RU2823246C2 - Pharmaceutical composition containing insulin and glucagon - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical industry, namely to a pharmaceutical combination, a composition for preventing or treating a disease associated with insulin. Pharmaceutical combination for preventing or treating a disease associated with insulin, containing insulin and glucagon, where insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate and glucagon is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by the following chemical formula 1: XL_a-F, where X is insulin or glucagon; L is polyethylene glycol; is 0 or a natural number, provided that when is 2 or more, each L is independent; F is an immunoglobulin Fc region capable of increasing the half-life of X in vivo; and "-" is a covalent or non-covalent bond, and wherein said combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a weight ratio of 0.1:1 to 100:1. Pharmaceutical combination for preventing or treating hypoglycaemia, containing insulin and glucagon, where insulin is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate and glucagon is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is presented by chemical formula 1 (see above) and where said pharmaceutical combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a weight ratio of 0.1:1 to 100:1. Combination for reducing the side effects of insulin, comprising insulin and glucagon, wherein the insulin is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate and glucagon is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is presented by chemical formula 1 (see above) and where said combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a weight ratio of 0.1:1 to 100:1. Complex composition for reducing hypoglycaemia in a patient with an insulin-related disease, containing insulin and glucagon, where insulin is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate and glucagon is bound to a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by chemical formula 1 (see above), and wherein said complex composition contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a weight ratio of 0.1:1 to 100:1.

EFFECT: above-described combinations and composition effectively relieve hypoglycaemia, inhibit body weight gain, while providing a therapeutic effect on diseases associated with insulin, reduce side effects of the long-acting insulin conjugate.

39 cl, 3 dwg, 8 tbl, 11 ex

Description

FIELD OF THE INVENTION

The present invention relates to a composition containing insulin and glucagon, and to a complex composition containing insulin and glucagon.

BACKGROUND ART

The pancreas produces and secretes glucagon when blood glucose levels begin to fall due to various reasons such as drug treatment, disease, hormone or enzyme deficiency, and so on. Secreted glucagon acts on the liver to promote the breakdown of glycogen, inducing the release of glucose and ultimately leading to the restoration of normal blood glucose levels.

Similar to glucagon, insulin, which is a type of hormone produced by the pancreas, is a hormone secreted from the pancreas and plays a role in maintaining blood glucose levels at proper levels when they become elevated. Insulin deficiency, absence or dysfunction can lead to various diseases. Therefore, insulin administration is known to be a key therapy for the treatment of insulin-related diseases, including diabetes, in which blood glucose levels are high.

It is widely known that glucagon and insulin have opposite effects, that is, antagonistic effects, on blood glucose, and they are used as therapeutic agents for various diseases. In particular, Korean Patent Publication No. 10-2017-0023066 discloses a method for treating diabetes with maintenance insulin while simultaneously antagonizing glucagon action by blocking glucagon receptors with an antagonistic antigenic protein.

In other words, since insulin and glucagon have opposite functions in the body, there are no known examples of drug therapy with their joint administration.

At the same time, when administering insulin, which is a therapeutic agent for various insulin-related diseases, to a patient, there is a risk of side effects such as weight gain, overdose and hypoglycemia.

Accordingly, there is a need to develop a drug capable of reducing these side effects while maintaining the effectiveness of insulin administration.

DESCRIPTION OF THE INVENTION

Technical problem

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of an insulin-related disease, comprising insulin and glucagon.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of hypoglycemia containing insulin and glucagon.

Another object of the present invention is to provide a composition for reducing the side effects of insulin containing insulin and glucagon.

Another object of the present invention is to provide a complex composition for reducing hypoglycemia in a patient with an insulin-related disease, containing insulin and glucagon.

Another object of the present invention is to provide the use of said composition in the manufacture of a medicinal product.

Another object of the present invention is to provide a method for preventing or treating an insulin-related disease, comprising the step of administering the composition or complex composition to an individual.

Another object of the present invention is to provide a method of reducing the side effects of insulin, comprising the step of administering said composition or said complex composition to an individual.

Technical solution

To achieve the present invention, according to one aspect, a pharmaceutical composition for preventing or treating an insulin-related disease is provided, comprising insulin and glucagon.

The composition according to one specific embodiment is characterized in that the insulin is associated with a biocompatible substance capable of increasing its half-life in vivo , in the form of a long-acting conjugate.

