Pharmaceutical compositions comprising insulin and triple agonists active at both glucagon and GLP-1 and GIP receptors
阅读说明：本技术 包括胰岛素和对胰高血糖素和glp-1和gip受体均具有活性的三重激动剂的药物组合物 (Pharmaceutical compositions comprising insulin and triple agonists active at both glucagon and GLP-1 and GIP receptors ) 是由 李钟锡 金正国 李相炖 李钟守 金尚允 于 2019-12-23 设计创作，主要内容包括：本发明涉及包括胰岛素和对胰高血糖素受体、GLP-1受体和GIP受体均具有活性的三重激动剂的组合物,以及包括该组合物的复合制剂。(The present invention relates to a composition comprising insulin and a triple agonist active at glucagon receptor, GLP-1 receptor and GIP receptor, and a complex formulation comprising the same.)
1. A pharmaceutical composition for preventing or treating an insulin-related disease, the composition comprising:
(i) insulin; and
(ii) isolated peptides having activity at glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) receptors.
2. The composition of claim 1, wherein 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 to the insulin.
3. The composition of claim 1, wherein the isolated peptide active at 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.
4. The composition of claim 1, wherein 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 to the insulin; and is
Wherein the isolated peptide active at 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.
5. The composition of claim 1, wherein the composition is administered to a subject in need of insulin administration.
6. The composition of claim 1, wherein the insulin-related disease is selected from the group consisting of an insulin resistance disorder, diabetes, hyperglycemia, and obesity.
7. The composition according to any one of claims 1 to 6, wherein the composition has both a blood glucose lowering effect and an effect of inhibiting body weight gain caused by insulin administration alone.
8. The composition according to any one of claims 1 to 6, wherein the composition comprises: insulin or a long-acting conjugate thereof; and an isolated peptide or long-acting conjugate thereof having activity at glucagon, GLP-1 and GIP receptors; and is
Wherein said insulin or said conjugate thereof and said isolated peptide active on glucagon, GLP-1 and GIP receptors or said long acting conjugate thereof are contained in a molar ratio of 1:1 to 100: 1.
9. The composition according to any one of claims 1 to 6, wherein the composition comprises: insulin or a long-acting conjugate thereof; and an isolated peptide or long-acting conjugate thereof having activity at glucagon, GLP-1 and GIP receptors; and is
Wherein said insulin or said conjugate thereof and said isolated peptide active on glucagon, GLP-1 and GIP receptors or said long-acting conjugate thereof are contained in a molar ratio of 1:1 to 1: 100.
10. The composition of any one of claims 1 to 6, wherein the isolated peptide having activity at glucagon, GLP-1 and GIP receptors is an analog of native glucagon with alterations selected from the group consisting of: substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in the sequence of said native glucagon.
11. The composition according to claim 10, wherein the amino acid sequence to be added is derived from the amino acid sequence of native GLP-1, native GIP or native exenatide-4.
12. The composition of claim 1, wherein the isolated peptide active at glucagon, GLP-1 and GIP receptors comprises an amino acid sequence represented by the following general formula 1:
xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (general formula 1, SEQ ID NO:103),
wherein, in the general formula 1,
xaa1 is histidine (His, H), 4-imidazoleacetyl (CA) or tyrosine (Tyr, Y);
xaa2 is glycine (Gly, G), α -methyl-glutamic acid, or aminoisobutyric acid (Aib);
xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);
xaa7 is threonine (Thr, T) or isoleucine (Ile, I);
xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or valine (Val, V);
xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);
xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine (Cys, C);
xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);
xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E) or leucine (Leu, L);
xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);
xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);
xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);
xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C) or valine (Val, V);
xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);
xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);
xaa23 is isoleucine (Ile, I) or valine (Val, V);
XXaa24 is alanine (Ala, a), glutamine (gin, Q), cysteine (Cys, C), asparagine (Asn, N), aspartic acid (Asp, D) or glutamic acid (Glu, E);
xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);
xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N) or aspartic acid (Asp, D);
xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);
xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H) or absent; and
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 absent, where m is Cys, Pro or Gly-Pro and n is Cys, Gly, Ser or His-Gly or absent.
13. The composition of claim 12, wherein Xaa14 is leucine or methionine; and Xaa15 is cysteine, aspartic acid or leucine.
14. The composition according to claim 12, wherein in formula 1,
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa7 is a threonine and Xaa7 is,
xaa10 is tyrosine, cysteine, or valine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, glutamine or cysteine;
xaa14 is leucine, cysteine, or methionine;
xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;
xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, valine, or cysteine;
xaa20 is lysine, arginine, or glutamine;
xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and
xaa27 is leucine or lysine.
15. The composition of claim 12, wherein the peptide comprises an amino acid sequence represented by the following general formula 2:
xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO:104),
wherein, in the general formula 2,
xaa1 is 4-imidazoleacetyl, histidine or tyrosine;
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa10 is tyrosine or cysteine;
xaa13 is alanine, glutamine, tyrosine, or cysteine;
xaa14 is leucine, methionine, or tyrosine;
xaa15 is aspartic acid, glutamic acid, or leucine;
xaa16 is glycine, glutamic acid, or serine;
xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, cysteine, or valine;
xaa20 is lysine, glutamine or arginine;
xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;
xaa28 is lysine, cysteine, asparagine, or aspartic acid;
xaa29 is glycine, glutamine, cysteine, or histidine;
xaa30 is cysteine, glycine, lysine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
16. The composition according to claim 12, wherein in formula 1,
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa7 is threonine;
xaa10 is tyrosine, cysteine, or valine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, or cysteine;
xaa14 is leucine or methionine;
xaa15 is cysteine or aspartic acid;
xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;
xaa18 is alanine, arginine, or histidine;
xaa19 is alanine, glutamine or cysteine;
xaa20 is lysine or glutamine;
xaa21 is glutamic acid, cysteine, or aspartic acid;
xaa23 is valine;
xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and Xaa27 is leucine or lysine.
17. The composition according to claim 15, wherein in formula 2,
xaa13 is alanine, tyrosine, or cysteine;
xaa15 is aspartic acid or glutamic acid;
xaa17 is glutamine, arginine, cysteine, or lysine;
xaa18 is alanine, arginine, or histidine;
xaa21 is cysteine, glutamic acid, glutamine or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, glutamine or asparagine;
xaa28 is cysteine, asparagine, or aspartic acid;
xaa29 is glutamine, cysteine, or histidine; and
xaa30 is cysteine, lysine or histidine.
18. The composition according to claim 12, wherein in formula 1,
xaa2 is α -methyl-glutamic acid or Aib;
xaa7 is threonine;
xaa10 is tyrosine or cysteine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, or cysteine;
xaa14 is leucine or methionine;
xaa15 is cysteine or aspartic acid;
xaa16 is glutamic acid;
xaa17 is arginine, isoleucine, cysteine or lysine;
xaa18 is alanine, arginine, or histidine;
xaa19 is alanine, glutamine or cysteine;
xaa20 is lysine or glutamine;
xaa21 is glutamic acid or aspartic acid;
xaa23 is valine;
xaa24 is glutamine, asparagine, or aspartic acid;
xaa27 is leucine; and
xaa28 is cysteine, alanine, asparagine, or aspartic acid.
19. The composition according to claim 12, wherein in formula 1,
xaa1 is histidine or 4-imidazoleacetyl;
xaa2 is α -methyl-glutamic acid or Aib;
xaa3 is glutamine;
xaa7 is threonine;
xaa10 is tyrosine;
xaa12 is isoleucine;
xaa13 is alanine or cysteine;
xaa14 is methionine;
xaa15 is aspartic acid;
xaa16 is glutamic acid;
xaa17 is isoleucine or lysine;
xaa18 is alanine or histidine;
xaa19 is glutamine or cysteine;
xaa20 is lysine;
xaa21 is aspartic acid;
xaa23 is valine;
xaa24 is asparagine;
xaa27 is leucine;
xaa28 is alanine or asparagine;
xaa29 is glutamine or threonine; and
xaa30 is cysteine or lysine or absent.
20. The composition of claim 11, wherein the peptide comprises an amino acid sequence represented by the following general formula 3:
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-Xaa 31-Ser-Ser-Gly-Gln-Pro-Pro-Ser-Xaa 40 (general formula 3, SEQ ID NO:105),
wherein, in the general formula 3,
xaa1 is histidine or tyrosine;
xaa2 is α -methyl-glutamic acid or Aib;
xaa13 is alanine, tyrosine, or cysteine;
xaa17 is arginine, cysteine, or lysine;
xaa18 is alanine or arginine;
xaa19 is alanine or cysteine;
xaa21 is glutamic acid or aspartic acid;
xaa24 is glutamine or asparagine;
xaa28 is cysteine or aspartic acid;
xaa29 is cysteine, histidine or glutamine;
xaa30 is cysteine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
21. The composition of claim 12, wherein 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 absent.
22. The composition of claim 12, wherein the isolated peptide active on glucagon, GLP-1 and GIP receptors comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 102.
23. The composition of claim 12, wherein the isolated peptide active on glucagon, GLP-1 and GIP receptors comprises the amino acid sequence set forth in SEQ ID No. 42.
24. The composition according to any one of claims 12 to 21, wherein in the general formula, amino acids at positions 16 and 20 from the N-terminus form a ring with each other.
25. The composition of any one of claims 12-21, wherein the C-terminal of the isolated peptide active on glucagon, GLP-1 and GIP receptors is amidated.
26. The composition of claim 1, wherein the insulin is a native insulin, or an altered insulin analog selected from the group consisting of: substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in the native insulin.
27. The composition of claim 26, wherein the insulin analogue has an alteration 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 B-chain of the native insulin and amino acids at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19 and 21 of the A-chain of the native insulin, the alteration being a substitution, deletion or combination thereof with another amino acid.
28. The composition of claim 226, wherein the insulin analog comprises: the A-chain of SEQ ID NO 119 represented by the general formula 4; and a B-chain of SEQ ID NO:120 represented by the general formula 5:
general formula 4
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)，
Wherein, in the general formula 4,
xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;
xaa2 is alanine or isoleucine;
xaa 5is alanine, glutamic acid, glutamine, histidine, or asparagine;
xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine;
xaa14 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid, or asparagine;
xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine;
xaa19 is alanine, tyrosine, serine, glutamic acid, histidine, threonine, or asparagine; and
xaa21 is asparagine, glycine, histidine or alanine,
general formula 5
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)
Wherein, in the general formula 5,
xaa8 is alanine or glycine;
xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or is absent;
xaa23 is glycine or alanine;
xaa24 is alanine or phenylalanine;
xaa25 is alanine, phenylalanine, aspartic acid, or glutamic acid, or is absent;
xaa27 is threonine or absent; and
xaa28 is proline, glutamic acid or aspartic acid or is absent
(wherein the peptide comprising the A-chain of SEQ ID NO:121 and the B-chain of SEQ ID NO:122 is not included).
29. The composition of claim 26, wherein the insulin analogue has a substitution of at least one amino acid selected from the group consisting of amino acids at positions 8, 23, 24 and 25 in the natural insulin B-chain and amino acids at positions 1, 2 and 19 in the natural insulin a-chain with alanine, or a substitution of an amino acid at position 14 in the natural insulin a-chain with glutamic acid or asparagine.
30. The composition of claim 29, wherein the insulin analogue contains an amino acid sequence selected from the group consisting of SEQ ID NOs 124, 126, 128, 130, 132, 134, 136, 138 and 140.
31. The composition of claim 26, wherein the insulin analog has an alteration by: substituting glutamic acid for the amino acid at position 16 in the B-chain of said native insulin; a deletion of amino acid 25 in the B-chain of native insulin; substituting glutamic acid or alanine for the amino acid at position 14 in the natural insulin A-chain; or a combination thereof.
32. The composition of claim 31, wherein the insulin analog contains the amino acid sequence of SEQ ID NO:142 or 144.
33. The composition of claim 26, wherein the insulin analog has an alteration by: substituting glutamic acid, serine, threonine or aspartic acid for the amino acid at position 16 in the natural insulin B-chain; substituting aspartic acid or glutamic acid for the amino acid at position 25 in the B-chain of native insulin; substituting histidine, lysine, alanine or aspartic acid for the amino acid at position 14 in the natural insulin a-chain; substituting glutamic acid, serine or threonine for the amino acid at position 19 in the a-chain of said native insulin; or a combination thereof.
34. The composition of claim 33, wherein the insulin analogue contains an amino acid sequence selected from the group consisting of: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 and 170.
35. The composition of claim 28, wherein the insulin analogue is in the form of two polypeptide chains consisting of the a-chain of SEQ ID No. 119 represented by general formula 4 and the B-chain of SEQ ID No. 120 represented by general formula 5.
36. The composition of claim 35, wherein the a-chain and the B-chain are linked by a disulfide bond.
37. The composition according to any one of claims 2 to 4, wherein the conjugate is represented by chemical formula 1:
chemical formula 1
X-La-F，
Wherein the content of the first and second substances,
x is the insulin or the isolated peptide active on glucagon, GLP-1 and GIP receptors;
l is a linker;
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
f is a substance capable of increasing the half-life of X; and
"-" is a covalent or non-covalent bond.
38. The composition of claim 37, wherein F is selected from 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.
39. The composition of claim 38, wherein the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.
40. The composition of claim 37, wherein F is an immunoglobulin Fc region.
41. The composition of claim 40, wherein F is an IgG Fc region.
42. The composition of claim 41, wherein the immunoglobulin Fc region is aglycosylated.
43. The composition of claim 40, wherein the immunoglobulin Fc region is selected from the group consisting of:
(a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain;
(b) a CH1 domain and a CH2 domain;
(c) a CH1 domain and a CH3 domain;
(d) a CH2 domain and a CH3 domain;
(e) a combination of at least one of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region or portion of a hinge region; and
(f) a dimer of each domain of the heavy and light chain constant regions.
44. The composition of claim 40, wherein the immunoglobulin Fc region has a deletion of a site capable of forming a disulfide bond, a deletion of some amino acids from the N-terminus of a native Fc, an addition of a methionine residue from the N-terminus of a native Fc, a deletion of a complement binding site, or a deletion of an antibody-dependent cell-mediated cytotoxicity (ADCC) site.
