositions according to the present invention are not particularly limited in terms of formulation, route of administration, and method of administration, as long as the pharmaceutical composition exhibits the effects of the present invention.

[0660] Furthermore, the pharmaceutical compositions of the present invention may contain a combination of, i.e., 0.01% by weight / volume to 99% by weight / volume, a triple agonist or a long-acting conjugate thereof; and insulin or a long-acting conjugate thereof (or each component of the combination).

[0661] In one aspect of the invention, a compound formulation for weight loss in patients administering insulin is provided, the compound formulation comprising: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0662] All the above descriptions apply to insulin, isolated peptides active against glucagon, GLP-1 and GIP receptors, and components contained in the composition.

[0663] Specifically, the compound preparation may contain:

[0664] (a) Insulin and triple agonists;

[0665] (b) Long-acting conjugates of insulin, wherein insulin is conjugated with a biocompatible substance that can increase the in vivo half-life of insulin, and a triple agonist.

[0666] (c) A long-acting conjugate of insulin and a triple agonist, wherein the triple agonist is conjugated to a biocompatible substance that can increase the in vivo half-life of the triple agonist; or

[0667] (d) Long-acting conjugates of insulin and long-acting conjugates of triple agonists.

[0668] Specifically, the combination formulation may contain: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein the molar ratio of insulin and the triple agonist may be from 1:1 to 100:1; the molar ratio of insulin and the triple agonist may be from 1:1 to 1:100; the molar ratio of insulin or a long-acting conjugate thereof and the triple agonist or a long-acting conjugate thereof may be from 1:1 to 1:100; or the molar ratio of insulin or a long-acting conjugate thereof and the triple agonist or a long-acting conjugate thereof may be from 1:1 to 1:100, but the combination formulation is not limited to these.

[0669] One aspect of the present invention provides a kit comprising: (i) insulin; and (ii) a isolated peptide active against glucagon, GLP-1 and GIP receptors.

[0670] Insulin and isolated peptides active against glucagon, GLP-1, and GIP receptors are described above.

[0671] The kit may contain instructions for use of the following substances: (i) insulin; and (ii) isolated peptides active against glucagon, GLP-1 and GIP receptors.

[0672] Specifically, the set may include:

[0673] (a) Insulin and triple agonists;

[0674] (b) Long-acting conjugates of insulin, wherein insulin is conjugated with a biocompatible substance that can increase the in vivo half-life of insulin, and a triple agonist.

[0675] (c) A long-acting conjugate of insulin and a triple agonist, wherein the triple agonist is conjugated to a biocompatible substance that can increase the in vivo half-life of the triple agonist; or

[0676] (d) Long-acting conjugates of insulin and long-acting conjugates of triple agonists.

[0677] Specifically, the kit may include: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein the kit may include instructions for administering insulin and the triple agonist to the subject at a molar ratio of 1:1 to 100:1 or 1:1 to 1:100, administering insulin or a long-acting conjugate thereof and the triple agonist or a long-acting conjugate thereof to the subject at a molar ratio of 1:1 to 100:1, or administering insulin or a long-acting conjugate thereof and the triple agonist or a long-acting conjugate thereof to the subject at a molar ratio of 1:1 to 1:100, but the kit is not limited thereto.

[0678] One aspect of the present invention provides the use of a composition or compound preparation in the preparation of a medicinal material, said composition or compound preparation comprising: (i) insulin; and (ii) a separated peptide active against glucagon, GLP-1 and GIP receptors.

[0679] The composition or compound preparation is as described above. All medicinal materials can be used for the above purposes.

[0680] One aspect of the present invention provides a method for preventing or treating insulin-related diseases, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1, and GIP receptors.

[0681] One aspect of the present invention provides a method for reducing weight gain caused by insulin administration, the method comprising administering to a subject a isolated peptide that is active against glucagon, GLP-1 and GIP receptors.

[0682] Specifically, one aspect of the invention provides a method for reducing weight gain caused by insulin administration, the method comprising administering (i) insulin to a subject; and (ii) a separated peptide active against glucagon, GLP-1, and GIP receptors.

[0683] Subjects may be at risk of developing insulin-related disease or may develop insulin-related disease. Subjects may also be subjects who require insulin administration, or subjects who require insulin administration and are experiencing weight loss. However, subjects are not specifically limited to these categories.

[0684] The administration procedure can be carried out by combining the following substances: (a) insulin and a triple agonist; (b) a long-acting conjugate of insulin and a triple agonist; (c) a long-acting conjugate of insulin and a triple agonist; or (d) a long-acting conjugate of insulin and a long-acting conjugate of a triple agonist.

[0685] In this document, the term "combination" should be understood to mean simultaneous, individual, or sequential administration. When administration is sequential or individual, delaying the administration of the second component should not negate the beneficial effects of the combination.

[0686] The substance may be applied simultaneously, alone, successively or in reverse order, and may be applied simultaneously in appropriate combinations of effective amounts, but such application is not limited to a specific method or order of application.

[0687] Furthermore, the prevention or treatment methods of the present invention may include administering to a subject a composition or compound preparation containing (a) to (d) above, but are not limited thereto.

[0688] The compositions or complex formulations of the present invention containing insulin or its long-acting conjugates and triple agonists or their long-acting conjugates supplement the activity or function of insulin, thereby inhibiting the side effects of insulin such as weight gain, while significantly reducing blood glucose, and thus exhibiting excellent efficacy in the treatment of insulin-related diseases.

[0689] Furthermore, according to typical methods in the pharmaceutical field, the composition or compound preparation can be formulated into a unit dose formulation suitable for administration to a patient, specifically a formulation for administering protein drugs, and can be administered via oral or parenteral routes using methods commonly used in the art, including, but not limited to, skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, pulmonary, percutaneous, subcutaneous, intraperitoneal, intranasal, intragastric, local, sublingual, vaginal, or rectal routes.

[0690] The method of the present invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition containing an active ingredient. The appropriate total daily dose of the pharmaceutical composition may be determined by a licensed physician within the bounds of sound medical judgment, and the pharmaceutical composition may be administered in divided doses once or multiple times. However, for the purposes of the present invention, it is preferred to apply a specific therapeutically effective amount for a particular patient based on the following differences: the type and extent of the desired response; the use or non-use of another formulation in certain circumstances; various factors, including the specific composition, the patient's age, weight, general condition, sex and diet, time of administration, route of administration, secretion rate of the composition, duration of treatment, and drugs used in combination with or concurrently with the specific composition; and similar factors well known in the medical field.