The composition according to the above specific embodiment is characterized in that the glucagon is associated with a biocompatible substance capable of increasing its half-life in vivo , in the form of a long-acting conjugate.

The composition according to any of the above specific embodiments is characterized in that insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo , in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo , in the form long-acting conjugate.

The composition according to any of the above specific embodiments is characterized in that the insulin-related disease is selected from the group consisting of insulin-resistant disease, diabetes, hyperglycemia and obesity.

The composition according to any of the above specific embodiments is characterized in that it alleviates hypoglycemia, which is a side effect of insulin, and suppresses weight gain.

The composition according to any of the above specific embodiments is characterized in that it contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a mass ratio of from 0.1:1 to 100:1.

The composition according to any of the above specific embodiments is characterized in that the glucagon is native glucagon or a glucagon analogue obtained by altering one or more amino acids of native glucagon selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

The composition according to any of the above specific embodiments is characterized in that the glucagon analog contains the amino acid sequence of the following general formula 1:

X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-T-X30 ( general formula 1, SEQ ID NO:46),

in general formula 1: X1 is histidine (H), desaminohistidyl, dimethylhistidyl (N-dimethylhistidyl), beta-hydroxyimidazopropionyl, 4-imidazoacetyl, beta-carboxyimidazopropionyl, tryptophan (W) or tyrosine (Y) or absent;

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

X7 is threonine (T), valine (V), or cysteine (C);

X10 is tyrosine (Y) or cysteine (C);

X12 is lysine (K) or cysteine (C);

X13 is tyrosine (Y) or cysteine (C);

X14 is leucine (L) or cysteine (C);

X15 is aspartic acid (D), glutamic acid (E) or cysteine (C);

X16 is glutamic acid (E), aspartic acid (D), series (S), alpha-methylglutamic acid or cysteine (C), or none;

X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), series (S), cysteine (C) or valine (V) or absent;

X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V) or cysteine (C), or none;

X19 is alanine (A), arginine (R), series (S), valine (V) or cysteine (C) or absent;

X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid or cysteine (C), or none;

X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V) or cysteine (C) or absent;

X23 is isoleucine (I), valine (V) or arginine (R) or absent;

X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid or leucine (L) or none ;

X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q) or arginine (R) or absent;

X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R) or absent; And

X30 is cysteine (C) or absent (provided that when the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1, it is excluded).

The composition according to any of the above specific embodiments is characterized in that in general formula 1: XI represents histidine (H), tryptophan (W) or tyrosine (Y) or is absent;

X2 represents series (S) or Aib (aminoisobutyric acid);

X7 is threonine (T), valine (V), or cysteine (C);

X10 is tyrosine (Y) or cysteine (C);

X12 is lysine (K) or cysteine (C);

X13 is tyrosine (Y) or cysteine (C);

X14 is leucine (L) or cysteine (C);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E), series (S) or cysteine (C);

X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V);

X18 is aspartic acid (D), glutamic acid (E), arginine (R) or cysteine (C);

X19 is alanine (A) or cysteine (C);

X20 is glutamine (Q), aspartic acid (D), lysine (K) or cysteine (C);

X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V) or cysteine (C);

X23 is isoleucine (I), valine (V) or arginine (R);

X24 is valine (V), arginine (R), alanine (A), glutamic acid (E), lysine (K), glutamine (Q) or leucine (L);

X27 is isoleucine (I), valine (V), alanine (A), methionine (M), glutamine (Q) or arginine (R);

X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R); And

X30 is cysteine (C) or absent.

The composition according to any of the above specific embodiments is characterized in that, in general formula 1:

X1 is histidine (H), tryptophan (W), or tyrosine (Y);

X2 represents series (S) or Aib (aminoisobutyric acid);

X7 is cysteine (C), threonine (T), or valine (V);

X10 is tyrosine (Y) or cysteine (C);

X12 is lysine (K) or cysteine (C);

X13 is tyrosine (Y) or cysteine (C);

X14 is leucine (L) or cysteine (C);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E), series (S) or cysteine (C);

X17 is glutamic acid (E), lysine (K), arginine (R), cysteine (C) or valine (V);

X18 is arginine (R) or cysteine (C);

X19 is alanine (A) or cysteine (C);

X20 is glutamine (Q) or lysine (K);

X21 is aspartic acid (D), glutamic acid (E), valine (V) or cysteine (C);

X23 is valine (V);

X24 is valine (V) or glutamine (Q);

X27 represents methionine (M);

X28 is asparagine (N) or arginine (R); And

X30 is cysteine (C) or absent.