45. The composition of claim 40, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.
46. The composition of claim 40, 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.
47. The composition of claim 37, wherein L is selected from the group consisting of peptides, fatty acids, sugars, polymers, low molecular weight compounds, nucleotides, and combinations thereof.
48. The composition of claim 47, wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.
49. The composition of claim 37, wherein L is polyethylene glycol.
50. A kit for preventing or treating an insulin-related disorder, the kit comprising:
(i) insulin; and
(ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
51. A pharmaceutical composition for reducing weight gain resulting from insulin administration, said composition comprising:
(i) insulin; and
(ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
52. The composition of claim 51, wherein 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 to the insulin.
53. The composition of claim 51, wherein the isolated peptide active at 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.
54. The composition of claim 51, wherein 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 to the insulin; and is
Wherein the isolated peptide active at 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.
55. A complex formulation for weight loss in an insulin-administered patient, the complex formulation comprising:
(i) insulin; and
(ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
56. The complex formulation of claim 55, wherein 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 to the insulin.
57. The co-formulation of claim 55, wherein the isolated peptide active at 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.
58. The complex formulation of claim 55, wherein 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 to the insulin; and is
Wherein the isolated peptide active at 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.
59. The complex formulation of any one of claims 55-58, wherein said composition comprises: insulin or a long-acting conjugate thereof; and an isolated peptide or long-acting conjugate thereof having activity at glucagon, GLP-1 and GIP receptors; and is
Wherein said insulin and said isolated peptide active on glucagon, GLP-1 and GIP receptors are contained in a molar ratio of 1:1 to 100: 1.
60. The complex formulation of any one of claims 55-58, wherein said composition comprises: insulin or a long-acting conjugate thereof; and an isolated peptide or long-acting conjugate thereof having activity at glucagon, GLP-1 and GIP receptors; and is
Wherein said insulin or said conjugate thereof and said isolated peptide active on GLP-1 receptor and GIP receptor or said conjugate thereof are contained in a molar ratio of 1:1 to 1: 100.
Technical Field
The present invention relates to compositions comprising insulin and a triple agonist active at glucagon, GLP-1 and GIP receptors and to complex formulations comprising the same.
Background
Insulin is a blood glucose controlling hormone secreted by the human pancreas and used to transport excess glucose in the blood to cells, thereby supplying the cells with an energy source and maintaining blood glucose levels within a normal range. However, in diabetic patients, insulin does not function properly due to insulin deficiency, insulin resistance and loss of β -cell function. As a result, diabetic patients cannot utilize glucose in blood as an energy source and exhibit hyperglycemia symptoms with high blood glucose levels, resulting in excretion of glucose in urine, which causes various complications. Therefore, diabetic patients with insulin production abnormalities (type I) or insulin resistance (type II) require insulin therapy, and blood glucose levels can be adjusted to the normal range by insulin administration.
In recent years, 25% or more of type I diabetes mellitus cases and 80% or more of type II diabetes mellitus cases have high blood sugar levels and are obese. It is known that insulin administered alone, although having an excellent blood sugar control effect, causes a side effect of weight gain. Therefore, there is a need to develop a drug capable of effectively reducing the side effect of weight gain of insulin while exhibiting the blood glucose lowering effect of insulin.
The present applicant has developed various insulin analogues that retain insulin activity (WO 2014/133324a1 and WO2017/039267) and has developed triple agonists that are active at both glucagon, GLP-1 and GIP receptors (WO 2017/116204 and WO 2017/116205). However, these have not been used in combination as a combined preparation.
Disclosure of Invention
Technical problem
In the present application, it was confirmed that the combined use of insulin and a triple agonist shows significantly superior glycemic control ability and effectively reduces the weight gain caused by insulin, as compared to the administration of insulin or a triple agonist alone.
Technical scheme
It is an object of the present invention to provide a composition comprising: (i) insulin; and (ii) an isolated peptide having activity at glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) receptors.
Specifically, an object of the present invention is to provide a pharmaceutical composition for preventing or treating insulin-related diseases, the composition comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
It is another object of the present invention to provide a pharmaceutical composition for reducing weight gain caused by insulin administration: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
It is another object of the present invention to provide a complex formulation for weight loss in insulin-administered patients, which comprises: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
It is another object of the present invention to provide a kit (kit) for preventing or treating insulin-related diseases, the kit comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
It is a further object of the present invention to provide the use of said composition in the preparation of a medicinal material.
It is a further object of the present invention to provide a method for preventing or treating an insulin-related disease, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
It is a further object of the present invention to provide a method for reducing weight gain resulting from insulin administration comprising administering to a subject an isolated peptide having activity at glucagon, GLP-1 and GIP receptors.
In particular, it is a further object of the present invention to provide a method for reducing weight gain caused by insulin administration, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
Advantageous effects
The composition or complex formulation of the present invention comprising insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof provides a novel combination therapy capable of not only exhibiting a preventive or therapeutic effect on insulin-related diseases such as diabetes, but also reducing side effects such as weight gain caused by insulin administration.
Drawings
Figures 1 and 2 show the effect of glycemic control and weight change, respectively, in type II diabetes model mice due to the combined administration of long-acting conjugates of insulin and of triple agonists.
Detailed Description
One aspect of the present invention relates to a composition comprising: (i) insulin; and (ii) an isolated peptide having activity at glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) receptors.
In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating an insulin-related disease, the composition comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
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) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
In the composition according to any one of the preceding embodiments, 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 to the insulin.
In the composition according to any one of the preceding embodiments, the isolated peptide having activity at 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.
In the composition according to any one of the preceding embodiments, 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 to the insulin; and
isolated peptides having activity at glucagon, GLP-1 and GIP receptors are in the form of long-acting conjugates, wherein a biocompatible substance capable of increasing the in vivo half-life of the isolated peptide is conjugated to the isolated peptide.
In the composition according to any one of the preceding embodiments, the composition is administered to a subject in need of insulin administration.
In the composition according to any one of the preceding embodiments, the insulin-related disease is selected from the group consisting of insulin resistance disorders, diabetes, hyperglycemia, and obesity.
In the composition according to any one of the preceding embodiments, the composition has both a blood glucose lowering effect and an effect of inhibiting body weight gain caused by insulin administration alone.
In a composition according to any one of the preceding embodiments, the composition comprises: insulin or a long-acting conjugate thereof; and isolated peptides or long-acting conjugates thereof having activity at glucagon, GLP-1 and GIP receptors,
wherein insulin or a conjugate thereof and an isolated peptide having activity at glucagon, GLP-1 and GIP receptors or a long-acting conjugate thereof are contained in a molar ratio of 1:1 to 100:1, or wherein insulin or a conjugate thereof and an isolated peptide having activity at glucagon, GLP-1 and GIP receptors or a long-acting conjugate thereof are contained in a molar ratio of 1:1 to 1: 100.
In the composition according to any one of the preceding embodiments, the isolated peptide having activity at glucagon, GLP-1 and GIP receptors is an analog of native glucagon with alterations selected from the group consisting of: substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in the native glucagon sequence.
In the composition according to any of the preceding embodiments, the amino acid sequence to be added is derived from the amino acid sequence of native GLP-1, native GIP or native exenatide-4.
In a composition according to any of the preceding embodiments, an isolated peptide having activity at glucagon, GLP-1 and GIP receptors comprises an amino acid sequence represented by the following general formula 1:
xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (general formula 1, SEQ ID NO:103),
wherein, in the general formula 1,
xaa1 is histidine (His, H), 4-imidazoleacetyl (CA) or tyrosine (Tyr, Y);
xaa2 is glycine (Gly, G), α -methyl-glutamic acid, or aminoisobutyric acid (Aib);
xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);
xaa7 is threonine (Thr, T) or isoleucine (Ile, I);
xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or valine (Val, V);
xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);
xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine (Cys, C);
xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);
xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E) or leucine (Leu, L);
xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);
xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);
xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H);
xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C) or valine (Val, V);
xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);
xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);
xaa23 is isoleucine (Ile, I) or valine (Val, V);
XXaa24 is alanine (Ala, a), glutamine (gin, Q), cysteine (Cys, C), asparagine (Asn, N), aspartic acid (Asp, D) or glutamic acid (Glu, E);
xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met, M);
xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N) or aspartic acid (Asp, D);
xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);
xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H) or absent; and
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 absent,
wherein m is Cys, Pro or Gly-Pro, and n is Cys, Gly, Ser or His-Gly or absent.
In the composition according to any of the preceding embodiments, Xaa14 is leucine or methionine; and Xaa15 is cysteine, aspartic acid or leucine.
In the composition according to any one of the preceding embodiments, in formula 1,
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa7 is a threonine and Xaa7 is,
xaa10 is tyrosine, cysteine, or valine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, glutamine or cysteine;
xaa14 is leucine, cysteine, or methionine;
xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;
xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, valine, or cysteine;
xaa20 is lysine, arginine, or glutamine;
xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and
xaa27 is leucine or lysine.
In the composition according to any one of the preceding embodiments, the peptide comprises an amino acid sequence represented by the following general formula 2:
xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO:104),
wherein, in the general formula 2,
xaa1 is 4-imidazoleacetyl, histidine or tyrosine;
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa10 is tyrosine or cysteine;
xaa13 is alanine, glutamine, tyrosine, or cysteine;
xaa14 is leucine, methionine, or tyrosine;
xaa15 is aspartic acid, glutamic acid, or leucine;
xaa16 is glycine, glutamic acid, or serine;
xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, cysteine, or valine;
xaa20 is lysine, glutamine or arginine;
xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;
xaa28 is lysine, cysteine, asparagine, or aspartic acid;
xaa29 is glycine, glutamine, cysteine, or histidine;
xaa30 is cysteine, glycine, lysine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
In the composition according to any one of the preceding embodiments, in formula 1,
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa7 is threonine;
xaa10 is tyrosine, cysteine, or valine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, or cysteine;
xaa14 is leucine or methionine;
xaa15 is cysteine or aspartic acid;
xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;
xaa18 is alanine, arginine, or histidine;
xaa19 is alanine, glutamine or cysteine;
xaa20 is lysine or glutamine;
xaa21 is glutamic acid, cysteine, or aspartic acid;
xaa23 is valine;
xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and
xaa27 is leucine or lysine.
In the composition according to any one of the preceding embodiments, in formula 2,
xaa13 is alanine, tyrosine, or cysteine;
xaa15 is aspartic acid or glutamic acid;
xaa17 is glutamine, arginine, cysteine, or lysine;
xaa18 is alanine, arginine, or histidine;
xaa21 is cysteine, glutamic acid, glutamine or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, glutamine or asparagine;
xaa28 is cysteine, asparagine, or aspartic acid;
xaa29 is glutamine, cysteine, or histidine; and
xaa30 is cysteine, lysine or histidine.
In the composition according to any one of the preceding embodiments, in formula 1,
xaa2 is α -methyl-glutamic acid or Aib;
xaa7 is threonine;
xaa10 is tyrosine or cysteine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, or cysteine;
xaa14 is leucine or methionine;
xaa15 is cysteine or aspartic acid;
xaa16 is glutamic acid;
xaa17 is arginine, isoleucine, cysteine or lysine;
xaa18 is alanine, arginine, or histidine;
xaa19 is alanine, glutamine or cysteine;
xaa20 is lysine or glutamine;
xaa21 is glutamic acid or aspartic acid;
xaa23 is valine;
xaa24 is glutamine, asparagine, or aspartic acid;
xaa27 is leucine; and
xaa28 is cysteine, alanine, asparagine, or aspartic acid.
In the composition according to any one of the preceding embodiments, in formula 1,
xaa1 is histidine or 4-imidazoleacetyl;
xaa2 is α -methyl-glutamic acid or Aib;
xaa3 is glutamine;
xaa7 is threonine;
xaa10 is tyrosine;
xaa12 is isoleucine;
xaa13 is alanine or cysteine;
xaa14 is methionine;
xaa15 is aspartic acid;
xaa16 is glutamic acid;
xaa17 is isoleucine or lysine;
xaa18 is alanine or histidine;
xaa19 is glutamine or cysteine;
xaa20 is lysine;
xaa21 is aspartic acid;
xaa23 is valine;
xaa24 is asparagine;
xaa27 is leucine;
xaa28 is alanine or asparagine;
xaa29 is glutamine or threonine; and
xaa30 is cysteine or lysine or absent.
In the composition according to any one of the preceding embodiments, the peptide comprises an amino acid sequence represented by the following general formula 3:
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-Xaa 31-Ser-Ser-Gly-Gln-Pro-Pro-Ser-Xaa 40 (general formula 3, SEQ ID NO:105),
wherein, in the general formula 3,
xaa1 is histidine or tyrosine;
xaa2 is α -methyl-glutamic acid or Aib;
xaa13 is alanine, tyrosine, or cysteine;
xaa17 is arginine, cysteine, or lysine;
xaa18 is alanine or arginine;
xaa19 is alanine or cysteine;
xaa21 is glutamic acid or aspartic acid;
xaa24 is glutamine or asparagine;
xaa28 is cysteine or aspartic acid;
xaa29 is cysteine, histidine or glutamine;
xaa30 is cysteine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
In a composition according to any one of the preceding 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 absent.
In the composition according to any of the preceding embodiments, the isolated peptide having activity at glucagon, GLP-1 and GIP receptors comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 102.
In the composition according to any of the preceding embodiments, the isolated peptide having activity at glucagon, GLP-1 and GIP receptors comprises the amino acid sequence shown in SEQ ID NO: 42.
In the composition according to any one of the preceding embodiments, in the general formula, the amino acids at positions 16 and 20 from the N-terminus form a ring with each other.
In the composition according to any of the preceding embodiments, the C-terminal of the isolated peptide active on glucagon, GLP-1 and GIP receptors is amidated.
In the composition according to any one of the preceding embodiments, the insulin is a native insulin, or an altered insulin analog having a sequence selected from the group consisting of: substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in native insulin.
In the composition according to any one of the preceding embodiments, the insulin analogue has an alteration 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, the alteration being a substitution, deletion or combination thereof with another amino acid.