[0691] Specifically, insulin or its long-acting conjugates and triple agonists or their long-acting conjugates may be administered to subjects at a molar ratio of 1:1 to 100:1 or 1:1 to 1:100; or insulin or its long-acting conjugates and triple agonists or their long-acting conjugates may be administered to subjects at a molar ratio of 1:1 to 100:1; or insulin or its long-acting conjugates and triple agonists or their long-acting conjugates may be administered to subjects at a molar ratio of 1:1 to 1:100, but not limited thereto.

[0692] The present invention will be described in detail below with reference to the following exemplary embodiments. However, the following exemplary embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0693] Example 1: Preparation of Triple Agonists and Their Long-Acting Conjugates

[0694] (1) Preparation of triple agonists

[0695] Triple agonists exhibiting activity against glucagon, GLP-1, and GIP receptors were prepared, and their sequences are shown in Table 1 below.

[0696] Table 1

[0697]

[0698]

[0699]

[0700]

[0701] In the sequences shown in Table 1, amino acids marked with X represent aminoisobutyric acid (Aib), which is a non-natural amino acid, and underlined amino acids represent amino acids that form a ring together. In Table 1, CA represents 4-imidazolyl acetyl, and Y represents tyrosine.

[0702] (2) Preparation of long-acting triple agonist conjugates

[0703] To PEGylate the cysteine ​​residues of a 10kDa PEG with maleimide and aldehyde groups at both ends, namely maleimide-PEG-aldehyde (10kDa, NOF, Japan) and the triple agonists (SEQ ID NO: 21, 22, 42, 43, 50, 77, and 96) in Example 1, the triple agonists and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1-3 at low temperature for 0.5-3 hours, with a protein concentration of 1-5 mg / mL. The reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was complete, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the cysteine-monopolyglycolated triple agonists.

[0704] The purified polyethylene glycol-modified triple agonist and immunoglobulin Fc were then reacted at a molar ratio of 1:1-5 at 4-8°C for 12-18 hours, with a protein concentration of 10-50 mg / mL. The reaction was carried out in an environment where 10-50 mM sodium cyanoborohydride as a reducing agent and 10-30% isopropanol were added to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was complete, the reaction solution was purified using a Butyl Sepharose FF purification column (GE Healthcare, USA) and a SourceISO purification column (GE Healthcare, USA) to purify the triple agonist-immunoglobulin Fc conjugate.

[0705] After preparation, the purity was analyzed by reversed-phase chromatography, size exclusion chromatography and ion exchange chromatography and was 95% or higher.

[0706] The conjugates of the triple agonist and immunoglobulin Fc linked by PEG in SEQ ID NO:21 are named “conjugates containing SEQ ID NO:21 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:21”, or “long-acting conjugates of SEQ ID NO:22”, and these terms are used interchangeably herein.

[0707] The conjugates of the triple agonist of SEQ ID NO:22 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:22 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:22”, or “long-acting conjugates of SEQ ID NO:22”, and these terms are used interchangeably herein.

[0708] The conjugates of the triple agonist of SEQ ID NO:42 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:42 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:42”, or “long-acting conjugates of SEQ ID NO:42”, and these terms are used interchangeably herein.

[0709] The conjugates of the triple agonist of SEQ ID NO:43 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:43 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:43”, or “long-acting conjugates of SEQ ID NO:43”, and these terms are used interchangeably herein.

[0710] The conjugates of the triple agonist of SEQ ID NO:50 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:50 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:50”, or “long-acting conjugates of SEQ ID NO:50”, and these terms are used interchangeably herein.

[0711] The conjugates of the triple agonist of SEQ ID NO:77 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:77 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:77”, or “long-acting conjugates of SEQ ID NO:77”, and these terms are used interchangeably herein.

[0712] The conjugates of the triple agonist of SEQ ID NO:96 and immunoglobulin Fc linked by PEG are named “conjugates containing SEQ ID NO:96 and immunoglobulin Fc”, “long-acting conjugates of SEQ ID NO:96”, or “long-acting conjugates of SEQ ID NO:96”, and these terms are used interchangeably herein.

[0713] Example 2: Preparation of long-acting natural insulin conjugates

[0714] To PEGylate 3.4K propion-ALD(2)PEG (3.4kDa PEG with a propionaldehyde group at each end, NOF, Japan) at the N-terminus of the B-chain of human natural insulin (India, Biocon), natural insulin and PEG were reacted at a molar ratio of 1:4 at 25°C for 2 hours, with the natural insulin concentration being 5 mg / mL. The reaction was carried out by adding 3 mM sodium cyanoborohydride (NaCNBH3) as a reducing agent to a mixed solvent of 50 mM sodium citrate buffer (pH 5.0) and 45% isopropanol. The reaction solution was purified using an SP-HP (GE Healthcare) column with a KCl concentration gradient containing a buffer of sodium citrate (pH 3.0) and 45% EtOH.

[0715] Next, to ligate the PEG linked to natural insulin to the N-terminus of the immunoglobulin Fc fragment, purified polyethylene glycol-modified insulin and the immunoglobulin Fc fragment were reacted at a molar ratio of 1:1.2 at 25°C for 15 hours, with a total protein concentration of 20 mg / mL. For the reaction solution, 20 mM sodium cyanoborohydride as a reducing agent was added to 100 mM HEPES buffer (pH 8.2) and sodium chloride.

[0716] After the reaction was completed, the reaction solution was applied to a Q-HP column (GE, USA) using a concentration gradient of Tris-HCl buffer (pH 7.5) and NaCl, and then applied to a Source 15ISO column (GE, USA) using a concentration gradient of ammonium sulfate and Tris-HCl (pH 7.5) to purify the natural insulin-3.4K PEG-immunoglobulin Fc conjugate.

[0717] Test Example 1: Determination of the in vitro activity of triple agonists and their long-acting conjugates

[0718] To determine the activity of the triple agonist and its long-acting conjugate prepared in Example 1, cell activity was measured in vitro using cell lines transformed with GLP-1, glucagon (GCG), and GIP receptors, respectively.

[0719] This cell line was obtained by transforming Chinese hamster ovary (CHO) cells to express human GLP-1, human GCG, and human GIP receptors, respectively, and was suitable for measuring the activities of GLP-1, GCG, and GIP. Therefore, the activities of the receptors were measured using the transformed cell line.