The composition according to any of the above specific embodiments is characterized in that, in general formula 1:

X1 is tyrosine (Y);

X2 represents Aib (aminoisobutyric acid);

X7 is cysteine (C), threonine (T), or valine (V);

X10 is tyrosine (Y) or cysteine (C);

X12 represents lysine (K);

X13 is tyrosine (Y) or cysteine (C);

X14 is leucine (L) or cysteine (C);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E), series (S) or cysteine (C);

X17 is lysine (K), arginine (R), cysteine (C) or valine (V);

X18 is arginine (R) or cysteine (C);

X19 is alanine (A) or cysteine (C);

X20 is glutamine (Q) or lysine (K);

X21 is aspartic acid (D), glutamic acid (E) or cysteine (C);

X23 is valine (V);

X24 is glutamine (Q);

X27 represents methionine (M);

X28 is asparagine (N) or arginine (R); And

X30 is cysteine (C) or absent.

The composition according to any of the above specific embodiments is characterized in that, in general formula 1:

X1 is histidine (H), tryptophan (W), or tyrosine (Y) or absent;

X2 represents series (S) or Aib (aminoisobutyric acid);

X7 is threonine (T), valine (V), or cysteine (C);

X10 is tyrosine (Y) or cysteine (C);

X12 is lysine (K) or cysteine (C);

X13 is tyrosine (Y) or cysteine (C);

X14 is leucine (L) or cysteine (C);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E), series (S) or cysteine (C);

X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C), or valine (V);

X18 is aspartic acid (D), glutamic acid (E), arginine (R) or cysteine (C);

X19 is alanine (A) or cysteine (C);

X20 is glutamine (Q), aspartic acid (D) or lysine (K);

X21 is aspartic acid (D) or glutamic acid (E);

X23 is valine (V);

X24 is valine (V) or glutamine (Q);

X27 is isoleucine (I) or methionine (M);

X28 is asparagine (N) or arginine (R); And

X30 is cysteine (C) or absent.

The composition according to any of the above specific embodiments is characterized in that, in general formula 1:

X1 is tyrosine (Y);

X2 represents Aib (aminoisobutyric acid);

X7 is threonine (T);

X10 represents tyrosine (Y);

X12 represents lysine (K);

X13 is tyrosine (Y);

X14 is leucine (L);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E), series (S) or cysteine (C);

X17 is lysine (K) or arginine (R);

X18 represents arginine (R);

X19 represents alanine (A);

X20 is glutamine (Q), cysteine (C) or lysine (K);

X21 is aspartic acid (D), cysteine (C), valine (V) or glutamic acid (E);

X23 is valine (V) or arginine (R);

X24 is glutamine (Q) or leucine (L);

X27 represents methionine (M);

X28 is asparagine (N) or arginine (R); And

X30 is missing.

The composition according to any of the above specific embodiments is characterized in that the glucagon analog contains the amino acid sequence of the following general formula 2:

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

in general formula 2:

X7 is threonine (T), valine (V), or cysteine (C);

X10 is tyrosine (Y) or cysteine (C);

X12 is lysine (K) or cysteine (C);

X15 is aspartic acid (D) or cysteine (C);

X16 represents glutamic acid (E) or series (S);

X17 is lysine (K) or arginine (R);

X20 is glutamine (Q) or lysine (K);

X21 is aspartic acid (D) or glutamic acid (E);

X24 is valine (V) or glutamine (Q); and X30 is cysteine (C) or absent.

The composition according to any of the above specific embodiments is characterized in that at least one pair of amino acids from the pairs of amino acids X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of the general formula 1 forms a ring , respectively.

The composition according to any of the above specific embodiments is characterized in that each amino acid of at least one pair of amino acids from the pairs of amino acids X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of the general formula 1 replaced by glutamic acid or lysine, which are capable of forming a ring.

The liquid composition according to any of the above specific embodiments is characterized in that the long-acting glucagon analog conjugate has a pi of the glucagon analog of 6 to 7 and its in vitro activity, compared with that of native glucagon, is 200% or more. The composition according to any of the above specific embodiments is characterized in that the glucagon analog contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-45.