In a composition according to any one of the preceding embodiments, the insulin analogue comprises: the A-chain of SEQ ID NO 119 represented by the general formula 4; and a B-chain of SEQ ID NO:120 represented by the general formula 5:
general formula 4
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)，
Wherein, in the general formula 4,
xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;
xaa2 is alanine or isoleucine;
xaa 5is alanine, glutamic acid, glutamine, histidine, or asparagine;
xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine;
xaa14 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid, or asparagine;
xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine;
xaa19 is alanine, tyrosine, serine, glutamic acid, histidine, threonine, or asparagine; and
xaa21 is asparagine, glycine, histidine or alanine,
general formula 5
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)，
Wherein, in the general formula 5,
xaa8 is alanine or glycine;
xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or is absent;
xaa23 is glycine or alanine;
xaa24 is alanine or phenylalanine;
xaa25 is alanine, phenylalanine, aspartic acid, or glutamic acid, or is absent;
xaa27 is threonine or absent; and
xaa28 is proline, glutamic acid or aspartic acid or is absent
(wherein peptides comprising the A-chain of SEQ ID NO:121 and the B-chain of SEQ ID NO:122 are not included).
In the composition according to any one of the preceding embodiments, the insulin analogue has a substitution of at least one amino acid selected from the group consisting of amino acids at positions 8, 23, 24 and 25 in the natural insulin B-chain and amino acids at positions 1, 2 and 19 in the natural insulin a-chain with alanine, or a substitution of the amino acid at position 14 in the natural insulin a-chain with glutamic acid or asparagine.
In a composition according to any one of the preceding embodiments, the insulin analogue contains an amino acid sequence selected from the group consisting of SEQ ID NOs 124, 126, 128, 130, 132, 134, 136, 138 and 140.
In a composition according to any one of the preceding embodiments, the insulin analogue has an alteration by: glutamic acid for the 16 th amino acid in the natural insulin B-chain; a deletion of amino acid 25 in the B-chain of native insulin; substituting glutamic acid or alanine for amino acid at position 14 in the natural insulin A-chain; or a combination thereof.
In a composition according to any one of the preceding embodiments, the insulin analogue contains the amino acid sequence of SEQ ID NO:142 or 144.
In a composition according to any one of the preceding embodiments, the insulin analogue has an alteration by: substitution of glutamic acid, serine, threonine or aspartic acid for the amino acid at position 16 of the natural insulin B-chain; substituting aspartic acid or glutamic acid for the amino acid at position 25 in the B-chain of native insulin; substituting histidine, lysine, alanine or aspartic acid for the amino acid at position 14 in the natural insulin A-chain; substitution of glutamic acid, serine or threonine for the amino acid at position 19 in the natural insulin a-chain; or a combination thereof.
In a composition according to any one of the preceding embodiments, the insulin analogue contains an amino acid sequence selected from the group consisting of: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 and 170.
In a composition according to any one of the preceding embodiments, the insulin analogue is in the form of two polypeptide chains consisting of the A-chain of SEQ ID NO 119 represented by general formula 4 and the B-chain of SEQ ID NO 120 represented by general formula 5.
In the composition according to any one of the preceding embodiments, the a-chains and B-chains are linked by disulfide bonds.
In the composition according to any one of the preceding embodiments, the conjugate is represented by chemical formula 1:
chemical formula 1
X-La-F，
Wherein the content of the first and second substances,
x is insulin or an isolated peptide active at glucagon, GLP-1 and GIP receptors;
l is a linker;
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
f is a substance capable of increasing the half-life of X; and
"-" is a covalent or non-covalent bond.
In the composition according to any one of the preceding 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 specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, connective tissue in vivo, nucleotides, fibronectin, transferrin, carbohydrates, heparin, and elastin.
In the composition according to any of the preceding embodiments, the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.
In the composition according to any one of the preceding embodiments, F is an immunoglobulin Fc region.
In the composition according to any one of the preceding embodiments, F is an IgG Fc region.
In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region is aglycosylated.
In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region is selected from the group consisting of:
(a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain;
(b) a CH1 domain and a CH2 domain;
(c) a CH1 domain and a CH3 domain;
(d) a CH2 domain and a CH3 domain;
(e) a combination of at least one of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region or portion of a hinge region; and
(f) a dimer of each domain of the heavy and light chain constant regions.
In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region has a deletion of a site capable of forming a disulfide bond, a deletion of some amino acids at the N-terminus of the native Fc, an addition of a methionine residue at the N-terminus of the native Fc, a deletion of a complement binding site, or a deletion of an antibody-dependent cell-mediated cytotoxicity (ADCC) site.
In the composition according to any one of the preceding embodiments, the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM.
In the composition according to any one of the preceding embodiments, 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.
In the composition according to any one of the preceding embodiments, L is selected from the group consisting of peptides, fatty acids, sugars, polymers, low molecular weight compounds, nucleotides, and combinations thereof.
In the composition according to any of the preceding embodiments, the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides and combinations thereof.
In the composition according to any one of the preceding embodiments, L is polyethylene glycol.
One aspect of the present invention relates to a complex formulation for weight loss in a patient to whom insulin is administered, the complex formulation comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
One aspect of the present invention relates to a kit for preventing or treating an insulin-related disease, the kit comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
One aspect of the present invention relates to the use of said composition for the preparation of a medicinal material.
One aspect of the present invention relates to a method for preventing or treating an insulin-related disease, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
One aspect of the invention relates to a method for reducing weight gain resulting from insulin administration, the method comprising administering to a subject an isolated peptide having activity at glucagon, GLP-1 and GIP receptors.
One aspect of the invention relates to a method for reducing weight gain resulting from insulin administration, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
Modes for carrying out the invention
Hereinafter, the present invention will be described in detail.
Each description and embodiment disclosed in the present disclosure may also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure are within the scope of this disclosure. Furthermore, the scope of the present disclosure is not limited by the following detailed description.
Further, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the present invention.
Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as other three-letter codes commonly permitted for amino acids, such as α -aminoisobutyric acid (Aib), N-methylglycine (Sar), and α -methyl-glutamic acid. The amino acids mentioned in the abbreviations herein are described according to the IUPAC-IUB rules:
alanine (Ala, A), arginine (Arg, R)
Asparagine (Asn, N), aspartic acid (Asp, D)
Cysteine (Cys, C), glutamic acid (Glu, E)
Glutamine (Gln, Q), glycine (Gly, G)
Histidine (His, H), isoleucine (Ile, I)
Leucine (Leu, L), lysine (Lys, K)
Methionine (Met, M), phenylalanine (Phe, F)
Proline (Pro, P), serine (Ser, S)
Threonine (Thr, T), tryptophan (Trp, W)
Tyrosine (Tyr, Y), valine (Val, V)
One aspect of the present invention provides a composition comprising: (i) insulin; and (ii) an isolated peptide (also referred to as a "triple agonist") having activity at glucagon, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) receptors.
In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating an insulin-related disease, the composition comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
In another embodiment, the present invention provides a pharmaceutical composition for reducing weight loss resulting from insulin administration, the composition comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
In particular, the composition may comprise:
(a) insulin; and isolated peptides active at glucagon, GLP-1 and GIP receptors;
(b) a long-acting conjugate of insulin, wherein insulin and a biocompatible substance capable of increasing the half-life in insulin bodies are conjugated to each other; and isolated peptides active at glucagon, GLP-1 and GIP receptors;
(c) insulin; and a long-acting conjugate of a triple agonist, wherein an isolated peptide having activity to glucagon, GLP-1 and GIP receptors and a biocompatible substance capable of increasing the in vivo half-life of the isolated peptide are conjugated to each other; or
(d) Long-acting conjugates of insulin; and long-acting conjugates of triple agonists.
The pharmaceutical composition for preventing or treating insulin-related diseases of the present invention comprises: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein the composition can reduce side effects (e.g., weight gain) caused by insulin administration while exhibiting the medicinal effects of insulin, and has the prophylactic or therapeutic effects on insulin-related diseases due to insulin deficiency, insufficiency or dysfunction.
The exemplary embodiments of the present invention demonstrate the blood glucose lowering effect and the body weight gain inhibiting effect in db/db mice administered with 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 drug efficacy of insulin.
As used herein, the term "insulin-related disease" refers to a disease caused by abnormal glycemic control function inherent to insulin, such as insulin deficiency, insufficiency, or dysfunction, and any disease in which a prophylactic or therapeutic effect by administration of insulin is desired is included in the scope of the present invention. In particular, the insulin-related disease may be selected from the group consisting of insulin resistance disorders, diabetes, hyperglycemia, and obesity.
Administration of insulin for the treatment of insulin-related disorders can achieve the desired therapeutic effect but can cause unexpected side effects, such as weight gain, which can lead to other types of disorders and patient distress. The pharmaceutical composition according to the present invention can exhibit a therapeutic effect on insulin-related diseases by using insulin and a triple agonist in combination while inhibiting body weight gain as a side effect of insulin.
The composition of the present invention may contain insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof at a content ratio that can exhibit a therapeutic effect on insulin-related diseases and can reduce side effects, particularly weight gain.
In particular, 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 the triple agonist in a molar ratio of 1:1 to 100:1 or 1:1 to 1:100, but is not limited thereto.
In one embodiment of the invention, the therapeutic use of the composition may be provided by the combined use of insulin or a conjugate thereof and a substance or conjugate thereof active at glucagon, GLP-1 and GIP receptors. The term "insulin" is as described above.
The term "composition" is used interchangeably herein with "combination" and contains insulin or a conjugate thereof and a substance active at glucagon, GLP-1 and GIP receptors. The composition may be provided in the form of a kit.
As used herein, "combination" has the use of administering insulin or a conjugate thereof in combination with a substance or conjugate thereof that is active at glucagon, GLP-1 and GIP receptors, and the term may be understood as synonymous with "combined use". The composition may be administered in the form of:
a) in the form of a mixture in which (i) insulin or a conjugate thereof and (ii) a substance active on glucagon, GLP-1 and GIP receptors or a conjugate thereof are mixed; or
b) In the form of isolating (i) insulin or a conjugate thereof and (ii) a substance or conjugate thereof active on glucagon, GLP-1 and GIP receptors, but is not limited thereto.
When the composition is in the form of an isolated insulin or conjugate thereof and a substance or conjugate thereof having activity at glucagon, GLP-1 and GIP receptors, the insulin or conjugate thereof and the substance or conjugate thereof having activity at glucagon, GLP-1 and GIP receptors may be prepared as separate formulations and administered simultaneously, separately, sequentially or in reverse order.
In the present invention, combined administration is understood to mean not only simultaneous administration, but also administration forms in which insulin or a conjugate thereof and a substance active on glucagon, GLP-1 and GIP receptors or a conjugate thereof act together on a subject such that each acts at a level equal to or higher than its original function. Thus, the use of the term "combination" is understood to mean simultaneous, separate, sequential or reverse administration. When administration is sequential, reverse or separate, the order of administration is not particularly limited, but delaying administration of the second component should not result in loss of the beneficial effect of the combined use.
In the present invention, the term "composition" refers to a combination itself, which contains insulin or a conjugate thereof and a substance having activity to glucagon, GLP-1 and GIP receptors or a conjugate thereof, or may be a composition containing a combination and having a therapeutic use, but is not limited thereto. Examples thereof may be a composition having a prophylactic or therapeutic use for insulin-related diseases, but are not limited thereto.
The compositions according to the invention are used for combined administration: insulin or a conjugate thereof; and a substance having activity to glucagon, GLP-1 and GIP receptors or a conjugate thereof, wherein insulin or a conjugate thereof and the substance having activity to glucagon, GLP-1 and GIP receptors or a conjugate thereof may be prepared as one preparation or separately. In particular, insulin or a conjugate thereof and a substance having activity to glucagon, GLP-1 and GIP receptors or a conjugate thereof may be administered simultaneously, separately, sequentially or in reverse order, but not limited thereto.
In the present invention, the term "kit" may contain a combination or composition according to the invention for the combined administration of insulin or a conjugate thereof and a substance active on glucagon, GLP-1 and GIP receptors or a conjugate thereof. In particular, the kit according to the invention may contain: a formulation prepared from insulin or a conjugate thereof and a substance or conjugate thereof having activity at glucagon, GLP-1 and GIP receptors; or a separate preparation prepared from insulin or a conjugate thereof and a substance having activity to glucagon, GLP-1 and GIP receptors or a conjugate thereof, and the kit may further contain substances necessary for combined administration of the two substances, but is not limited thereto.
Insulin and substances active at the glucagon, GLP-1 and GIP receptors include insulin and various substances, such as compounds or peptides, having significant levels of activity at the glucagon, GLP-1 and GIP receptors.
In the present invention, "peptides active at glucagon, GLP-1 and GIP receptors" may be used interchangeably with triple agonists. As to triple agonists and long-acting conjugates thereof, the entire disclosures of international patent publication nos. WO 2017/116204 and WO 2017/116205 are incorporated herein by reference.
Examples of triple agonists include various substances, such as various peptides, having significant levels of activity at glucagon, GLP-1 and GIP receptors.
For a substance having a significant level of activity at the glucagon, GLP-1 and GIP receptors, although not particularly limited, the substance has an in vitro activity of 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 100% or more, for the corresponding receptor, as compared to the native ligand (native glucagon, native GLP-1 and native GIP), and more particularly all three.
The method of measuring the in vitro activity of these triple agonists can refer to test example 1 herein, but is not particularly limited thereto.
At the same time, the triple agonists are characterized by having one or more, two or more, in particular three, of the following activities i) to iii), in particular having significant levels of activity:
i) activation of the GLP-1 receptor; ii) activation of the glucagon receptor; and iii) activation of the GIP receptor.
Activation of the receptor may comprise, 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 on the receptor as compared to the native form. However, the activation is not limited thereto.
The triple agonist may have an increased half-life in vivo as compared to any one of native GLP-1, native glucagon, and native GIP, but is not particularly limited thereto.
Although not particularly limited, these peptides may be non-naturally occurring peptides.
Specifically, the triple agonist may be an analog of native glucagon, but is not particularly limited thereto.
The native glucagon analogues according to the present invention may comprise peptides having at least one difference in amino acid sequence compared to native glucagon, peptides obtained by modification of the native glucagon sequence, and mimetics of native glucagon.