[0720] To determine the GLP-1 activity of the triple agonists and their long-acting conjugates prepared in Examples 1 and 2, human GLP-1 and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted. Cultures were removed from CHO cells expressing the human GLP-1 receptor, and each serially diluted substance was added to the cells at 5 μL, followed by 5 μL of buffer containing cAMP antibody, and then incubated at room temperature for 15 min. Then, 10 μL of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 min. After incubation, the cell lysates were applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were then compared with each other. The relative titers compared to human GLP-1 are shown in Tables 2 and 3 below.

[0721] To determine the GGG activity of the triple agonists and their long-acting conjugates prepared in Examples 1 and 2, human GGG and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted. Cultures were removed from cultured CHO cells expressing the human GGG receptor, and each serially diluted substance was added to the cells at 5 μL, followed by 5 μL of buffer containing cAMP antibody, and then incubated at room temperature for 15 min. Then, 10 μL of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 min. After incubation, the cell lysates were applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were then compared with each other. The relative titers compared to human GGG are shown in Tables 2 and 3 below.

[0722] To determine the GIP activity of the triple agonists and their long-acting conjugates prepared in Examples 1 and 2, human GIP and the triple agonists and their long-acting conjugates prepared in Examples 1 and 2 were serially diluted. Cultures were removed from cultured CHO cells expressing the human GIP receptor, and each serially diluted substance was added to the cells at 5 μL, followed by 5 μL of buffer containing cAMP antibody, and then incubated at room temperature for 15 min. Then, 10 μL of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 min. After incubation, the cell lysates were applied to a LANCE cAMP kit (PerkinElmer, USA) to calculate EC50 by accumulating cAMP. 50 The values ​​were then compared with each other. The relative titers compared to human GIP are shown in Tables 2 and 3 below.

[0723] Table 2

[0724] The relative titer ratio of triple agonists

[0725]

[0726]

[0727]

[0728] Table 3

[0729] The relative titer ratio of triple agonists

[0730]

[0731] Test Example 2: Combined administration of long-acting insulin conjugates and long-acting triple agonist conjugates for type 2 diabetes Blood glucose control and Δbody weight in model mice

[0732] To determine the in vivo effects of administration of a combination or combination of a long-acting conjugate containing the triple agonist prepared in Example 1 (SEQ ID NO:42) or a long-acting conjugate of natural insulin prepared in Example 2, or a combination of administration of a long-acting conjugate containing the triple agonist prepared in Example 1 and a long-acting conjugate of natural insulin, type II diabetic model mice (db / db mice, Charles River, Japan) were used. db / +Lepr db / OlaHsd mice were used in this test case because the mice exhibited symptoms of diabetes due to the removal of leptin receptors.

[0733] Blood glucose levels in 8-week-old db / db mice were measured using a blood glucose meter (OneTouch Ultra, LifeScan, Inc., USA) from one to two drops of blood collected from the tail vein using a 26-G syringe. The induction of diabetes was determined by the measured blood glucose levels (350 mg / dL to 600 mg / dL). The diabetic mice were divided into four groups: G1, G2, G3, and G4, with seven mice in each group.

[0734] These groups were assigned as follows: a control group (substrate), a group receiving a long-acting natural insulin conjugate (15.8 nmol / kg / Q2D), a group receiving a long-acting triple agonist conjugate (1.4 nmol / kg / Q2D), and a group receiving a combination of a long-acting natural insulin conjugate (15.8 nmol / kg / Q2D) and a long-acting triple agonist conjugate (1.4 nmol / kg / Q2D). After two weeks of repeated administration of the test substance, glycosylated hemoglobin (HbA1c) levels were measured in each group. Glycosylated hemoglobin is the form in which glucose is bound to hemoglobin normally present in red blood cells, and its levels increase when blood glucose levels remain high. The change in body weight (ΔBW) of the test animals before drug administration and on the last day of the test was calculated.

[0735] As a result, the group receiving combined administration of a long-acting conjugate of natural insulin and a long-acting conjugate of a triple agonist showed a decrease in glycosylated hemoglobin levels. Figure 1 Specifically, such as Figure 1 As shown, compared with the control group, the change in glycosylated hemoglobin level in the group receiving the long-acting insulin conjugate was -0.1; the change in glycosylated hemoglobin level in the group receiving the long-acting triple agonist conjugate was -0.3; and the change in glycosylated hemoglobin level in the group receiving the combination of the long-acting insulin conjugate and the long-acting triple agonist conjugate was -0.5.

[0736] Compared with the group that received either the long-acting natural insulin conjugate alone or the group that received either the long-acting triple agonist conjugate alone, the combination therapy showed significantly improved results.

[0737] The results of Δbody weight measurement showed that, compared with the group receiving long-acting insulin conjugate alone, the group receiving the combination of natural long-acting insulin conjugate and triple agonist long-acting conjugate showed improved weight gain and significant weight loss compared with the control group. Figure 2 Specifically, such as Figure 2 As shown, the weight change in the control group was +15.8%; the weight change in the group receiving the long-acting insulin conjugate was +23.1%; the weight change in the group receiving the long-acting triple agonist conjugate was -9.3%; and the weight change in the group receiving a combination of the long-acting insulin conjugate and the long-acting triple agonist conjugate was -3.8%.

[0738] These results indicate that, compared with the use of long-acting insulin conjugates or long-acting triple agonist conjugates alone, the combined use of long-acting insulin conjugates and long-acting triple agonist conjugates of the present invention can exhibit superior glycemic control, and the combined use of long-acting insulin conjugates and long-acting triple agonist conjugates of the present invention can significantly reduce the side effect of weight gain caused by the use of insulin alone.