The composition according to any of the above specific embodiments is characterized in that the glucagon analogue contains the amino acid sequence of SEQ ID NO: 37.

The composition according to any of the above specific embodiments is characterized in that the insulin is natural insulin or an insulin analog obtained by altering one or more amino acids of native insulin selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

The composition according to any of the above specific embodiments is characterized in that the insulin analog is an insulin analog obtained by substitution with another amino acid or deletion of one or more amino acids selected from the group consisting of an amino acid at position 1, an amino acid at position 2, amino acids at position 3, amino acids at position 5, amino acids at position 8, amino acids at position 10, amino acids at position 12, amino acids at position 16, amino acids at position 23, amino acids at position 24, amino acids at position 25, amino acids at position 26, amino acids at position 27, amino acids at position 28, amino acids at position 29 and amino acids at position 30 of the natural insulin B chain and amino acids at position 1, amino acids at position 2, amino acids at position 5, amino acids at position 8, amino acids at position 10, amino acids at position 12, amino acids at position 14, amino acids at position 16, amino acids at position 17, amino acids at position 18, amino acids at position 19 and amino acids at position 21 of the natural insulin A chain.

The composition according to any of the above specific embodiments is characterized in that the insulin analog contains an A chain of SEQ ID NO: 48, represented by the following general formula 3, and a B chain of SEQ ID NO: 49, represented by the following general formula 4:

[general formula 3]

Xaa1-Xaa2-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 48),

in general formula 3:

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

Xaa2 is alanine or isoleucine;

Xaa5 is 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 4]

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: 49),

in general formula 4:

Xaa8 is alanine or glycine;

Xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or absent;

Xaa23 is glycine or alanine;

Xaa24 is alanine or phenylalanine;

Xaa25 is alanine, phenylalanine, aspartic acid or glutamic acid or absent;

Xaa27 is threonine or absent; And

Xaa28 is proline, glutamic acid or aspartic acid or absent

(herein, it is provided that when the peptide contains the A chain of SEQ ID NO: 124 and the B chain of SEQ ID NO: 125, it is excluded).

The composition according to any of the above specific embodiments is characterized in that the insulin analog is obtained by replacing with alanine one amino acid selected from the group consisting of an amino acid at position 8, an amino acid at position 23, an amino acid at position 24 and an amino acid at position 25 of the B chain natural insulin and amino acid at position 1, amino acid at position 2 and amino acid at position 19 of the A-chain of natural insulin or by replacing the amino acid at position 14 of the A-chain of natural insulin with glutamic acid or asparagine.

The composition according to any of the above specific embodiments is characterized in that the insulin analog contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67.

The composition according to any of the above specific embodiments is characterized in that the insulin analog is obtained by replacing the amino acid at position 16 with glutamic acid, or deleting the amino acid at position 25 of the natural insulin B chain, or replacing the amino acid at position 14 A with glutamic acid or alanine. natural insulin chains.

The composition according to any of the above specific embodiments is characterized in that the insulin analog contains the amino acid sequence of SEQ ID NO: 69 or 71.

The composition according to any of the above specific embodiments is characterized in that the insulin analog is obtained by replacing the amino acid at position 16 of the B-chain with glutamic acid, serine, threonine or aspartic acid, replacing the amino acid at position 25 with B-chain of natural insulin with aspartic acid or glutamic acid chain of natural insulin, or by replacing the amino acid at position 14 of the A-chain of natural insulin with histidine, lysine, alanine or aspartic acid, or by replacing with glutamic acid, serine or threonine of the amino acid at position 19 of the A-chain of natural insulin.

The composition according to any of the above specific embodiments is characterized in that the insulin analog contains an amino acid sequence selected from the group consisting of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123.

The composition according to any of the above specific embodiments is characterized in that the insulin analog is presented in the form of two polypeptide chains consisting of an A chain of SEQ ID NO: 48, represented by the general formula 3, and a B chain of SEQ ID NO: 49, represented by general formula 4.

The composition according to any of the above specific embodiments is characterized in that the A chain and the B chain are linked to each other through a disulfide bond.

The composition according to any of the above specific embodiments is characterized in that the conjugate is represented by the following chemical formula 1:

[chemical formula 1]

XL _a -F,

where: X represents insulin or glucagon;

L represents a linker;

a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent;

F is a substance capable of increasing the half-life of X in vivo; And

"-" represents a covalent or non-covalent bond.