Meanwhile, although not particularly limited, native glucagon may have the following amino acid sequence:
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)
in particular, the triple agonist may be an altered native glucagon analog having an alteration selected from the group consisting of a substitution, addition, deletion, modification of at least one amino acid in the native glucagon sequence, and combinations thereof, but is not particularly limited thereto.
The substitution of the amino acid may include substitution with an amino acid and substitution with a non-natural compound.
Addition may be at the N-terminus and/or C-terminus of the triple agonist. The length of the amino acid to be added is not particularly limited, but 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 or more amino acids may be added, and in a broad sense, the addition may include the addition of a polypeptide, but is not particularly limited thereto.
The amino acid to be introduced into native glucagon may 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 is not particularly limited thereto.
For example, the amino acid sequence to be added may be at least one amino acid sequence derived from the amino acid sequence of native GLP-1, native GIP, or native exenatide-4.
Such a triple agonist may comprise an intramolecular bridge (e.g., a covalent bridge or a non-covalent bridge), and particularly, the triple agonist is in the form of a ring-containing form, for example, a ring-forming form between amino acids at positions 16 and 20 in the triple agonist, but is not particularly limited thereto.
Non-limiting examples of rings may include a lactam bridge (or lactam ring).
Further, examples of the triple agonist include all triple agonists modified to contain an amino acid capable of forming a loop at a target position so as to include the loop.
For example, in the triple agonist, a pair of amino acids at positions 16 and 25 may be substituted with glutamic acid or lysine, respectively, which can form a ring, but is not limited thereto.
The ring may be formed between amino acid side chains within the 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.
Examples of triple agonists prepared by combining these methods include: a peptide different from native glucagon in at least one amino acid sequence, in which the α -carbon of the N-terminal amino acid residue is removed, and which has activity on glucagon, GLP-1, GIP receptor and the like, but is not limited thereto, and the triple agonist to be applied to the present invention can be prepared by a combination of various methods of analog preparation.
Although not particularly limited, 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 substituted with other amino acids or unnatural compounds so as to avoid recognition by agonist-degrading enzymes.
Specifically, the triple agonist of the present invention may be a peptide having an increased half-life in vivo by avoiding recognition by a degrading enzyme by substituting the second amino acid sequence in the amino acid sequence of the triple agonist, but includes, but is not limited to, any amino acid substitution or modification for avoiding recognition by an in vivo degrading enzyme.
Such modifications for preparing native glucagon analogs include: changes using amino acids in the L or D form and/or unnatural amino acids; and/or all changes made by modifying the sequence of the native form, for example, changing side chain functionalities, intramolecular covalent bonds such as ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminocaproylation, biotinylation, and the like.
Furthermore, such alterations also include all additions of one or more amino acids at the amino and/or carboxy terminus of native glucagon.
Examples of the amino acid to be substituted or added may include not only 20 amino acids commonly found in human proteins but also atypical amino acids or non-naturally occurring amino acids. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPerp, and Genzyme Pharmaceuticals. Peptide sequences and typical peptides containing these amino acids can be synthesized by and purchased from commercial suppliers, such as American Peptide Company and Bachem in the United states, or Antigen in Korea.
Amino acid derivatives can also be obtained in a similar manner, and for example, 4-imidazoleacetic acid and the like can be used.
The peptides active at the glucagon, GLP-1 and GIP receptors according to the invention may be in the form of: the N-terminal and/or C-terminal is chemically modified or protected by an organic group or an amino acid is added to the terminal of a peptide or the like to prevent the influence of a protein cleaving enzyme in vivo and to increase stability.
In particular, the chemically synthesized peptide has charged N and C terminals, and thus the N terminal may be acetylated and/or the C terminal may be amidated to eliminate these charges, but these are not particularly limited thereto. Further, a form in which the C-terminal is not changed, that is, a form having a carboxyl group is also included.
The peptides having activity at glucagon, GLP-1 and GIP receptors according to the present invention, i.e. triple agonists, include all of the peptide itself, salts thereof (e.g. pharmaceutically acceptable salts of the peptide), or solvates thereof. Peptides active at the glucagon, GLP-1 and GIP receptors can be in the form of any pharmaceutically acceptable salt.
The kind of the salt is not particularly limited. However, the salt is preferably in a form safe and effective for a subject such as a mammal, but is not particularly limited thereto.
The term "pharmaceutically acceptable" refers to materials that are, within the scope of pharmaceutical decision, effective for the intended use without causing undue toxicity, irritation, allergic response, and the like.
As used herein, "pharmaceutically acceptable salts" include salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Suitable examples of acids may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Examples of salts derived from suitable bases may include: alkali metals such as sodium and potassium; alkaline earth metals, such as magnesium; ammonium; and the like.
As used herein, the term "solvate" refers to a complex formed between a peptide or salt thereof according to the present invention and a solvent molecule.
Obviously, all the above description applies to insulin described later.
In a particular aspect, peptides active at the glucagon, GLP-1 and GIP receptors may comprise an amino acid sequence represented by the following general formula 1:
xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (general formula 1, SEQ ID NO:103),
wherein, in the general formula 1,
xaa1 is histidine, 4-imidazole acetyl or tyrosine;
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa3 is glutamic acid or glutamine;
xaa7 is threonine or isoleucine;
xaa10 is leucine, tyrosine, lysine, cysteine, or valine;
xaa12 is lysine, serine, or isoleucine;
xaa13 is glutamine, tyrosine, alanine, or cysteine;
xaa14 is leucine, methionine, or tyrosine;
xaa15 is cysteine, aspartic acid, glutamic acid, or leucine;
xaa16 is glycine, glutamic acid, or serine;
xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, cysteine, or valine;
xaa20 is lysine, glutamine or arginine;
xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;
xaa23 is isoleucine or valine;
XXaa24 is alanine, glutamine, cysteine, asparagine, aspartic acid or glutamic acid;
xaa27 is valine, leucine, lysine, or methionine;
xaa28 is cysteine, lysine, alanine, asparagine, or aspartic acid;
xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine;
xaa30 is cysteine, glycine, lysine or histidine or absent; and
r1 is cysteine, GKKNDWKHNIT (SEQ ID NO:106), m-SSGAPPPS-n (SEQ ID NO:107) or m-SSGQPPPS-n (SEQ ID NO:108) or absent,
wherein m is Cys, Pro or Gly-Pro, and
n is Cys, Gly, Ser or His-Gly or absent.
Examples of the triple agonist may include a triple agonist comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 102, or a triple agonist (substantially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 102, but are not limited thereto.
Although described herein as "a peptide consisting of a specific SEQ ID NO", the addition of a nonsense sequence, a naturally occurring mutation and a silent mutation thereof upstream or downstream of the amino acid sequence of the corresponding sequence number is not excluded as long as there is an activity equivalent to or corresponding to the peptide consisting of the amino acid sequence of the corresponding sequence number, and it is apparent that a peptide having such sequence addition or mutation is also within the scope of the present invention.
The above description may be applied to other embodiments or aspects of the invention, but is not limited thereto.
Specifically, in the above formula 1, Xaa14 can be leucine or methionine; and Xaa15 can be cysteine, aspartic acid, or leucine. In this case, the remaining variables other than Xaa14 and Xaa15 described in formula 1 can have combinations of the above amino acids.
Examples of such peptides may be, but are not limited to, peptides comprising or (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 12, 14 to 17, and 21 to 102.
These peptides can significantly activate at least one of glucagon, GLP-1 and GIP receptors, but are not particularly limited thereto. Specifically, the peptide may significantly activate the GLP-1 receptor, or may further significantly activate the glucagon and/or GIP receptor, but is not particularly limited thereto.
Still more particularly, in still yet another aspect,
in the general formula 1, the compound represented by the formula,
xaa2 can be glycine, alpha-methyl-glutamic acid, or Aib;
xaa7 can be a threonine,
xaa10 can be tyrosine, cysteine, or valine;
xaa12 can be lysine or isoleucine;
xaa13 can be tyrosine, alanine, glutamine, or cysteine;
xaa14 can be leucine, cysteine, or methionine;
xaa15 can be cysteine, leucine, glutamic acid, or aspartic acid;
xaa17 can be glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine;
xaa18 can be alanine, glutamine, arginine, or histidine;
xaa19 can be alanine, glutamine, valine, or cysteine;
xaa20 can be lysine, arginine, or glutamine;
xaa21 can be glutamic acid, glutamine, leucine, cysteine, or aspartic acid;
xaa23 can be isoleucine or valine;
xaa24 can be cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid; and
xaa27 can be leucine or lysine, but the variables are not particularly limited thereto.
In addition to the variables described in this aspect, the remaining variables of formula 1 can have combinations of the amino acids described above.
Still more particularly, in still yet another aspect,
in the general formula 1, the compound represented by the formula,
xaa2 can be glycine, alpha-methyl-glutamic acid, or Aib;
xaa7 can be a threonine,
xaa10 can be tyrosine, cysteine, or valine;
xaa12 can be lysine or isoleucine;
xaa13 can be tyrosine, alanine, or cysteine;
xaa14 can be leucine or methionine;
xaa15 can be cysteine or aspartic acid;
xaa17 can be glutamine, arginine, isoleucine, cysteine, or lysine;
xaa18 can be alanine, arginine, or histidine;
xaa19 can be alanine, glutamine, or cysteine;
xaa20 can be lysine or glutamine;
xaa21 can be glutamic acid, cysteine, or aspartic acid;
xaa23 can be valine;
xaa24 can be alanine, glutamine, cysteine, asparagine, or aspartic acid; and
xaa27 can be leucine or lysine, but the variables are not particularly limited thereto.
In addition to the variables described in this aspect, the remaining variables of formula 1 can have combinations of the amino acids described above.
Still more particularly, in still yet another aspect,
in the general formula 1, the compound represented by the formula,
xaa2 is α -methyl-glutamic acid or Aib;
xaa7 is a threonine and Xaa7 is,
xaa10 is tyrosine or cysteine;
xaa12 is lysine or isoleucine;
xaa13 is tyrosine, alanine, or cysteine;
xaa14 is leucine or methionine;
xaa15 is cysteine or aspartic acid;
xaa16 is glutamic acid;
xaa17 is arginine, isoleucine, cysteine or lysine;
xaa18 is alanine, arginine, or histidine;
xaa19 is alanine, glutamine or cysteine;
xaa20 is lysine or glutamine;
xaa21 is glutamic acid or aspartic acid;
xaa23 is valine;
xaa24 is glutamine, asparagine, or aspartic acid;
xaa27 is leucine; and
xaa28 is cysteine, alanine, asparagine, or aspartic acid.
In addition to the variables described in this aspect, the remaining variables of formula 1 can have combinations of the amino acids described above.
In particular, the amount of the solvent to be used,
in the general formula 1, the compound represented by the formula,
xaa1 can be histidine or 4-imidazoleacetyl
Xaa2 can be α -methyl-glutamic acid or Aib;
xaa3 can be glutamine;
xaa7 can be a threonine,
xaa10 can be tyrosine;
xaa12 can be isoleucine;
xaa13 can be alanine or cysteine;
xaa14 can be methionine;
xaa15 can be aspartic acid;
xaa16 can be glutamic acid;
xaa17 can be isoleucine or lysine;
xaa18 can be alanine or histidine;
xaa19 can be glutamine or cysteine;
xaa20 can be lysine;
xaa21 can be aspartic acid;
xaa23 can be valine;
xaa24 can be asparagine;
xaa27 can be leucine;
xaa28 can be alanine or asparagine;
xaa29 can be glutamine or threonine; and
xaa30 may be cysteine or lysine, or may be absent.
In addition to the variables described in this aspect, the remaining variables of formula 1 can have combinations of the amino acids described above.
More specifically, the present invention is to provide a novel,
in the general formula 1, the compound represented by the formula,
xaa2 can be glycine, alpha-methyl-glutamic acid, or Aib;
xaa3 can be glutamine;
xaa7 can be a threonine,
xaa10 can be tyrosine, cysteine, or valine;
xaa12 can be lysine;
xaa13 can be tyrosine;
xaa14 can be leucine;
xaa15 can be aspartic acid;
xaa16 can be glycine, glutamic acid, or serine;
xaa17 can be glutamine, arginine, cysteine, or lysine;
xaa18 can be alanine, arginine, or histidine;
xaa19 can be alanine or glutamine;
xaa20 can be lysine or glutamine;
xaa21 can be glutamic acid, cysteine, or aspartic acid;
xaa23 can be valine;
xaa24 can be alanine, glutamine, or cysteine;
xaa27 can be leucine or lysine; and
xaa29 can be glycine, glutamine, threonine, or histidine, but the variables are not particularly limited thereto.
In addition to the variables described in this aspect, the remaining variables of formula 1 can have combinations of the amino acids described above.
These peptides active at the glucagon, GLP-1 and GIP receptors may correspond to: peptides having significant and higher activity at the GLP-1 and glucagon receptors than at the GIP receptor; peptides with significant activity at GLP-1, glucagon, and GIP receptors; and peptides having significant and higher activity at the GLP-1 and GIP receptors than at the glucagon receptor, but are not limited thereto.
Peptides having significant activity at the GLP-1 and glucagon receptors and higher than the GIP receptor provide greater weight loss and glycemic control, and peptides having significant activity at all of the GLP-1, glucagon, and GIP receptors provide the greatest weight loss. However, the peptide is not limited thereto.
Examples of these peptides may include, but are not particularly limited to, peptides comprising or (substantially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs 8, 9, 21 to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88, 89, 91 to 93, and 95 to 102.
In a particular aspect, peptides active at the glucagon, GLP-1 and GIP receptors may comprise an amino acid sequence represented by the following general formula 2:
xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO:104),
wherein, in the general formula 2,
xaa1 is 4-imidazoleacetyl, histidine or tyrosine;
xaa2 is glycine, α -methyl-glutamic acid, or Aib;
xaa10 is tyrosine or cysteine;
xaa13 is alanine, glutamine, tyrosine, or cysteine;
xaa14 is leucine, methionine, or tyrosine;
xaa15 is aspartic acid, glutamic acid, or leucine;
xaa16 is glycine, glutamic acid, or serine;
xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine;
xaa18 is alanine, glutamine, arginine, or histidine;
xaa19 is alanine, glutamine, cysteine, or valine;
xaa20 is lysine, glutamine or arginine;
xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;
xaa23 is isoleucine or valine;
xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;
xaa28 is lysine, cysteine, asparagine, or aspartic acid;
xaa29 is glycine, glutamine, cysteine, or histidine;
xaa30 is cysteine, glycine, lysine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
More specifically, in the general formula 2,
xaa13 can be alanine, tyrosine, or cysteine;
xaa15 can be aspartic acid or glutamic acid;
xaa17 can be glutamine, arginine, cysteine, or lysine;
xaa18 can be alanine, arginine, or histidine;
xaa21 can be cysteine, glutamic acid, glutamine or aspartic acid;
xaa23 can be isoleucine or valine;
xaa24 can be cysteine, glutamine or asparagine;
xaa28 can be cysteine, asparagine, or aspartic acid;
xaa29 can be glutamine, cysteine, or histidine; and
xaa30 can be cysteine, lysine or histidine.