[0739] Although the invention has been described with reference to specific illustrative embodiments, those skilled in the art will understand that the invention can be implemented in other specific forms without departing from its technical spirit or essential characteristics. Therefore, the above examples should be interpreted as illustrative rather than limiting. The scope of the invention is not limited by the foregoing detailed description, but by the appended claims, and it should also be understood that all variations or modifications derived from the definitions and scope of the claims and their equivalents fall within the scope of the invention. <110> Hanmi Pharmaceutical Co., Ltd. <120> Pharmaceutical compositions including insulin and trigonaccharin / GLP-1 / GIP receptor agonists <130> OPA19304 <150> KR 10-2018-0167698 <151> 2018-12-21 <160> 170 <170> KoPatentIn 3.0 <210> 1 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 1 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15 Gln Ala Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25 30 <210> 3 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Leu Gly 1 5 10 15 Gln Ala Ala Lys Gln Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser Cys 35 40 <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Leu Gly 1 5 10 15 Gln Gln Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25 30 <210> 5 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Leu Gly 1 5 10 15 Gln Gln Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser Cys 35 40 <210> 6 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 6 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Val Ala Trp Leu Leu Lys Gly Cys 20 25 30 <210> 7 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Val Ala Trp Leu Leu Lys Gly Cys 20 25 30 <210> 8 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 8 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Lys Gly Cys 20 25 30 <210> 9 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 9 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Cys 20 25 30 <210> 10 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 10 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser Cys 35 40 <210> 11 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 11 Xaa Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Gln Gln Leu Phe Val Gln Trp Leu Lys Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 12 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 12 Xaa Gly Glu Gly Thr Phe Ile Ser Asp Leu Ser Lys Tyr Met Asp Glu 1 5 10 15 Gln Ala Val Gln Leu Phe Val Glu Trp Leu Met Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 13 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 13 Xaa Gly Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Gln Leu Asp Glu 1 5 10 15 Ile Ala Val Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 14 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 14 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Gln Leu Asp Glu 1 5 10 15 Ile Ala Val Arg Asp Phe Val Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 15 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 15 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 16 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 16 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser 1 5 10 15 Glu Ala Gln Gln Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 17 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 17 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Glu 1 5 10 15 Glu Arg Ala Arg Glu Phe Ile Glu Trp Leu Leu Ala Gln Lys Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 18 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 18 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser 1 5 10 15 Glu Arg Ala Arg Glu Phe Ile Glu Trp Leu Lys Asn Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 19 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 19 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Tyr Asp Ser 1 5 10 15 Glu His Gln Arg Asp Phe Ile Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> 20 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <400> 20 Xaa Gly Gln Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Tyr Glu Glu 1 5 10 15 Glu Ala Gln Gln Asp Phe Val Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser His Gly 35 40 <210> twenty one <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> twenty one Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> twenty two <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> twenty two Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> twenty three <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> twenty three Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30 Lys Asn Asp Trp Lys His Asn Ile Thr 35 40 <210> twenty four <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> twenty four Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 25 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 25 His Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 26 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 26 His Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 27 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 27 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Cys Gln Asp Phe Val Gln Trp Leu Leu Asp Gln Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> 28 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 28 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Gln Glu Phe Val Cys Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30 Lys Asn Asp Trp Lys His Asn Ile Thr 35 40 <210> 29 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 29 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 30 <210> 30 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 30 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 25 30 <210> 31 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 31 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Cys Lys Glu Phe Val Gln Trp Leu Leu Ala Gln 20 25 <210> 32 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 32 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Cys Lys Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 33 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 33 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 34 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 34 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 35 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 35 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys Cys 20 25 30 <210> 36 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 36 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Cys Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly 35 40 <210> 37 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 37 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly 35 40 <210> 38 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 38 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Ser Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Ser 35 40 <210> 39 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 39 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln His Ala Gln Cys Phe Val Ala Trp Leu Leu Ala Gly Gly Gly Pro 20 25 30 Ser Ser Gly Ala Pro Pro Pro Ser 35 40 <210> 40 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 40 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Cys Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 41 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 41 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 42 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 42 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 43 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 43 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Cys Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 44 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <400> 44 His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Gln Leu Asp Gly 1 5 10 15 Gln Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 45 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <400> 45 His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Met Asp Gly 1 5 10 15 Gln Ala Ala Gln Asp Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 46 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <400> 46 His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Gln His Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 47 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <400> 47 His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Arg Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 48 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <400> 48 His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Gly 1 5 10 15 Gln Arg Ala Gln Asp Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 49 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 49 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Cys Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 30 <210> 50 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 50 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 51 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 51 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 30 <210> 52 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 52 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 25 30 <210> 53 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 53 His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 54 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 54 His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Cys 20 25 30 <210> 55 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 55 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 56 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 56 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 57 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 57 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Cys 20 25 30 <210> 58 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 58 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Lys Cys 20 25 30 <210> 59 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 59 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Cys Met Asp Glu 1 5 10 15 Lys His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 30 <210> 60 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 60 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Lys His Cys Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 30 <210> 61 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 61 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile Ala Cys Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 30 <210> 62 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 62 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 63 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <400> 63 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 64 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 64 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 65 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 65 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 66 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 66 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 67 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 67 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 68 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 68 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 69 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 69 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 70 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 70 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 71 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 71 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Cys Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 72 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 