The composition according to any of the above specific embodiments is characterized in that F is selected from the group consisting of a high molecular weight polymer, a fatty acid, cholesterol, albumin and a fragment thereof, an albumin-binding substance, a polymer of repeating units with a specific amino acid sequence, an antibody, an antibody fragment , a substance that binds to FcRn (neonatal Fc receptor), connective tissue in vivo, nucleotide, fibronectin, transferrin, saccharide, heparin and elastin.

The composition according to any of the above specific embodiments is characterized in that the high molecular weight polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid , oligonucleotide and combinations thereof.

The composition according to any of the above specific embodiments is characterized in that F represents the Fc region of an immunoglobulin.

The composition according to any of the above specific embodiments is characterized in that F represents the Fc region of IgG (immunoglobulin G).

The composition according to any of the above specific embodiments is characterized in that the Fc region of the immunoglobulin is aglycosylated.

The composition according to any of the above specific embodiments is characterized in that the Fc region of the immunoglobulin 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) CH1 domain and CH3 domain; (d) a CH2 domain and a CH3 domain; (e) combinations of one or two or more domains of a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain and an immunoglobulin hinge region or part of said hinge region; and (e) a dimer of each heavy chain constant region and light chain constant region domain.

The composition according to any of the above specific embodiments is characterized in that the Fc region of the immunoglobulin is an Fc region of the immunoglobulin where the region capable of forming a disulfide bond is removed, where part of the amino acid(s) at the N-terminus is removed, where to the N-terminus of the native Fc is attached to a methionine residue where the complement fixing site is removed or where the antibody-dependent cell-mediated cytotoxicity (ADCC) site is removed.

The composition according to any of the above specific embodiments is characterized in that the immunoglobulin Fc region is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE or IgM.

The composition according to any of the above specific embodiments is characterized in that the Fc region of the immunoglobulin is a hybrid of a domain having different origins derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

The composition according to any of the above specific embodiments is characterized in that L is selected from the group consisting of a peptide, a fatty acid, a saccharide, a high molecular weight polymer, a low molecular weight compound, a nucleotide, and combinations thereof.

The composition according to any of the above specific embodiments is characterized in that the high molecular weight polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid , oligonucleotide and combinations thereof.

The composition according to any of the above specific embodiments is characterized in that L represents polyethylene glycol.

To achieve the present invention, according to another aspect, there is provided a pharmaceutical composition for preventing or treating hypoglycemia containing insulin and glucagon.

The composition according to one specific embodiment is characterized in that the insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The composition according to the above specific embodiment is characterized in that the glucagon is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The composition according to any of the above specific embodiments is characterized in that insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form long-acting conjugate.

To achieve the present invention, according to another aspect, there is provided a composition for reducing the side effects of insulin containing insulin and glucagon.

The composition according to one specific embodiment is characterized in that the insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The composition according to the above specific embodiment is characterized in that the glucagon is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The composition according to any of the above specific embodiments is characterized in that insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form long-acting conjugate.

To achieve the present invention, according to another aspect, there is provided a complex composition for reducing hypoglycemia in a patient with an insulin-related disease, containing insulin and glucagon.

The complex composition according to one specific embodiment is characterized in that the insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The complex composition according to the above specific embodiment is characterized in that the glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate.

The complex composition according to any of the above specific embodiments is characterized in that insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in form of a long-acting conjugate.

The complex composition according to any of the above specific embodiments is characterized in that hypoglycemia is a side effect of insulin.

The complex composition according to any of the above specific embodiments is characterized in that it suppresses body weight gain.

The complex composition according to any of the above specific embodiments is characterized in that it contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a mass ratio of from 0.1:1 to 100:1.

Beneficial effects

The composition or complex composition, each containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, of the present invention provides a new combination therapy capable of ameliorating side effects caused by insulin administration, such as weight gain or hypoglycemia, by providing at the same time, a preventive or therapeutic effect on an insulin-related disease, such as diabetes.

DESCRIPTION OF GRAPHIC MATERIALS

In FIG. 1 shows changes in blood glucose levels following the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate;

in FIG. 2 shows the changes (AUC) in blood glucose levels following the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate; And

in FIG. Figure 3 shows changes in body weight following the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate.