In addition to the variables described in this aspect, the remaining variables of formula 2 can have combinations of the amino acids described above.
Examples of peptides having activity at the glucagon, GLP-1 and GIP receptors may be peptides comprising or (consisting essentially of) a sequence selected from the group consisting of SEQ ID NO: 21. 22, 42, 43, 50, 64 to 77, and 95 to 102, and more specifically SEQ ID NO: 21. 22, 42, 43, 50, 64 to 77, and 96 to 102, but is not particularly limited thereto.
In a particular aspect, peptides active at the glucagon, GLP-1 and GIP receptors may comprise an amino acid sequence represented by the following general formula 3:
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-Xaa 31-Ser-Ser-Gly-Gln-Pro-Pro-Ser-Xaa 40 (general formula 3, SEQ ID NO:105),
wherein, in the general formula 3,
xaa1 is histidine or tyrosine;
xaa2 is α -methyl-glutamic acid or Aib;
xaa13 is alanine, tyrosine, or cysteine;
xaa17 is arginine, cysteine, or lysine;
xaa18 is alanine or arginine;
xaa19 is alanine or cysteine;
xaa21 is glutamic acid or aspartic acid;
xaa24 is glutamine or asparagine;
xaa28 is cysteine or aspartic acid;
xaa29 is cysteine, histidine or glutamine;
xaa30 is cysteine or histidine;
xaa31 is proline or cysteine; and
xaa40 is cysteine or absent.
Examples of peptides having activity at the glucagon, GLP-1 and GIP receptors may be peptides comprising or (consisting essentially of) a sequence 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 is not particularly limited thereto.
In general formula 1, R1 may be cysteine, GKKNDWKHNIT (SEQ ID NO:106), CSSGQPPPS (SEQ ID NO:109), GPSSGAPPPS (SEQ ID NO:110), GPSSGAPPPSC (SEQ ID NO:111), PSSGAPPPS (SEQ ID NO:112), PSSGAPPPSG (SEQ ID NO:113), PSSGAPPPSHG (SEQ ID NO:114), PSSGAPPPSS (SEQ ID NO:115), PSSGQPPPS (SEQ ID NO:116), or PSSGQPPPSC (SEQ ID NO:117), or may be absent, but is not particularly limited thereto.
In addition, the peptide of the present invention can be synthesized by a method known in the art according to its length, such as an automated peptide synthesizer, and can be produced using genetic engineering techniques.
In particular, the peptides of the invention may be prepared by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the peptides according to the invention can be synthesized by a variety of methods, including, for example, the following methods:
(a) methods for synthesizing peptides and isolating and purifying the final peptide product by solid or liquid phase methods or stepwise or assembly from fragments;
(b) methods of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture;
(c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product therefrom; or
A method of obtaining a peptide fragment by any combination of the methods (a), (b) and (c), obtaining a peptide by linking the fragments, and then recovering the peptide.
As used herein, the term "insulin" refers to a hormone secreted in pancreatic beta cells and generally controls blood glucose in vivo by promoting intracellular glucose uptake and inhibiting lipolysis. The proinsulin precursor without blood sugar control function is processed into insulin with blood sugar control function. Insulin is composed of two polypeptide chains, the a-chain and the B-chain, which contain 21 and 30 amino acids, respectively, and are connected to each other by two disulfide bridges. The insulin may 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.
A-chain:
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)
b-chain:
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)
the term "proinsulin" as used herein refers to a precursor molecule of insulin. Proinsulin can contain the insulin A-chain and B-chain, and the C-peptide between them. The proinsulin can be human proinsulin.
In the present invention, the insulin may be a native insulin, or an analog, derivative or fragment thereof, with alterations selected from the group consisting of: substitution, addition, deletion, modification and combination thereof of at least one amino acid in the natural insulin, but not limited thereto.
As used herein, the term "insulin analog" refers to a non-natural insulin that is different from a natural insulin.
Examples of insulin analogues include analogues obtained by altering some of the amino acids of native insulin by addition, deletion or substitution. For example, an insulin analogue may be an insulin analogue in which at least one amino acid selected from the group consisting of the amino acids at positions 1, 2, 3, 5, 8, 10, 12, 16, 23, 24, 25, 26, 27, 28, 29 and 30 in the natural amino acid B-chain and the amino acids at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19 and 21 in the natural amino acid a-chain is substituted or deleted with another amino acid, but is not limited thereto. The insulin analogues of the present invention can be referred to the disclosures of korean patent publication No. 10-2014-0106452 or 10-2017-0026284 (or WO 2014/133324a1 or WO2017/039267), the respective specifications of which are incorporated herein by reference in their entirety.
The insulin analogues to be used in the present 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 can be composed of two polypeptides-a polypeptide corresponding to the native insulin a-chain and a polypeptide corresponding to the native insulin B-chain. When any one of the two polypeptide chains is compared with the a-chain or B-chain of native insulin in terms of sequence identity, expression of the a-chain or B-chain corresponding to native 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 can be easily 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 native insulin.
As used herein, the term "homology" is intended to mean a degree of similarity to the amino acid sequence of a wild-type protein or a nucleotide sequence encoding the same, and encompasses sequences having at least the above-described percentage level of sequence identity to the amino acid sequence or nucleotide sequence of the present invention. Homology can be determined by visual comparison of two given sequences, or can be determined using bioinformatics algorithms whereby the degree of homology is analyzed by aligning the subject sequences for comparison. The homology between two given amino acid sequences can be expressed as a percentage. Useful automated algorithms can be used in the GAP, BESTFIT, FASTA and TFASTA Computer software modules of the Wisconsin Genetics software package (Genetics Computer Group, Madison, Wis., USA). Automated alignment algorithms in the above modules include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Other useful algorithms for sequence alignment and homology determination are automated in software containing FASTP, BLAST2, psibllast and CLUSTAL W.
Information on the insulin sequences and the nucleotide sequences encoding them used in the present invention can be obtained from known databases, such as NCBI.
In a specific exemplary embodiment, the insulin analogues of the present invention contain: 119, represented by the following general formula 4; and a B-chain of SEQ ID NO 120 represented by the following formula 5:
general formula 4
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)，
Wherein, in the general formula 4,
xaa1 is alanine, glycine, glutamine, histidine, glutamic acid, or asparagine;
xaa2 is alanine or isoleucine;
xaa 5is alanine, glutamic acid, glutamine, histidine, or asparagine;
xaa12 is alanine, serine, glutamine, glutamic acid, histidine, or asparagine;
xaa14 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid, or asparagine;
xaa16 is alanine, leucine, tyrosine, histidine, glutamic acid, or asparagine;
xaa19 is alanine, tyrosine, serine, glutamic acid, histidine, threonine, or asparagine; and
xaa21 is asparagine, glycine, histidine or alanine,
general formula 5
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)
Wherein, in the general formula 5,
xaa8 is alanine or glycine;
xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or is absent;
xaa23 is glycine or alanine;
xaa24 is alanine or phenylalanine;
xaa25 is alanine, phenylalanine, aspartic acid, or glutamic acid, or is absent;
xaa27 is threonine or absent; and
xaa28 is proline, glutamic acid or aspartic acid or is absent
(wherein peptides comprising the A-chain of SEQ ID NO:121 and the B-chain of SEQ ID NO:122 are not included).
More specifically, the insulin analogue may have a substitution with alanine of at least one amino acid selected from the group consisting of the amino acids at positions 8, 23, 24 and 25 in the natural insulin B-chain and the amino acids at positions 1, 2 and 19 in the natural insulin a-chain, and/or a substitution with glutamic acid or asparagine of the amino acid at position 14 in the natural insulin a-chain, and in particular the insulin analogue may contain or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs 124, 126, 128, 130, 132, 134, 136, 138 and 140, but the insulin analogue is not limited thereto.
Alternatively, the insulin analogue may have a substitution of the amino acid at position 16 in the natural insulin B-chain with glutamic acid, and/or a deletion of the amino acid at position 25 in the natural insulin B-chain and/or a substitution of the amino acid at position 14 in the natural insulin a-chain with glutamic acid or alanine, and in particular the insulin analogue may contain or consist of the amino acid sequence of SEQ ID NO:142 or 144, but the insulin analogue is not limited thereto.
Alternatively, the insulin analogue may have a substitution of the amino acid at position 16 in the natural insulin B-chain with glutamic acid, serine, threonine or aspartic acid, and/or a substitution of the amino acid at position 25 in the natural insulin B-chain with aspartic acid or glutamic acid, and/or a substitution of the amino acid at position 14 in the natural insulin a-chain with histidine, lysine, alanine or aspartic acid, and/or a substitution of the amino acid at position 19 in the natural insulin a-chain with glutamic acid, serine or threonine, and in particular, the insulin analogue may contain or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 and 170, but the insulin analogue is not limited thereto.
Meanwhile, the insulin analog of the present invention may be a substance in the form of short-chain insulin without removing C-peptide, contains 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 has the activity and function of insulin.
Furthermore, insulin analogues of the invention may be prepared by removing the C-peptide from the proinsulin containing the C-peptide between the A-chain and the B-chain. Removal of the C-peptide can be performed by methods known in the art, and in particular, the insulin analog can be prepared by treatment with trypsin and carboxypeptidase B, but is not limited thereto.
In particular, the insulin analogue of the present invention may consist 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 particularly, the insulin analogue 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.
It is clear that insulin analogues shown with a specific sequence number and prepared in the form of two polypeptide chains by removing the C-peptide from the proinsulin form insulin analogues are also included in the scope of the present invention.
In particular, insulin analogues may have the following changes in the sequence of natural insulin, in particular natural human insulin, and may be selected from the following analogues 1 to 24.
Analogues
Amino acid changes
Analog 1
A1G->A
Analog 2
A2I->A
Analogue 3
A19Y->A
Analog 4
B8G->A
Analog 5
B23G->A
Analog 6
B24F->A
Analog 7
B25F->A
Analog 8
A14Y->E
Analog 9
A14Y->N
Analog 10
A14Tyr->Glu+B25Absence of
Analog 11
A14Tyr->Ala+B16Tyr->Glu.B25Absence of
Analog 12
A14Y->H
Analog 13
A14Y->K
Analog 14
A19Y->E
Analog 15
A19Y->S
Analog 16
A19Y->T
Analog 17
B16Y->E
Analogs 18
B16Y->S
Analog 19
B16Y->T
Analog 20
A14Y->A
Analog 21
A14Y->D
Analog 22
B16Y->D
Analog 23
B25F->D
Analogs 24
B25F->E
As used herein, the term "insulin derivative" includes peptides having at least one difference in amino acid sequence compared to native insulin, peptides prepared by modifying the sequence of native insulin, and native insulin mimetics that can modulate glycemic control function in vivo like native insulin. Such derivatives of native insulin may have glycemic control in vivo.
Specifically, the insulin derivative can be obtained by any one of or a combination of methods of changing substitution, addition, deletion, and modification of certain amino acids in natural insulin.
Specifically, the derivative 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 in a form in which one group of one amino acid residue of insulin is subjected to chemical substitution (e.g., α -methylation or α -hydroxylation), deletion (e.g., deamination), or modification (e.g., N-methylation), but is not limited thereto.
The natural insulin derivatives to be used in the present invention can be prepared by a combination of various methods for preparing the derivatives.
Such modifications for preparing insulin derivatives include: changes made using L-or D-form amino acids and/or unnatural amino acids; and/or by modification of the native form sequence or post-translational modification (e.g., methylation, acylation, ubiquitination, intermolecular covalent bonds, etc.).
Furthermore, such changes also include all additions of one or more amino acids at the amino and/or carboxy terminus of insulin.
The amino acids to be substituted or added may be not only the 20 kinds of amino acids commonly found in human proteins but also atypical amino acids or non-naturally occurring amino acids. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPerp, and Genzyme Pharmaceuticals. Peptide sequences and typical peptides containing these amino acids may be synthesized by and purchased from commercial suppliers, such as American Peptide Company and Bachem in the United states, or Antigen in Korea, but are not particularly limited.
As used herein, the term "fragment of natural insulin, insulin analog or derivative of insulin" refers to a form in which one or more amino acids are removed from the amino-or carboxy-terminus of the natural insulin, insulin analog or natural insulin derivative. These fragments may retain glycemic control function in vivo.
Furthermore, the insulin analogues of the present invention can be prepared by using the corresponding methods for preparing derivatives and fragments of native insulin, alone or in combination.
In particular, the insulin analogue according to the invention comprises alterations of specific amino acid residues in the a-chain and the B-chain of the natural insulin described above, and in particular the insulin analogue may have alterations of specific amino acid residues in the a-chain of the natural insulin and/or alterations of specific amino acid residues in the B-chain of the natural insulin.
The pharmaceutical composition according to the present invention may contain, as insulin, the following substances as one of the active ingredients: (a) natural insulin, (b) an insulin analog, (c) a derivative of insulin, (d) a fragment thereof, or (e) a combination thereof.
In a particular embodiment, the insulin or isolated peptide having activity at glucagon, GLP-1 and GIP receptors of the present invention 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.
As used herein, the term "long-acting conjugate" or "conjugate" of insulin or an isolated peptide active at glucagon, GLP-1 and GIP receptors has a structure in which a biocompatible substance is conjugated to insulin or an isolated peptide active at glucagon, GLP-1 and GIP receptors, and may exhibit an increased duration of efficacy compared to insulin or an isolated peptide active at glucagon, GLP-1 and GIP receptors to which the biocompatible substance is not conjugated.