72 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 73 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 73 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 74 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 74 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 75 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 75 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 76 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 76 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 77 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 77 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 78 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 78 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Lys Cys 20 25 30 <210> 79 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 79 His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 80 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 80 His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Lys Cys 20 25 30 <210> 81 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 81 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 30 <210> 82 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 82 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 25 30 <210> 83 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 83 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 84 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 84 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Cys 20 25 30 <210> 85 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 85 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 86 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 86 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 30 <210> 87 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 87 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Cys 20 25 30 <210> 88 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 88 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Lys Cys 20 25 30 <210> 89 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 89 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Lys Cys 20 25 30 <210> 90 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 90 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 91 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 91 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Lys Cys 20 25 30 <210> 92 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 92 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 93 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 93 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln Lys Cys 20 25 30 <210> 94 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazolium acetyl (CA) <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 94 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys Cys 20 25 30 <210> 95 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 95 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Cys His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 96 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 96 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His Cys Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 97 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 97 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Cys His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser 35 <210> 98 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 98 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Asn Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 99 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 99 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Asn Trp Leu Leu Asp His His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 100 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 100 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Gln His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 101 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 101 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Asn Trp Leu Leu Asp Gln His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 102 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Triangle agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib). <220> <221> MISC_FEATURE <222> (16) (20) <223> The amino acids at positions 16 and 20 form a ring. <400> 102 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Asn Trp Leu Leu Asp Gln His Pro Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Cys 35 40 <210> 103 <211> 30 <212> PRT <213> Artificial sequence <220> <223> General Formula 1 <220> <221> MISC_FEATURE <222> (1) <223> Xaa can be His (H), 4-imidazolium acetyl (CA), or Tyr (Y). <220> <221> MISC_FEATURE <222> (2) <223> Xaa is Gly(G), α-methyl-glutamic acid, or Aib (Aminoisobutyric acid) <220> <221> MISC_FEATURE <222> (3) <223> Xaa is either Glu (E) or Gln (Q). <220> <221> MISC_FEATURE <222> (7) <223> Xaa is Thr (T) or Ile (I) <220> <221> MISC_FEATURE <222> (10) <223> Xaa is Leu (L), Tyr (Y), Lys (K), Cys (C), or Val (V). <220> <221> MISC_FEATURE <222> (12) <223> Xaa is Lys (K), Ser (S), or Ile (I). <220> <221> MISC_FEATURE <222> (13) <223> Xaa is Gln (Q), Tyr (Y), Ala (A), or Cys (C). <220> <221> MISC_FEATURE <222> (14) <223> Xaa can be Leu (L), Met (M), or Tyr (Y). <220> <221> MISC_FEATURE <222> (15) <223> Xaa is Cys (C), Asp (D), Glu (E), or Leu (L). <220> <221> MISC_FEATURE <222> (16) <223> Xaa can be Gly (G), Glu (E), or Ser (S). <220> <221> MISC_FEATURE <222> (17) <223> Xaa can be Gln (Q), Arg (R), Ile (I), Glu (E), Cys (C), or Lys (K). <220> <221> MISC_FEATURE <222> (18) <223> Xaa can be Ala (A), Gln (Q), Arg (R), or His (H). <220> <221> MISC_FEATURE <222> (19) <223> Xaa is Ala (A), Gln (Q), Cys (C), or Val (V). <220> <221> MISC_FEATURE <222> (20) <223> Xaa is Lys (K), Gln (Q), or Arg (R). <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa can be Glu (E), Gln (Q), Leu (L), Cys (C), or Asp (D). <220> <221> MISC_FEATURE <222> (twenty three) <223> Xaa is either Ile (I) or Val (V) <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa can be Ala (A), Gln (Q), Cys (C), Asn (N), Asp (D), or Glu (E). <220> <221> MISC_FEATURE <222> (27) <223> Xaa is Val (V), Leu (L), Lys (K), or Met (M). <220> <221> MISC_FEATURE <222> (28) <223> Xaa can be Cys (C), Lys (K), Ala (A), Asn (N), or Asp (D). <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be Cys (C), Gly (G), Gln (Q), Thr (T), Glu (E), or His (H). <220> <221> MISC_FEATURE <222> (30) <223> Xaa is Cys, Gly, Lys, or His, or does not exist; and Xaa can be further connected to R1, where R1 is Cys, GKKNDWKHNIT, m-SSGAPPPS-n, or m-SSGQPPPS-n, or does not exist, and where m is -Cys-, -Pro-, or -Gly-Pro-, and n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or does not exist. <400> 103 Xaa Xaa Xaa Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Xaa Xaa Xaa Xaa 20 25 30 <210> 104 <211> 40 <212> PRT <213> Artificial sequence <220> <223> General Formula 2 <220> <221> MISC_FEATURE <222> (1) <223> Xaa can be His (H), 4-imidazolium acetyl (CA), or Tyr (Y). <220> <221> MISC_FEATURE <222> (2) <223> Xaa is Gly(G), α-methyl-glutamic acid, or Aib (Aminoisobutyric acid) <220> <221> MISC_FEATURE <222> (10) <223> Xaa is Tyr (Y) or Cys (C). <220> <221> MISC_FEATURE <222> (13) <223> Xaa is Gln (Q), Tyr (Y), Ala (A), or Cys (C). <220> <221> MISC_FEATURE <222> (14) <223> Xaa can be Leu (L), Met (M), or Tyr (Y). <220> <221> MISC_FEATURE <222> (15) <223> Xaa can be Asp (D), Glu (E), or Leu (L). <220> <221> MISC_FEATURE <222> (16) <223> Xaa can be Gly (G), Glu (E), or Ser (S). <220> <221> MISC_FEATURE <222> (17) <223> Xaa can be Gln (Q), Arg (R), Ile (I), Glu (E), Cys (C), or Lys (K). <220> <221> MISC_FEATURE <222> (18) <223> Xaa can be Ala (A), Gln (Q), Arg (R), or His (H). <220> <221> MISC_FEATURE <222> (19) <223> Xaa is Ala (A), Gln (Q), Cys (C), or Val (V). <220> <221> MISC_FEATURE <222> (20) <223> Xaa is Lys (K), Gln (Q), or Arg (R). <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa can be Glu (E), Gln (Q), Leu (L), Cys (C), or Asp (D). <220> <221> MISC_FEATURE <222> (twenty three) <223> Xaa is either Ile (I) or Val (V) <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa can be Ala (A), Gln (Q), Cys (C), Asn (N), or Glu (E). <220> <221> MISC_FEATURE <222> (28) <223> Xaa is Cys (C), Lys (K), Asn (N), or Asp (D). <220> <221> MISC_FEATURE <222> (29) <223> Xaa can be Cys (C), Gly (G), Gln (Q), or His (H). <220> <221> MISC_FEATURE <222> (30) <223> Xaa can be Cys (C), Gly (G), Lys (K), or His (H). <220> <221> MISC_FEATURE <222> (31) <223> Xaa is either Pro (P) or Cys (C). <220> <221> MISC_FEATURE <222> (40) <223> Xaa is Cys (C), or does not exist. <400> 104 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Xaa Ser Lys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Xaa Xaa Xaa Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Xaa 35 40 <210> 105 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Formula 3 <220> <221> MISC_FEATURE <222> (1) <223> Xaa is His (H) or Tyr (Y). <220> <221> MISC_FEATURE <222> (2) <223> Xaa is α-methylglutamic acid, or Aib (aminoisobutyric acid). <220> <221> MISC_FEATURE <222> (13) <223> Xaa can be Tyr (Y), Ala (A), or Cys (C). <220> <221> MISC_FEATURE <222> (17) <223> Xaa can be Arg (R), Cys (C), or Lys (K). <220> <221> MISC_FEATURE <222> (18) <223> Xaa is either Ala (A) or Arg (R). <220> <221> MISC_FEATURE <222> (19) <223> Xaa is either Ala (A) or Cys (C). <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa is either Glu (E) or Asp (D). <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa is Gln(Q) or Asn(N). <220> <221> MISC_FEATURE <222> (28) <223> Xaa is either Cys (C) or Asp (D). <220> <221> MISC_FEATURE <222> (29) <223> Xaa is Cys (C), Gln (Q), or His (H). <220> <221> MISC_FEATURE <222> (30) <223> Xaa is either Cys (C) or His (H). <220> <221> MISC_FEATURE <222> (31) <223> Xaa is either Pro (P) or Cys (C). <220> <221> MISC_FEATURE <222> (40) <223> Xaa is Cys (C), or does not exist. <400> 105 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Xaa Leu Asp Glu 1 5 10 15 Xaa Xaa Xaa Lys Xaa Phe Val Xaa Trp Leu Leu Xaa Xaa Xaa Xaa Ser 20 25 30 Ser Gly Gln Pro Pro Pro Ser Xaa 35 40 <210> 106 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 106 Gly Lys Lys Asn Asp Trp Lys His Asn Ile Thr 1 5 10 <210> 107 <211> 8 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 107 Ser Ser Gly Ala Pro Pro Pro Ser 1 5 <210> 108 <211> 8 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 108 Ser Ser Gly Gln Pro Pro Pro Ser 1 5 <210> 109 <211> 9 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 109 Cys Ser Ser Gly Gln Pro Pro Pro Ser 1 5 <210> 110 <211> 10 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 110 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 10 <210> 111 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 111 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser Cys 1 5 10 <210> 112 <211> 9 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 112 Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 <210> 113 <211> 10 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 113 Pro Ser Ser Gly Ala Pro Pro Pro Ser Gly 1 5 10 <210> 114 <211> 11 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 114 Pro Ser Ser Gly Ala Pro Pro Pro Ser His Gly 1 5 10 <210> 115 <211> 10 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 115 Pro Ser Ser Gly Ala Pro Pro Pro Ser Ser 1 5 10 <210> 116 <211> 9 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 116 Pro Ser Ser Gly Gln Pro Pro Pro Ser 1 5 <210> 117 <211> 10 <212> PRT <213> Artificial sequence <220> <223> R1 <400> 117 Pro Ser Ser Gly Gln Pro Pro Pro Ser Cys 1 5 10 <210> 118 <211> 29 <212> PRT <213> Homo sapiens <400> 118 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 119 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Insulin analogs, A-chain <220> <221> MISC_FEATURE <222> (1) <223> Xaa is