BEST OPTION FOR IMPLEMENTING THE INVENTION

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

At the same time, each of the explanations and embodiments disclosed in the present invention can be applied to other explanations and embodiments. That is, the scope of the present invention includes all combinations of the various elements disclosed in the present invention. Moreover, the scope of the present invention should not be limited to the specific descriptions given below.

Throughout the description of the present invention, not only the traditional one-letter codes and three-letter codes for naturally occurring amino acids are used, but also the three-letter codes commonly used for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), and alpha methylglutamic acid (α-methylglutamic acid). In addition, the amino acids abbreviated in the present invention are described in accordance with the IUPAC-IUB nomenclature.

To achieve the present invention, according to one aspect, a pharmaceutical composition for preventing or treating an insulin-related disease is provided, comprising insulin and glucagon.

Specifically, the composition may contain:

(1) insulin and glucagon;

(2) a long-acting insulin conjugate, wherein insulin is associated with a biocompatible substance capable of increasing its half-life in vivo, and glucagon;

(3) insulin and long-acting glucagon conjugate, where the glucagon is associated with a biocompatible substance capable of increasing its half-life in vivo; or

(4) long-acting insulin conjugate and long-acting glucagon conjugate.

The pharmaceutical composition for preventing or treating an insulin-related disease of the present invention contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof and can alleviate the side effects caused by the administration of insulin (for example, weight gain or hypoglycemia) by providing at the same time, a preventive or therapeutic effect on an insulin-related disease caused by insulin deficiency, absence or dysfunction.

In one exemplary embodiment of the present invention, the effects of lowering blood glucose, suppressing body weight gain, and alleviating hypoglycemia were confirmed in db/db mice that were administered a composition containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and thus It has been demonstrated that the pharmaceutical composition of the present invention can maintain the effectiveness of insulin while reducing its side effects.

In one specific embodiment of the present invention, the composition may provide a therapeutic use of a combination of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof.

As used herein, “composition” may be used interchangeably with “combination” comprising insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof. The composition may be presented in the form of a set.

As used herein, the term “combination” may be used to mean the combined administration of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and the combination may be intended to have the same meaning as “combined use.” The combination can be administered: (a) in the form of a mixture wherein (1) insulin or a long-acting conjugate thereof and (2) glucagon or a long-acting conjugate thereof are mixed; or (b) in a separate form, wherein (1) insulin or a long-acting conjugate thereof and (2) glucagon or a long-acting conjugate thereof are administered separately; but without limiting it.

When insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are provided in separate form, the insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be formulated as separate preparations for administration simultaneously, separately, sequentially or in in reverse order.

In the present invention, combination administration does not only mean simultaneous administration, but should also be understood as a dosage form capable of performing a function at a level equal to or greater than the natural function of each substance when combined with the action of insulin or its long-acting conjugate and glucagon or its long-acting conjugate per individual. Therefore, when the term “combined” is used in this application, it should be understood that they are administered simultaneously, separately, sequentially or in reverse order. When administration is carried out sequentially, in reverse order or separately, there are no specific restrictions regarding the order of administration. However, the interval before the second administration should be such that the beneficial effects of the combined administration are not lost.

As used herein, the term "composition" may be the combination itself, including insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, or a composition containing the combination, and a therapeutic use, without limitation. For example, the composition may be a composition having prophylactic or therapeutic use for, but not limited to, insulin-related diseases.

The composition of the present invention may be for combined administration of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and the insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be formulated as a single preparation or separate preparations. Specifically, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be administered simultaneously, separately, sequentially, or in reverse order, but without limitation thereof.

As used herein, the term “kit” may include the combination or composition of the present invention for the combined administration of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof. Specifically, the kit of the present invention may contain insulin or a conjugate thereof and glucagon or a long-acting conjugate thereof formulated as a single preparation, or insulin or a conjugate thereof and glucagon or a conjugate thereof formulated as separate preparations, and may further contain a substance necessary for the combined administration of two of these substances, but without limitation.

Insulin and glucagon include various substances with significant levels of insulin and glucagon activity, for example, a substance in the form of a compound or peptide.