The biocompatible substance in the long-acting conjugate may be linked to insulin or an isolated peptide having activity to glucagon, GLP-1 and GIP receptors through a covalent bond, but is not particularly limited thereto.
In the present invention, insulin which is one element of the conjugate may be insulin having a sequence of a native form, or may be an analog, derivative or fragment of insulin having a change of at least one amino acid in a sequence of a native form by substitution, addition, deletion, modification or a combination thereof, but any form of insulin may be used as one element of the conjugate of the present invention without limitation so long as it has a blood glucose lowering/increasing effect of native insulin.
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 on glucagon, GLP-2, and GIP receptors, insulin, etc.) to increase the duration of efficacy of the physiologically active substance as compared to a physiologically active substance that is not conjugated to a biocompatible substance moiety or carrier. The biocompatible substance may be covalently linked to the physiologically active substance, but is not particularly limited thereto.
Specifically, the conjugate is represented by chemical formula 1:
chemical formula 1
X-La-F，
Wherein the content of the first and second substances,
x is insulin, or an isolated peptide active at glucagon, GLP-1 and GIP receptors (i.e., a triple agonist);
l is a linker;
a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other; and
f is a substance capable of increasing the half-life of X.
The composition 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 triple agonist, wherein the long-acting conjugate form of insulin or a triple agonist exhibits an excellent blood glucose control effect on the basis of an increased duration of time in vivo and can alleviate the side effects of insulin.
In the conjugate, F is X, i.e. a substance capable of increasing the half-life of insulin or a triple agonist, and corresponds to one element constituting part of the conjugate of the invention.
F and X may be linked to each other by a covalent chemical bond or a non-covalent chemical bond, or F and X may be linked to each other via L by a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.
The substance capable of increasing the half-life of X may be a biocompatible substance and may be selected from, for example, the group consisting of a polymer, a fatty acid, cholesterol, albumin and a fragment thereof, an albumin binding substance, a polymer of a repeating unit of a specific amino acid sequence, an antibody fragment, an FcRn binding substance, an in vivo connective tissue, a nucleotide, fibronectin, transferrin, a saccharide, heparin, and elastin, but is not limited thereto.
The elastin may be human elastin which is a water-soluble precursor and may be a polymer of a part of its sequence or some repeating unit thereof, and examples thereof include all elastin-like polypeptides, but are not particularly limited thereto.
Examples of the high molecular polymer are high molecular polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, but are not particularly limited thereto.
Polyethylene glycol corresponds to all forms of terms including ethylene glycol homopolymer, PEG copolymer and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.
Examples of the biocompatible substance may include polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid, but are not limited thereto.
The fatty acid may have a binding affinity for albumin in vivo, but is not particularly limited thereto.
In a more specific embodiment, the FcRn binding substance may be an immunoglobulin Fc region, and more specifically an IgG Fc region, but is not particularly limited thereto.
One or more amino acid side chains within the peptides of the invention may be conjugated to these biocompatible substances to increase in vivo solubility and/or half-life, and/or to increase their bioavailability. Such changes may also reduce clearance of therapeutic proteins and peptides.
The biocompatible material may be water soluble (amphiphilic or hydrophilic) and/or non-toxic and/or pharmaceutically acceptable.
F and X may be directly linked to each other (i.e., a is 0 in chemical formula 1) or may be linked through a linker (L).
In the present invention, the "immunoglobulin Fc region" refers to a region comprising heavy chain constant region 2(CH2) and/or heavy chain constant region 3(CH3) in addition to the heavy and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be an element that forms part of the conjugate of the invention.
The immunoglobulin Fc region may comprise a hinge region in a heavy chain constant region, but is not limited thereto. The immunoglobulin Fc region of the present invention may be an extended Fc region comprising a portion or all of heavy chain constant region 1(CH1) and/or light chain constant region 1(CT1), and does not comprise the heavy and light chain variable regions of an immunoglobulin, so long as the immunoglobulin Fc region has substantially the same or improved effect as compared to the native form. Alternatively, the immunoglobulin Fc region of the present invention may be a region in which a relatively long amino acid sequence corresponding to CH2 and/or CH3 is deleted.
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 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region); and 6) dimers between each domain of the heavy and light chain constant regions. However, the immunoglobulin Fc region of the present invention is not limited thereto.
In a particular embodiment, the immunoglobulin Fc region may be in dimeric form and one molecule of X may be covalently linked to one dimeric form of the Fc region, wherein the immunoglobulin Fc and X may be linked to each other by a non-peptidic polymer. Alternatively, two X molecules may be symmetrically linked to a dimeric form of the Fc region. The immunoglobulins Fc and X may be linked to each other by a non-peptide linker. However, the immunoglobulin Fc region of the present invention is not limited thereto.
In addition, the immunoglobulin Fc region of the present invention contains not only the amino acid sequence in a natural form but also a derivative thereof. Amino acid sequence derivatives refer to amino acid sequences that differ from the natural form of the amino acid sequence by deletion, insertion, non-conservative or conservative substitution of at least one amino acid residue, or a combination thereof.
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 have an important role in conjugation, may be used as suitable sites for alteration.
Various derivatives are possible, for example, by removing sites capable of disulfide bond formation, lacking some amino acid residues at the N-terminus of the native Fc, or adding methionine residues at the N-terminus of the native Fc. Furthermore, to eliminate effector functions, complement binding sites, such as the C1q binding site, may be removed, and antibody-dependent cell-mediated cytotoxicity (ADCC) sites may be removed. Techniques for preparing such sequence derivatives of immunoglobulin Fc regions are disclosed in International patent publication Nos. WO 97/34631 and WO 96/32478, among others.
Amino acid exchanges in Proteins and peptides that do not completely alter The activity of The protein or peptide are known in The art (h.neurath, r.l.hill, "Proteins (The Proteins)," Academic Press, New York, 1979). The exchanges which occur most frequently are exchanges between the 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, the alteration can be by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amination, and the like.
The above-described Fc derivatives exhibit biological activities equivalent to the Fc region of the present invention, and can improve the structural stability of the Fc region against heat, pH, and the like.
In addition, the Fc region may be obtained in a native form isolated from a living body of a human or an animal such as a cow, a goat, a pig, a mouse, a rabbit, a hamster, a rat, and a guinea pig, or may be a recombinant form obtained from a transformed animal cell or a microorganism or a derivative thereof. In this case, the Fc region may be obtained from a native form by isolating the whole immunoglobulin from a living body of a human or an 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) by pepsin treatment2. These may be subjected to size exclusion chromatography or the like to separate Fc or pF' c. In a more specific embodiment, the immunoglobulin Fc region is a recombinant immunoglobulin Fc region, wherein the human Fc region is obtained from a microorganism.
Furthermore, the immunoglobulin Fc region may be in the form of a native glycan, a form in which glycans are increased as compared to the native form, or a form in which glycans are decreased as compared to the native form, or in a deglycosylated form. The increase, decrease or removal of immunoglobulin Fc glycan can be obtained by using conventional methods such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. The immunoglobulin Fc region, with glycan removed from the Fc, showed a significant deterioration in binding affinity to complement C1q, as well as a reduction or elimination of antibody-or complement-dependent cellular cytotoxicity, and thus did not elicit an unwanted immune response in vivo. In this regard, deglycosylated or aglycosylated immunoglobulin Fc regions may be a more suitable form for the primary purpose of the present invention as a pharmaceutical carrier.
As used herein, the term "deglycosylation" refers to the enzymatic removal of glycans from an Fc region, and the term "aglycosylation" refers to an aglycosylated Fc region produced in prokaryotes, more specifically, e.
Meanwhile, the immunoglobulin Fc region may be derived from a human, or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in more specific embodiments, the immunoglobulin Fc region is derived from a human.
Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination or mixture thereof. In still more particular embodiments, the immunoglobulin Fc region is derived from IgG or IgM that is most abundant in human blood, and in still more particular embodiments, the immunoglobulin Fc region is derived from IgG that is known to increase the half-life of ligand binding proteins. In still more particular embodiments, the immunoglobulin Fc region is an IgG4 Fc region, and in most particular embodiments, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4, but is not so limited.
As used herein, the term "combination" refers to the linkage of a polypeptide encoding a single chain immunoglobulin Fc region of the same origin to a single chain polypeptide of a different origin when forming a dimer or multimer. That is, the dimer or multimer may be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.
Furthermore, the above conjugates may have an increased duration of efficacy compared to native insulin or triple agonists, or compared to X not modified with F, and such conjugates include not only the above forms, but also forms encapsulated in biodegradable nanoparticles.
Further, L may be a peptide linker or a non-peptide linker.
When L is a peptide linker, L may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not particularly limited thereto. In the present invention, various known ones can be usedA peptide linker to link F and X, and examples thereof may include [ GS]xLinker, [ GGGS ]]xLinker, [ GGGGS]xA joint, etc., where x may be a natural number of at least 1. However, the peptide linker is not limited to the above examples.
In the present invention, the "non-peptide linker" comprises a biocompatible polymer having two or more repeating units linked to each other. The repeating units are linked to each other by any covalent bond other than a peptide bond. The non-peptide linker may be one element constituting part of the conjugate of the present invention, and corresponds to L in chemical formula 1. In the present invention, non-peptide linkers may be used interchangeably with non-peptide polymers.
In the present invention, the peptide linker contains a reactive group at its terminal, and thus a conjugate can be formed by reacting with another element constituting the conjugate. When the non-peptidic linker having reactive functional groups at both ends is combined with X and F in chemical formula 1 through the respective reactive groups to form a conjugate, the non-peptidic linker or the non-peptidic polymer may be referred to as a non-peptidic polymer linker moiety or a non-peptidic linker moiety.
At LaIn (e), a may be 1 or more, and when a is 2 or more, each L may be independent.
In a specific embodiment, in the conjugate, F and X are covalently linked to each other via a non-peptide linker comprising at both ends thereof a reactive group capable of linking to F (in particular an immunoglobulin Fc region) and X (in particular a peptide drug).
Specifically, the non-peptide linker may be selected from the group consisting of fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.
Although not particularly limited, the high molecular weight polymer in the present invention may be in the range of greater than 0kDa to about 100kDa, specifically in the range of about 1kDa to about 100kDa, and more specifically in the range of about 1kDa to about 20kDa, but is not particularly limited thereto.
As used herein, the term "about" is meant to encompass ranges of ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1, and the like, and therefore all values within ranges equal to or similar to those set forth after the term, but is not limited thereto.
Although not particularly limited, the high molecular weight polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and a combination thereof, but is not particularly limited thereto. In a more specific embodiment, L may be polyethylene glycol, but is not limited thereto. In addition, derivatives thereof known in the art and derivatives that can be easily prepared by those skilled in the art are also included in the scope of the present invention.
As the non-peptide linker that can be used in the present invention, any polymer having resistance to in vivo protease can be used without limitation. The molecular weight of the non-peptidic polymer is in the range of about 1kDa to about 100kDa, and specifically in the range of about 1kDa to about 20kDa, but is not limited thereto. Furthermore, as the non-peptide linker of the invention linked to the polypeptide corresponding to F, one polymer as well as a combination of different types of polymers may be used.
In a specific example, both ends of the non-peptide linker may be linked to an amine or thiol group of F, such as an immunoglobulin Fc region, and an amine or thiol group of X, respectively.
Specifically, the non-peptide polymer may contain, at both ends thereof, reactive groups capable of being linked to F (e.g., immunoglobulin Fc region) and X, respectively, and specifically reactive groups capable of being linked to X or a thiol group of an amine group or a cysteine at the N-terminus or lysine in F (e.g., immunoglobulin Fc region), but is not limited thereto.
Alternatively, the reactive group of the non-peptidic polymer capable of being linked to F such as an immunoglobulin Fc region and X may be selected from the group consisting of aldehyde group, maleimide group and succinimide derivative, but is not limited thereto.
Examples of the aldehyde group may include a propyl aldehyde group or a butyl aldehyde group, but are not limited thereto.
Examples of the succinimide derivative may include succinimide valerate, succinimide methyl butyrate, succinimide methyl propionate, succinimide butyrate, succinimide propionate, N-hydroxysuccinimide, succinimide carboxymethyl ester, or succinimide carbonate, but are not limited thereto.
The non-peptide linker may be linked to X and F through these reactive groups, but is not particularly limited thereto.
Furthermore, the end products produced by reductive alkylation by means of aldehyde bonds are significantly more stable than the linkage by means of amide bonds. The aldehyde-reactive group selectively reacts with the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, e.g., pH 9.0.
The reactive groups at both ends of the non-peptide linker may be the same as or different from each other, and for example, the non-peptide linker may have a maleimide group at one end; and has an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. However, the reactive group is not particularly limited thereto as long as F (specifically, immunoglobulin Fc region) and X can be linked to both ends of the non-peptide linker.
For example, the non-peptide linker may have a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like as a reactive group at the other end.
When polyethylene glycol having hydroxyl reactive groups at both ends is used as a non-peptide polymer, or by using commercially available polyethylene glycol having modified reactive groups, the long-acting protein conjugate of the present invention can be prepared by activating hydroxyl groups to various reactive groups by known chemical reactions.
In one particular embodiment, the non-peptidic polymer may be linked to a cysteine residue of the triple agonist and more particularly, the-SH group of cysteine, but is not so limited.
For example, the non-peptide polymer may be linked to the cysteine residue at position 10, the cysteine residue at position 13, the cysteine residue at position 15, the cysteine residue at position 17, the cysteine residue at position 19, the cysteine residue at position 21, the cysteine residue at position 24, the cysteine residue at position 28, the cysteine residue at position 29, the cysteine residue at position 30, the cysteine residue at position 31, the cysteine residue at position 40, or the cysteine residue at position 41 in the triple agonist, but is not particularly limited.
In particular, the reactive group of the non-peptidic polymer may be linked to the-SH group of a cysteine residue, and all of the above description applies to the reactive group. When maleimide-PEG-aldehyde is used, the maleimide group may be linked to the-SH group of X via a thioether bond, and the aldehyde group may be amidated by reduction with-NH of F, particularly immunoglobulin Fc2The group is attached, but not limited thereto, and this case corresponds to one example.