alanine, glycine, glutamine, histidine, glutamic acid, or Asparagine <220> <221> MISC_FEATURE <222> (2) <223> Xaa is either alanine or isoleucine. <220> <221> MISC_FEATURE <222> (5) <223> Xaa is alanine, glutamic acid, glutamine, histidine, or Asparagine <220> <221> MISC_FEATURE <222> (12) <223> Xaa is alanine, serine, glutamine, glutamic acid, histidine, or Asparagine <220> <221> MISC_FEATURE <222> (14) <223> Xaa consists of alanine, tyrosine, glutamic acid, histidine, and lysine. Aspartic acid, or asparagine <220> <221> MISC_FEATURE <222> (16) <223> Xaa is alanine, leucine, tyrosine, histidine, glutamic acid, or Asparagine <220> <221> MISC_FEATURE <222> (19) <223> Xaa consists of alanine, tyrosine, serine, glutamic acid, and histidine. Threonine, or asparagine <220> <221> MISC_FEATURE <222> (twenty one) <223> Xaa is asparagine, glycine, histidine, or alanine. <400> 119 Xaa Xaa Val Glu Xaa Cys Cys Thr Ser Ile Cys Xaa Leu Xaa Gln Xaa 1 5 10 15 Glu Asn Xaa Cys Xaa 20 <210> 120 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Insulin analogues, B-chain <220> <221> MISC_FEATURE <222> (8) <223> Xaa is either alanine or glycine. <220> <221> MISC_FEATURE <222> (16) <223> Xaa is tyrosine, glutamic acid, tryptophan, threonine, or aspartic acid, or it may not be present. <220> <221> MISC_FEATURE <222> (twenty three) <223> Xaa is glycine or alanine. <220> <221> MISC_FEATURE <222> (twenty four) <223> Xaa is alanine or phenylalanine. <220> <221> MISC_FEATURE <222> (25) <223> Xaa may be alanine, phenylalanine, aspartic acid, or glutamic acid, or it may not exist. <220> <221> MISC_FEATURE <222> (27) <223> Xaa is threonine, or it may not exist. <220> <221> MISC_FEATURE <222> (28) <223> Xaa may be proline, glutamic acid, or aspartic acid, or it may not be present. <400> 120 Phe Val Asn Gln His Leu Cys Xaa Ser His Leu Val Glu Ala Leu Xaa 1 5 10 15 Leu Val Cys Gly Glu Arg Xaa Xaa Xaa Tyr Xaa Xaa Lys Thr 20 25 30 <210> 121 <211> twenty one <212> PRT <213> Homo sapiens <400> 121 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> 122 <211> 30 <212> PRT <213> Homo sapiens [[ID=!9]]<400> 122 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 <210> 123 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> Analogue 1 <400> 123 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtgcgat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 124 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 1 <400> 124 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Ala Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 125 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 2 <400> 125 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcgc ggtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 126 <211> 86 <212> PRT <213> Synthetic sequence <220> <223> Analogue 2 <400> 126 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ala Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 127 <211> 258 <212> DNA <213> Synthetic sequence <220> <223> Analogue 3 <400> 127 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaacg cgtgcaac 258 <210> 128 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Analogue 3 <400> 128 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Ala Cys Asn 85 <210> 129 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 4 <400> 129 ttcgttaacc aacacttgtg tgcgtcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 130 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 4 <400> 130 Phe Val Asn Gln His Leu Cys Ala Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 131 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 5 <400> 131 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgagcgt tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 132 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 5 <400> 132 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Ala Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 133 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> Analogue 6 <400> 133 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggcg cgttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 134 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analog 6 <400> 134 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Ala Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 135 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analog 7 <400> 135 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcgcgtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 136 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Analogue 7 <400> 136 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 137 <211> 261 <212> DNA <213> Artificial sequence <220> <223> Analogue 8 <400> 137 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaacag 240 ctggagaact actgcaactg a 261 <210> 138 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 8 <400> 138 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 139 <211> 261 <212> DNA <213> Artificial sequence <220> <223> Analogue 9 <400> 139 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaaccag 240 ctggagaact actgcaactg a 261 <210> 140 ...<211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 9 <400> 140 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Asn Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 141 <211> 258 <212> DNA <213> Synthetic sequence <220> <223> Analogue 10 <X00> 141 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tctacacacc caagacccgc cgggaggcag aggacctgca ggtggggcag 120 gtggagctgg gcgggggccc tggtgcaggc agcctgcagc ccttggccct ggaggggtcc 180 ctgcagaagc gtggcattgt ggaacaatgc tgtaccagca tctgctccct cgaacagctg 240 gagaactact gcaactga 258 <210> 142 <211> 85 <212> PRT <213> Artificial sequence <220> <223> Analog 10 <400> 142 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu 20 25 30 Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly 35 40 45 Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg 50 55 60 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu 65 70 75 80 Glu Asn Tyr Cys Asn 85 <210> 143 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analog 11 <400> 143 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgagct agtgtgcggg 60 gaacgaggct tctacacacc caagacccgc cgggaggcag aggacctgca ggtggggcag 120 gtggagctgg gcgggggccc tggtgcaggc agcctgcagc ccttggccct ggaggggtcc 180 ctgcagaagc gtggcattgt ggaacaatgc tgtaccagca tctgctccct cgcccagctg 240 gagaactact gcaactga 258 <210> 144 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> Analogue 11 <400> 144 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu 20 25 30 Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly 35 40 45 Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg 50 55 60 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Ala Gln Leu 65 70 75 80 Glu Asn Tyr Cys Asn 85 <210> 145 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 12 <400> 145 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctccatcag 240 ctggagaact actgcaac 258 <210> 146 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 12 <400> 146 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu His Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 147 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 13 <400> 147 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaagcag 240 ctggagaact actgcaac 258 <210> 148 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 13 <400> 148 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Lys Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 149 <211> 258​​​​​​​​​​​​​​​​​tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaacg agtgcaac 258 <210> 150 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analog 14 <400> 150 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Glu Cys Asn 85 <210> 151 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analog 15 <400> 151 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact cctgcaac 258 <210> 152 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Analogue 15 <400> 152 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Ser Cys Asn 85 <210> 153 <211> 258 <212> DNA <213> The snowstorm <220> <223> 16. Sections <400> 153 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 120. tcttctacac acccaagacc cgccggggagg cagaggacct gcaggtgggg caggtggagc tggggcgggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 240. tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag ctggagaaca cctgcaac <210> 154 <211> 86 <212> PRT <213> The snowstorm <220> <223> 16. Sections <400> 154 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Thr Cys Asn 85 <210> 155 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 17 <400> 155 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgagct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 156 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 17 <400> 156 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 157 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 18 <400> 157 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctccct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 158 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analog 18 <400> 158 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ser 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 159 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analog 19 <400> 159 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcaccct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 160 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Analogue 19 <400> 160 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Thr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 161 <211> 258 <212> DNA <213> The snowstorm <220> <223> 20. Section <400> 161 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 120. tcttctacac acccaagacc cgccggggagg cagaggacct gcaggtgggg caggtggagc tggggcgggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgcccag ctggagaact actgcaac <210> 162 <211> 86 <212> PRT <213> The snowstorm <220> <223> 20. Section <400> 162 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Ala Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 163 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analogue 21 <400> 163 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaccag 240 ctggagaact actgcaac 258 <210> 164 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analogue 21 <400> 164 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Asp Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 165 <211> 258 <212> DNA <213> Synthetic Sequence <220> <223> Analogue 22 <400> 165 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgacct agtgtgcggg 60 gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 166 <211> 86 <212> PRT <213> Artificial sequence <220> <223> Analog 22 <400> 166 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Asp 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85 <210> 167 <211> 258 <212> DNA <213> Artificial sequence <220> <223> Analog 23 <400> 167 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60 gaacgaggct tcgactacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120 caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240 ctggagaact actgcaac 258 <210> 168 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Analogue 23 <400> 168 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Asp Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 Leu Glu Asn Tyr Cys Asn 85 <210> 169 <211> 258 <212> DNA <213> The snowstorm <220> <223> 24. Section <400> 169 ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 120. gacgaggct tcgagtacac acccaagacc cgccggggagg cagaggacct gcaggtgggg caggtggagc tggggcgggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180 240. tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag ctggagaact actgcaac <210> 170 <211> 86 <212> PRT <213> The snowstorm <220> <223> 24. Section <400> 170 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Glu Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Asn 85