In a specific example, the non-peptide polymer may be linked to an amine group (more specifically, an amine group located at the N-terminus or an amine group located at a lysine side chain) of insulin or an analog thereof, but is not limited thereto, and this case corresponds to an example.
In the conjugate, the reactive group of the non-peptidic polymer may react with-NH at the N-terminus of the Fc region of an immunoglobulin2Groups are attached, but this case corresponds to one example.
As used herein, the term "preventing" refers to all acts of inhibiting or delaying the onset of an insulin-related disorder by administering a composition, and the term "treating" refers to all acts of reducing or beneficially altering the symptoms of an insulin-related disorder as a result of administering a composition.
As used herein, the term "administering" refers to introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition may be, but is not limited to, any general route by which the composition can reach a target in vivo, and examples of the route of administration may include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, and the like.
The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means having a property of having a sufficient amount to exhibit a therapeutic effect without causing side effects, and the amount can be easily determined by those skilled in the art according to factors well known in the medical field, including the type of disease, the age, body weight, health condition and sex of a patient, the sensitivity of a patient to a drug, the administration route, the administration manner, the number of administrations, the duration of treatment, the drug used in combination or at the same time, and the like.
As for the pharmaceutically acceptable carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, a flavoring agent, etc. may be used for oral administration; buffers, preservatives, analgesics, solubilizers, isotonizing agents, stabilizers and the like may be used in the injectable mixture; and for topical application may be used substrates, excipients, lubricants, preservatives, and the like. The formulation of the composition according to the present invention may be variously prepared by mixing with the above-mentioned pharmaceutically acceptable carrier. For example, for oral administration, the compositions may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like; and for injections, the compositions may be prepared in unit dose ampoules or in multi-dose containers. In addition, the composition can be formulated in the form of a solution, suspension, tablet, pill, capsule, sustained release formulation, and the like.
Meanwhile, examples of carriers, excipients and diluents suitable for the formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In addition, the composition may further contain fillers, anticoagulants, lubricants, humectants, flavoring agents, preservatives, and the like.
The pharmaceutical composition of the present invention may have any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid medicines for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations and suppositories.
The conjugate can be used by mixing with various carriers approved as medicinal materials such as physiological saline or organic solvents, and in order to increase stability or absorbability, carbohydrates such as glucose, sucrose or dextran; antioxidants, such as ascorbic acid or glutathione; a chelating agent; a low molecular weight protein; other stabilizers; etc. can be used as medicinal materials.
The dose and frequency of the pharmaceutical composition of the present invention are determined according to the type of the drug as an active ingredient, and various factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease.
A total effective amount of a composition of the present invention may be administered to a patient in a single dose, or may be administered in multiple doses over a prolonged period of time by a fractionated treatment regimen. The pharmaceutical composition of the present invention may contain an active ingredient in an amount that may vary depending on the severity of the disease. Specifically, the total dose of the conjugate of the present invention may be about 0.0001mg to 500mg per 1kg body weight of the patient per day. However, the effective dose of the conjugate to a patient is determined in consideration of various factors such as age, body weight, health condition and sex of the patient, severity of disease, diet and excretion rate in addition to the administration route and treatment frequency of the pharmaceutical composition, and thus, in consideration of these, those skilled in the art can easily determine an effective dose suitable for a specific use of the pharmaceutical composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited with respect to formulation, administration route and administration manner as long as the pharmaceutical composition exhibits the effects of the present invention.
Furthermore, the pharmaceutical compositions of the invention may contain a combination, i.e. 0.01 to 99 weight%/volume of a triple agonist or a long-acting conjugate thereof; and insulin or a long-acting conjugate thereof (or each component of the combination).
In one aspect of the present invention, there is provided a complex formulation for weight loss in a patient to whom insulin is administered, the complex formulation comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
All of the above description applies to insulin, isolated peptides active at glucagon, GLP-1 and GIP receptors, and the ingredients contained in the compositions.
Specifically, the complex formulation may contain:
(a) insulin and triple agonists;
(b) long-acting conjugates of insulin, wherein the insulin is conjugated to a biocompatible substance capable of increasing the half-life in the insulin body, and triple agonists;
(c) a long-acting conjugate of insulin and a triple agonist, wherein the triple agonist is conjugated to a biocompatible substance capable of increasing the in vivo half-life of the triple agonist; or
(d) Long-acting conjugates of insulin and long-acting conjugates of triple agonists.
Specifically, the complex formulation may contain: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein insulin and the triple agonist may be contained in a molar ratio of 1:1 to 100: 1; may contain insulin and a triple agonist in a molar ratio of 1:1 to 1: 100; may contain insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof in a molar ratio of 1:1 to 100: 1; or may contain insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof in a molar ratio of 1:1 to 1:100, but the complex formulation is not limited thereto.
One aspect of the invention provides a kit comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
Insulin and isolated peptides active at the glucagon, GLP-1 and GIP receptors are described above.
The kit may comprise instructions for administration of: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
Specifically, the kit may comprise:
(a) insulin and triple agonists;
(b) long-acting conjugates of insulin, wherein the insulin is conjugated to a biocompatible substance capable of increasing the half-life in the insulin body, and triple agonists;
(c) a long-acting conjugate of insulin and a triple agonist, wherein the triple agonist is conjugated to a biocompatible substance capable of increasing the in vivo half-life of the triple agonist; or
(d) Long-acting conjugates of insulin and long-acting conjugates of triple agonists.
Specifically, the kit may comprise: insulin or a long-acting conjugate thereof; and a triple agonist or a long-acting conjugate thereof, wherein the kit may comprise instructions for administering insulin and the triple agonist to the subject in a molar ratio of 1:1 to 100:1 or 1:1 to 1:100, administering insulin or the long-acting conjugate thereof and the triple agonist or the long-acting conjugate thereof to the subject in a molar ratio of 1:1 to 100:1, or administering insulin or the long-acting conjugate thereof and the triple agonist or the long-acting conjugate thereof to the subject in a molar ratio of 1:1 to 1:100, but the kit is not limited thereto.
One aspect of the present invention provides a use of a composition or a complex formulation for preparing a medicinal material, the composition or the complex formulation comprising: (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
The composition or complex formulation is as described above. The medicinal materials can be used for the above purposes.
One aspect of the present invention provides a method for preventing or treating an insulin-related disease, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
One aspect of the invention provides a method of reducing weight gain resulting from insulin administration, the method comprising administering to a subject an isolated peptide having activity at glucagon, GLP-1 and GIP receptors.
Specifically, one aspect of the invention provides a method of reducing weight gain resulting from insulin administration, the method comprising administering to a subject (i) insulin; and (ii) isolated peptides having activity at glucagon, GLP-1 and GIP receptors.
The subject may be at risk for or may develop an insulin-related disease. The subject may also be a subject in need of insulin administration, or a subject in need of insulin administration and weight loss. However, the subject is not particularly limited thereto.
The administering step may be carried out by a combined administration of: (a) insulin and triple agonists; (b) long-acting conjugates of insulin and triple agonists; (c) long-acting conjugates of insulin and a triple agonist; or (d) long-acting conjugates of insulin and triple agonists.
In the present context, the use of the term "combination" is understood to mean simultaneous, separate or sequential administration. When the administration is sequential or separate, the delayed administration of the second component should not lose the beneficial effect of the combination.
The agents may be administered simultaneously, separately, sequentially or in reverse order, and may be administered simultaneously in an appropriate combination of effective amounts, but such administration is not limited to a particular method or order of administration.
Further, the prophylactic or therapeutic method of the present invention may comprise administering a composition or a complex formulation comprising the above (a) to (d) to a subject, but is not limited thereto.
The composition or complex formulation of the present invention comprising insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof supplements the activity or function of insulin, thereby inhibiting side effects of insulin such as weight gain while remarkably lowering blood glucose, and thus exhibits excellent effects on the treatment of insulin-related diseases.
Further, the composition or complex formulation may be formulated into a unit dose formulation suitable for administration into a patient according to typical methods in the pharmaceutical field, particularly a formulation useful for administration of protein drugs, and may be administered by oral administration route or parenteral administration route by means of administration methods generally used in the art, including, but not limited to, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal route.
The methods of the invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising an active ingredient. The appropriate total daily dosage of the pharmaceutical composition may be determined by the practitioner within the scope of sound medical judgment, and the pharmaceutical composition may be administered in divided doses one or more times. However, for the purposes of the present invention, it is preferred to apply a specific therapeutically effective amount for a particular patient according to the following differences: the type and extent of the reaction to be achieved; in some cases, another formulation is used or not; various factors including the specific composition, the age, body weight, general physical condition, sex and diet of the patient, the time of administration, the route of administration, the secretion rate of the composition, the duration of treatment, and the drug combined or used simultaneously with the specific composition; and similar factors well known in the medical arts.
Specifically, for insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof, insulin and a triple agonist may be administered to a subject at a molar ratio of 1:1 to 100:1 or a molar ratio of 1:1 to 1: 100; or insulin or a long-acting conjugate thereof and a triple agonist or a long-acting conjugate thereof may be administered to the subject at a molar ratio of 1:1 to 100: 1; or insulin or a long-acting conjugate thereof and a triple agonist or a long-acting agonist thereof may be administered to the subject at a molar ratio of 1:1 to 1:100, but is not limited thereto.
Hereinafter, the present invention will be described in detail with reference to the following exemplary embodiments. However, the following exemplary embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1: preparation of triple agonists and long-acting conjugates thereof
(1) Preparation of triple agonists
Triple agonists exhibiting activity at all glucagon, GLP-1 and GIP receptors were prepared and their sequences are shown in table 1 below.
TABLE 1
In the sequences shown in table 1, the amino acid labeled X represents aminoisobutyric acid (Aib), which is an unnatural amino acid, and the underlined amino acids represent amino acids that together form a ring. In Table 1, CA represents 4-imidazoleacetyl group, and Y represents tyrosine.
(2) Preparation of triple agonist long-acting conjugates
In order to pegylate cysteine residues of 10kDa PEG having maleimide groups and aldehyde groups at both ends thereof, i.e., maleimide-PEG-aldehyde (10kDa, NOF, Japan) and the triple agonist (SEQ ID NOS: 21, 22, 42, 43, 50, 77 and 96) in example 1, the triple agonist and the maleimide-PEG-aldehyde were each reacted at a molar ratio of 1:1-3 at a low temperature for 0.5-3 hours, wherein the protein concentration was 1-5 mg/mL. The reaction was carried out in an environment in which 20-60% isopropanol was added to 50mM Tris buffer (pH 7.5). After completion of the reaction, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the cysteine-monopegylated triple agonist.
Then, the purified mono-pegylated triple agonist and immunoglobulin Fc were reacted at a molar ratio of 1:1-5 at 4-8 ℃ for 12-18 hours, wherein the protein concentration was 10-50 mg/mL. The reaction was carried out in an environment in which 10 to 50mM of sodium cyanoborohydride as a reducing agent and 10 to 30% of isopropanol were added to 100mM of potassium phosphate buffer (pH 6.0). After completion of the reaction, the reaction solution was applied to Butyl Sepharose FF purification column (GE Healthcare, USA) and Source ISO purification column (GE Healthcare, USA) to purify the conjugate of the triple agonist and the immunoglobulin Fc.
Purity after preparation was 95% or higher as analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography.
Conjugates in which the triple agonist of SEQ ID NO:21 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:21 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 21" or "long-acting conjugate of SEQ ID NO: 22", and these are used interchangeably herein.
Conjugates in which the triple agonist of SEQ ID NO:22 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:22 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 22" or "long-acting conjugate of SEQ ID NO: 22", and these are used interchangeably herein.
Conjugates in which the triple agonist of SEQ ID NO:42 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:42 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 42" or "long-acting conjugate of SEQ ID NO: 42", and these are used interchangeably herein.
Conjugates in which the triple agonist of SEQ ID NO:43 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:43 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 43" or "long-acting conjugate of SEQ ID NO: 43", and these are used interchangeably herein.
Conjugates in which the triple agonist of SEQ ID NO:50 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:50 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 50" or "long-acting conjugate of SEQ ID NO: 50", and these are used interchangeably herein.
The conjugate in which the triple agonist of SEQ ID NO:77 and immunoglobulin Fc are linked by PEG is named "conjugate comprising SEQ ID NO:77 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 77" or "long-acting conjugate of SEQ ID NO: 77", and these may be used interchangeably herein.
Conjugates in which the triple agonist of SEQ ID NO:96 and immunoglobulin Fc are linked by PEG are named "conjugate comprising SEQ ID NO:96 and immunoglobulin Fc", "long-acting conjugate of SEQ ID NO: 96" or "long-acting conjugate of SEQ ID NO: 96", and these are used interchangeably herein.
Example 2: preparation of long-acting conjugates of natural insulin
In order to PEGylate 3.4K propion-ALD (2) PEG (3.4kDa PEG, one malonyl group at each end, NOF, Japan) at the N-terminus of the B-chain of human native insulin (India, Biocon), native insulin and PEG were reacted at a molar ratio of 1:4 at 25 ℃ for 2 hours, with a native insulin concentration of 5 mg/mL. By adding 3mM sodium cyanoborohydride (NaCNBH) as a reducing agent to a mixed solvent of 50mM sodium citrate buffer (pH 5.0) and 45% isopropyl alcohol3) To carry out the reaction. The reaction solution was purified using an SP-HP (GE healthcare) column using a buffer containing sodium citrate (pH 3.0) and 45% EtOH and a KCl concentration gradient.
Next, in order to link native insulin-linked PEG to the N-terminus of the immunoglobulin Fc fragment, the purified monopegylated insulin and the immunoglobulin Fc fragment were reacted at 25 ℃ for 15 hours at a molar ratio of 1:1.2 while the total protein concentration was 20 mg/mL. For the reaction solution, 20mM sodium cyanoborohydride as a reducing agent was added to 100mM HEPES buffer (pH 8.2) and sodium chloride.
After completion of the reaction, the reaction solution was applied to a Q-HP column (GE, USA) using a Tris-HCl buffer (pH7.5) and a NaCl concentration gradient, and to Source 15ISO (GE, USA) using a concentration gradient of ammonium sulfate and Tris-HCl (pH7.5), thereby purifying a native insulin-3.4K PEG-immunoglobulin Fc conjugate.