Claims

1. A pharmaceutical composition for the prevention or treatment of insulin-related diseases, said composition comprising: (i) Insulin; and (ii) Isolated peptides active against glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) receptors. The composition described herein has both a blood glucose-lowering effect and an inhibitory effect on weight gain induced by insulin administration alone. The isolated peptide, which is active against glucagon, GLP-1, and GIP receptors, comprises the amino acid sequence of SEQ ID NO:

42. The insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the insulin is conjugated with the insulin; and The isolated peptides, which are active against glucagon, GLP-1, and GIP receptors, are in the form of long-acting conjugates, wherein biocompatible substances capable of increasing the in vivo half-life of the isolated peptides are conjugated with the isolated peptides. The insulin-related diseases mentioned above are selected from the group consisting of insulin resistance, diabetes, hyperglycemia, and obesity.

2. The composition of claim 1, wherein the composition is administered to a subject who requires insulin administration.

3. The composition according to claim 1 or 2, wherein the composition comprises: Both long-acting conjugates of insulin and long-acting conjugates of isolated peptides active against glucagon, GLP-1 and GIP receptors; and The conjugate of said insulin and the long-acting conjugate of said isolated peptides active against glucagon, GLP-1 and GIP receptors are contained in a molar ratio of 1:1 to 100:

1.

4. The composition according to claim 1 or 2, wherein the composition comprises: Both long-acting conjugates of insulin and long-acting conjugates of isolated peptides active against glucagon, GLP-1 and GIP receptors; and The conjugate contains insulin in a molar ratio of 1:1 to 1:100 and the long-acting conjugate contains isolated peptides active against glucagon, GLP-1 and GIP receptors.

5. The composition according to claim 1, wherein in the amino acid sequence of SEQ ID NO: 42, the amino acids at positions 16 and 20 from the N-terminus form a ring with each other.

6. The composition of claim 1, wherein the C-terminus of the isolated peptide active against glucagon, GLP-1 and GIP receptors is amidated.