Test example 1: determination of in vitro Activity of triple agonists and Long-acting conjugates thereof
To determine the activity of the triple agonist and its long-acting conjugate prepared in example 1, a method of determining the activity of cells in vitro by using cell lines transformed with GLP-1, glucagon (GCG) and GIP receptors, respectively, was used.
The cell line is obtained by transforming Chinese Hamster Ovary (CHO) cells to express human GLP-1 receptor, human GCG receptor and human GIP receptor, respectively, and is suitable for determining activities of GLP-1, GCG and GIP. Thus, the activity of the receptor was determined using the transformed cell lines, respectively.
To determine GLP-1 activity of the triple agonists and long-acting conjugates thereof prepared in examples 1 and 2, human GLP-1 was serially diluted, and the triple agonists and long-acting conjugates thereof prepared in examples 1 and 2 were serially diluted. Cultures were taken from cultured human GLP-1 receptor-expressing CHO cells, and each serially diluted substance was added to the cells at 5. mu.L, and then 5. mu.L of buffer containing cAMP antibody was added thereto, followed by incubation at room temperature for 15 minutes. Then, 10 μ L of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 minutes. Cell lysates were applied to the LANCE cAMP kit (PerkinElmer, USA) after incubation was complete to calculate EC from accumulated cAMP50Values, which are then compared with each other. The relative titers compared to human GLP-1 are shown in tables 2 and 3 below.
To determine GGG activity of the triple agonists and long-acting conjugates thereof prepared in examples 1 and 2, human GGG was serially diluted, and the triple agonists and long-acting conjugates thereof prepared in examples 1 and 2 were serially diluted. Cultures were taken from cultured CHO cells expressing human GGG receptors, and each serially diluted substance was added to the cells at 5. mu.L, and then 5. mu.L of a buffer containing cAMP antibody was added thereto, followed by incubation at room temperature for 15 minutes. Then, 10 μ L of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 minutes. Cell lysates were applied to the LANCE cAMP kit (PerkinElmer, USA) after incubation was complete to calculate EC from accumulated cAMP50Values, which are then compared with each other. The relative titers compared to human GGG are shown in tables 2 and 3 below.
To determine GIP activity of the triple agonists and long-acting conjugates thereof prepared in examples 1 and 2, human GIP was serially diluted, and the triple agonists prepared in examples 1 and 2 were appliedSerial dilutions of both the animal and its long-acting conjugate. Cultures were taken from cultured CHO cells expressing the human GIP receptor, and each serially diluted substance was added to the cells at 5 μ L, and then 5 μ L of buffer containing cAMP antibody was added thereto, followed by incubation at room temperature for 15 minutes. Then, 10 μ L of the assay mixture containing cell lysis buffer was added to lyse the cells, followed by incubation at room temperature for 90 minutes. Cell lysates were applied to the LANCE cAMP kit (PerkinElmer, USA) after incubation was complete to calculate EC from accumulated cAMP50Values, which are then compared with each other. The relative titers compared to human GIP are shown in tables 2 and 3 below.
TABLE 2
Relative titer ratio of triple agonists
TABLE 3
Relative titer ratio of triple agonists
Test example 2: combined administration of insulin long-acting conjugates and triple agonist long-acting conjugates for type II diabetes Blood glucose control and delta body weight in model mice
To determine the long-acting effect of triple agonist administration due to administration of a composition or combination comprising a long-acting conjugate of triple agonist prepared in example 1 (SEQ ID NO:42) or a long-acting conjugate of native insulin prepared in example 2The in vivo effects caused by the long-acting conjugate of the conjugate (SEQ ID NO:42) and native insulin were obtained using the type II diabetes model mouse db/db mouse (Charles River, Japan). db/db mouse (BKS. Cg- + Lepr)db/+LeprdbOlaHsd mice) were used in this test example, since mice showed diabetic symptoms due to the removal of leptin receptor.
Blood glucose levels were measured in 8-week-old db/db mice using a glucometer (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 (350mg/dL to 600 mg/dL). Diabetes-inducing mice were divided into four groups: g1, G2, G3 and G4, with seven mice per group.
These groups were assigned to a control group (vehicle), a group administered with a natural insulin long-acting conjugate (15.8nmol/kg/Q2D), a group administered with a triple agonist long-acting conjugate (1.4nmol/kg/Q2D), and a group administered with a combination of a natural insulin long-acting conjugate (15.8nmol/kg/Q2D) and a triple agonist long-acting conjugate (1.4 nmol/kg/Q2D). Two weeks after repeated administration of the test substance, the glycosylated hemoglobin (HbA1c) level of each group was measured. Glycosylated hemoglobin is a form in which glucose is bound to hemoglobin normally present in red blood cells, and when the blood glucose level is maintained at a high level, the glycosylated hemoglobin level is also increased. Body weight change (Δ BW) was calculated for the test animals before drug administration and on the last day of testing.
As a result, the group administered with the long-acting conjugate of native insulin and the long-acting conjugate of triple agonist in combination showed a decrease in glycosylated hemoglobin level (fig. 1). Specifically, as shown in fig. 1, the change in the glycosylated hemoglobin level of the group administered with the insulin long-acting conjugate was-0.1, compared to the control group; change in glycosylated hemoglobin level of the group administered the triple agonist long-acting conjugate was-0.3; and the change in glycosylated hemoglobin level of the group administered the insulin long-acting conjugate in combination with the triple agonist long-acting conjugate was-0.5.
The combined administration showed significantly improved results compared to the group administered with the native insulin long-acting conjugate alone or the group administered with the triple agonist long-acting conjugate alone.
The measurement results of delta body weight showed that the group administered with the combination of the native insulin long-acting conjugate and the triple agonist long-acting conjugate showed improved body weight gain effect compared to the group administered with the insulin long-acting conjugate alone, and showed significant body weight loss effect compared to the control group (fig. 2). Specifically, as shown in fig. 2, the body weight change of the control group was + 15.8%; the change in body weight of the group administered with the insulin long-acting conjugate was + 23.1%; the change in body weight of the group administered with the triple agonist long-acting conjugate was-9.3%; and the change in body weight of the group administered with the insulin long-acting conjugate in combination with the triple agonist long-acting conjugate was-3.8%.
These results indicate that the combined administration of the insulin long-acting conjugate and the triple agonist long-acting conjugate of the present invention can exhibit superior glycemic control effects compared to the administration of the insulin long-acting conjugate or the triple agonist long-acting conjugate alone, and that the combined administration of the insulin long-acting conjugate and the triple agonist long-acting conjugate of the present invention can significantly reduce side effects of weight gain due to the administration of insulin alone.
Although the present invention has been described with reference to specific illustrative embodiments, it will be appreciated by those skilled in the art that the present invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics thereof. Accordingly, the foregoing examples should be construed as illustrative, and not limiting, of the present invention. The scope of the present invention is defined not by the above detailed description but by the appended claims of the invention, and it should also be understood that all changes or modifications derived from the definitions and scope of the claims and their equivalents fall within the scope of the present invention.
<110> Korean Med chemical Co., Ltd
<120> pharmaceutical compositions comprising insulin and a glucagon trigonal/GLP-1/GIP receptor agonist
<130> OPA19304
<150> KR 10-2018-0167698
<151> 2018-12-21
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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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> 21
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> trigonal agonists
<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 loop
<400> 21
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> 22
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> trigonal agonists
<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 loop
<400> 22
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> 23
<211> 41
<212> PRT
<213> Artificial sequence
<220>
<223> trigonal agonists
<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 loop
<400> 23
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> 24
<211> 39
<212> PRT
<213> Artificial sequence
<220>
<223> trigonal agonists
<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 loop
<400> 24
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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<220>
<221> MISC_FEATURE
<222> (1)
<223> Xaa is 4-imidazoleacetyl (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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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> trigonal agonists
<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 loop
<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 is His (H), 4-imidazoleacetyl (CA), or Tyr (Y)
<220>
<221> MISC_FEATURE
<222> (2)
<223> Xaa is Gly (G), alpha-methyl-glutamic acid, or Aib
(Aminoisobutyric acid)
<220>
<221> MISC_FEATURE
<222> (3)
<223> Xaa is 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 is 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 is Gly (G), Glu (E), or Ser (S)
<220>
<221> MISC_FEATURE
<222> (17)
<223> Xaa is Gln (Q), Arg (R), Ile (I), Glu (E), Cys (C), or Lys (K)
<220>
<221> MISC_FEATURE
<222> (18)
<223> Xaa is 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> (21)
<223> Xaa is Glu (E), Gln (Q), Leu (L), Cys (C), or Asp (D)
<220>
<221> MISC_FEATURE
<222> (23)
<223> Xaa is Ile (I) or Val (V)
<220>
<221> MISC_FEATURE
<222> (24)
<223> Xaa is 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 is Cys (C), Lys (K), Ala (A), Asn (N), or Asp (D)
<220>
<221> MISC_FEATURE
<222> (29)
<223> Xaa is 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 is absent, and Xaa may be further linked to R1, wherein R1 is Cys, GKKNDWKHNIT,
m-SSGAPPPS-n, or m-SSGQPPPS-n, or absent, and wherein m is
-Cys-, -Pro-, or-Gly-Pro-, and n is-Cys-, -Gly-, -Ser-, or
-His-Gly-, or is absent
<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 is His (H), 4-imidazoleacetyl (CA), or Tyr (Y)
<220>
<221> MISC_FEATURE
<222> (2)
<223> Xaa is Gly (G), alpha-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 is Leu (L), Met (M), or Tyr (Y)
<220>
<221> MISC_FEATURE
<222> (15)
<223> Xaa is Asp (D), Glu (E), or Leu (L)
<220>
<221> MISC_FEATURE
<222> (16)
<223> Xaa is Gly (G), Glu (E), or Ser (S)
<220>
<221> MISC_FEATURE
<222> (17)
<223> Xaa is Gln (Q), Arg (R), Ile (I), Glu (E), Cys (C), or Lys (K)
<220>
<221> MISC_FEATURE
<222> (18)
<223> Xaa is 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> (21)
<223> Xaa is Glu (E), Gln (Q), Leu (L), Cys (C), or Asp (D)
<220>
<221> MISC_FEATURE
<222> (23)
<223> Xaa is Ile (I) or Val (V)
<220>
<221> MISC_FEATURE
<222> (24)
<223> Xaa is 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 is Cys (C), Gly (G), Gln (Q), or His (H)
<220>
<221> MISC_FEATURE
<222> (30)
<223> Xaa is Cys (C), Gly (G), Lys (K), or His (H)
<220>
<221> MISC_FEATURE
<222> (31)
<223> Xaa is Pro (P) or Cys (C)
<220>
<221> MISC_FEATURE
<222> (40)
<223> Xaa is Cys (C), or absent
<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> general 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 alpha-methyl-glutamic acid, or Aib (aminoisobutyric acid)
<220>
<221> MISC_FEATURE
<222> (13)
<223> Xaa is Tyr (Y), Ala (A), or Cys (C)
<220>
<221> MISC_FEATURE
<222> (17)
<223> Xaa is Arg (R), Cys (C), or Lys (K)
<220>
<221> MISC_FEATURE
<222> (18)
<223> Xaa is Ala (A) or Arg (R)
<220>
<221> MISC_FEATURE
<222> (19)
<223> Xaa is Ala (A) or Cys (C)
<220>
<221> MISC_FEATURE
<222> (21)
<223> Xaa is Glu (E) or Asp (D)
<220>
<221> MISC_FEATURE
<222> (24)
<223> Xaa is Gln (Q) or Asn (N)
<220>
<221> MISC_FEATURE
<222> (28)
<223> Xaa is 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 Cys (C) or His (H)
<220>
<221> MISC_FEATURE
<222> (31)
<223> Xaa is Pro (P) or Cys (C)
<220>
<221> MISC_FEATURE
<222> (40)
<223> Xaa is Cys (C), or absent
<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> Intelligent people
<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> 21
<212> PRT
<213> Artificial sequence
<220>
<223> insulin analogues, 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 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 is alanine, tyrosine, glutamic acid, histidine, 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 is alanine, tyrosine, serine, glutamic acid, histidine,
threonine, or asparagine
<220>
<221> MISC_FEATURE
<222> (21)
<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 alanine, or glycine
<220>
<221> MISC_FEATURE
<222> (16)
<223> Xaa is tyrosine, glutamic acid, tryptophan, threonine, or aspartic acid or is absent
<220>
<221> MISC_FEATURE
<222> (23)
<223> Xaa is glycine, or alanine
<220>
<221> MISC_FEATURE
<222> (24)
<223> Xaa is alanine or phenylalanine
<220>
<221> MISC_FEATURE
<222> (25)
<223> Xaa is alanine, phenylalanine, aspartic acid, or glutamic acid, or is absent
<220>
<221> MISC_FEATURE
<222> (27)
<223> Xaa is threonine, or is absent
<220>
<221> MISC_FEATURE
<222> (28)
<223> Xaa is proline, glutamic acid, or aspartic acid, or is absent
<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> 21
<212> PRT
<213> Intelligent people
<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> Intelligent people
<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> Artificial 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> Artificial 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> analogue 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> analogue 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> Artificial sequence
<220>
<223> analogue 10
<400> 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> analogue 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> analogue 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
<212> DNA
<213> Artificial sequence
<220>
<223> analogue 14
<400> 149
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 agtgcaac 258
<210> 150
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> analogue 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> analogue 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> Artificial sequence
<220>
<223> analogue 16
<400> 153
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
ctggagaaca cctgcaac 258
<210> 154
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> analogue 16
<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 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> analogue 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> analogue 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> Artificial sequence
<220>
<223> analogue 20
<400> 161
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 cctcgcccag 240
ctggagaact actgcaac 258
<210> 162
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> analogue 20
<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 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> Artificial sequence
<220>
<223> analogues 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> analogues 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> analogue 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 75 80
Leu Glu Asn Tyr Cys Asn
85
<210> 169
<211> 258
<212> DNA
<213> Artificial sequence
<220>
<223> analogue 24
<400> 169
ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60
gaacgaggct tcgagtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120
caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180
tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240
ctggagaact actgcaac 258
<210> 170
<211> 86
<212> PRT
<213> Artificial sequence
<220>
<223> analogue 24
<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 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
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