7. The composition of claim 1, wherein the insulin is natural insulin or an insulin analog, wherein the insulin analog comprises: The A-chain with altered SEQ ID NO: 121; And the B-chain of SEQ ID NO: 122, the alteration being made by one of the following composition: i) Replace an amino acid selected from the 8th, 23rd, 24th and 25th amino acids in the B-chain and the 1st, 2nd and 19th amino acids in the A-chain with alanine; ii) Replace the 14th amino acid in the A-chain with glutamic acid or asparagine; iii) The 25th amino acid in the B-chain is deleted and the 14th amino acid in the A-chain is replaced with glutamic acid; iv) Replacing the 16th amino acid in the B-chain with glutamic acid, deleting the 25th amino acid in the B-chain, and replacing the 14th amino acid in the A-chain with alanine; v) Replace the 16th amino acid in the B-chain with glutamic acid, serine, threonine, or aspartic acid; vi) Replace the 25th amino acid in the B-chain with aspartic acid or glutamic acid; vii) Replace the 14th amino acid in the A-chain with histidine, lysine, alanine, or aspartic acid; and viii) Replace the 19th amino acid in the A-chain with glutamic acid, serine, or threonine.

8. The composition of claim 7, wherein the insulin analog consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 124, 126, 128, 130, 132, 134, 136, 138 and 140.

9. The composition according to claim 7, wherein the insulin analog consists of the amino acid sequence of SEQ ID NO: 142 or 144.

10. The composition of claim 7, wherein the insulin analog comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 and 170.

11. The composition of claim 7, wherein the insulin analog is in the form of two polypeptide chains consisting of an A-chain and a B-chain.

12. The composition of claim 11, wherein the A-chain and the B-chain are connected by disulfide bonds.

13. The composition according to claim 1, wherein the conjugate is represented by chemical formula 1: Chemical Formula 1 X-L a -F, in, X is the insulin or the isolated peptide that is active against glucagon, GLP-1 and GIP receptors; L stands for connector; a is 0 or a natural number, provided that each L is independent of the others when a is 2 or greater; F is a substance that can increase the half-life of X; and "-" indicates a covalent or non-covalent bond.

14. The composition of claim 13, wherein F is selected from the group consisting of: polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, carbohydrates, heparin, and elastin.

15. The composition of claim 14, wherein the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

16. The composition according to claim 13, wherein F is the Fc region of an immunoglobulin.

17. The composition according to claim 16, wherein F is the IgG Fc region.

18. The composition of claim 17, wherein the immunoglobulin Fc region is glycosylated.

19. The composition of claim 16, wherein the immunoglobulin Fc region is selected from the group consisting of: (a) CH1 domain, CH2 domain, CH3 domain and CH4 domain; (b) CH1 and CH2 domains; (c) CH1 and CH3 domains; (d) CH2 and CH3 domains; (e) At least one of the CH1, CH2, CH3, and CH4 domains combined with an immunoglobulin hinge region or a portion thereof; and (f) Dimers of each domain in the heavy chain constant region and the light chain constant region.

20. The composition of claim 16, wherein the immunoglobulin Fc region has the following defects: loss of a disulfide bond-forming site, loss of some amino acids at the N-terminus of the native Fc, addition of a methionine residue at the N-terminus of the native Fc, loss of a complement-binding site, or loss of an antibody-dependent cell-mediated cytotoxicity (ADCC) site.

21. The composition of claim 16, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

22. The composition of claim 16, wherein the immunoglobulin Fc region is a mixture of domains having different origins from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

23. The composition of claim 13, wherein L is selected from the group consisting of peptides, fatty acids, sugars, polymers, low molecular weight compounds, nucleotides, and combinations thereof.

24. The composition of claim 23, wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

25. The composition according to claim 13, wherein L is polyethylene glycol.

26. A kit for the prevention or treatment of insulin-related diseases, the kit comprising: (i) Insulin; and (ii) Isolated peptides active against glucagon, GLP-1, and GIP receptors, The aforementioned kit has both a blood glucose-lowering effect and an inhibitory effect on weight gain induced by insulin administration alone. The isolated peptide, which is active against glucagon, GLP-1, and GIP receptors, comprises the amino acid sequence of SEQ ID NO:

42. The insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the insulin is conjugated with the insulin; and The isolated peptides, which are active against glucagon, GLP-1, and GIP receptors, are in the form of long-acting conjugates, wherein biocompatible substances capable of increasing the in vivo half-life of the isolated peptides are conjugated with the isolated peptides. The insulin-related diseases mentioned above are selected from the group consisting of insulin resistance, diabetes, hyperglycemia, and obesity.

27. A pharmaceutical composition for reducing weight gain caused by insulin administration, said composition comprising: (i) Insulin; and (ii) Isolated peptides active against glucagon, GLP-1, and GIP receptors, The composition described herein has both a blood glucose-lowering effect and an inhibitory effect on weight gain induced by insulin administration alone. The isolated peptide, which is active against glucagon, GLP-1, and GIP receptors, comprises the amino acid sequence of SEQ ID NO:

42. The insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the insulin is conjugated with the insulin; and The isolated peptides, which are active against glucagon, GLP-1 and GIP receptors, are in the form of long-acting conjugates, wherein biocompatible substances capable of increasing the in vivo half-life of the isolated peptides are conjugated with the isolated peptides.

28. A combination formulation for weight loss in patients administering insulin, said combination formulation comprising: (i) Insulin; and (ii) Isolated peptides active against glucagon, GLP-1, and GIP receptors, The formulation described herein has both a blood glucose-lowering effect and an inhibitory effect on weight gain induced by insulin administration alone. The isolated peptide, which is active against glucagon, GLP-1, and GIP receptors, comprises the amino acid sequence of SEQ ID NO:

42. The insulin is in the form of a long-acting conjugate, wherein a biocompatible substance capable of increasing the in vivo half-life of the insulin is conjugated with the insulin; and The isolated peptides, which are active against glucagon, GLP-1 and GIP receptors, are in the form of long-acting conjugates, wherein biocompatible substances capable of increasing the in vivo half-life of the isolated peptides are conjugated with the isolated peptides.

29. The compound formulation according to claim 28, wherein the formulation comprises: Both long-acting conjugates of insulin and long-acting conjugates of isolated peptides active against glucagon, GLP-1 and GIP receptors; and The conjugate contains insulin and isolated peptides active against glucagon, GLP-1 and GIP receptors in a molar ratio of 1:1 to 100:

1.

30. The compound formulation according to claim 28, wherein the formulation comprises: Both long-acting conjugates of insulin and long-acting conjugates of isolated peptides active against glucagon, GLP-1 and GIP receptors; and The conjugate contains insulin and the isolated peptide active against GLP-1 and GIP receptors in a molar ratio of 1:1 to 1:100.

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