As used herein, the term "insulin-related disease" refers to a disease caused by abnormalities in the function of insulin in controlling blood glucose, such as insulin deficiency, absence or dysfunction, and the scope of the present invention includes any disease, provided that the administration of insulin at this disease will presumably have a preventive or therapeutic effect. Specifically, the insulin-related disease may be selected from the group consisting of, but not limited to, insulin-resistant diseases, diabetes, hyperglycemia, and obesity.

When insulin is administered to treat an insulin-related disease, undesirable side effects such as hypoglycemia or weight gain occur despite the possibility of obtaining the desired therapeutic effect, and thus there is a problem of the patient suffering from other disease and pain. The pharmaceutical composition of the present invention, wherein insulin and glucagon are used in combination, can alleviate hypoglycemia, which is a side effect of insulin, and can suppress weight gain, while providing a therapeutic effect on insulin-related diseases.

The pharmaceutical composition of the present invention may contain insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a quantitative ratio to alleviate side effects while providing a therapeutic effect on insulin-related diseases. Specifically, they may be included in a mass ratio of 0.1:1 to 100:1, or a molar ratio of 1:1 to 30:1, 1:1 to 20:1, 1:1 to 16:1, but without limiting it.

The pharmaceutical composition of the present invention may contain insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a certain ratio, thereby demonstrating an excellent blood glucose control effect while suppressing side effects. When the mass ratio is outside the above range, for example when insulin or its long-acting conjugate is included in excess amounts, there is a high risk of developing significant side effects of insulin, and when glucagon or its long-acting conjugate is included in excess amounts, there is a problem of possible reduction in therapeutic effect on insulin-related diseases.

As used herein, the term “insulin” refers to a type of hormone secreted by the beta cells of the pancreas and generally functions in the body to regulate blood glucose by promoting intracellular glucose uptake and inhibiting the breakdown of fat. Proinsulin, in the form of a precursor that does not have a blood glucose control function, undergoes processing to form insulin, which has a blood glucose control function. Insulin consists of two polypeptide chains, i.e., A chain and B chain, containing 21 and 30 amino acid residues, respectively, linked to each other through two disulfide bonds. The A chain and B chain of natural insulin contain the amino acid sequences represented by the following SEQ ID NOs: 124 and 125, 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: 124).

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: 125).

As used herein, the term “proinsulin” refers to a molecule that is a precursor to insulin. Proinsulin may contain an insulin A chain and a B chain and a C peptide in between. The proinsulin may be human proinsulin.

In the present invention, the insulin may be a natural insulin or may be an analogue, derivative or fragment obtained by altering one or more amino acids of a natural insulin selected from the group consisting of substitution, addition, deletion, modification and combinations thereof.

As used herein, the term “insulin analogue” refers to a non-natural insulin other than natural insulin.

An insulin analog includes an analog obtained by modifying a portion of the amino acids of natural insulin by addition, deletion or substitution. For example, an insulin analog may be an insulin analog produced by substitution with another amino acid or deletion of one or more amino acids selected from the group consisting of an amino acid at position 1, an amino acid at position 2, an amino acid at position 3, an amino acid at position 5 , amino acids at position 8, amino acids at position 10, amino acids at position 12, amino acids at position 16, amino acids at position 23, amino acids at position 24, amino acids at position 25, amino acids at position 26, amino acids at position 27, amino acids at position 28 , amino acids at position 29 and amino acids at position 30 of the natural insulin B chain and amino acids at position 1, amino acids at position 2, amino acids at position 5, amino acids at position 8, amino acids at position 10, amino acids at position 12, amino acids at position 14 , amino acids at position 16, amino acids at position 17, amino acids at position 18, amino acids at position 19, and amino acids at position 21 of, but not limited to, the natural insulin A chain. Amino acid substitutions can generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Conservative substitutions with amino acids having similar properties can be expected to exhibit the same or similar activity.

The insulin analogue of the present invention may refer to the one described in Korean Patent Publication No. 10-2014-0106452 or 10-2017-0026284.

The insulin analog used in the present invention may be in the form of a single chain or two polypeptide chains, and more preferably in the form of two polypeptide chains, but is not particularly limited thereto. The two polypeptide chains may consist of two polypeptides, a polypeptide corresponding to the A chain of natural insulin and a polypeptide corresponding to the B chain of natural insulin. Here, the sequence of the peptide corresponding to the A chain or B chain of natural insulin may be at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more identical when comparing the sequence identity of any one chain of two polypeptide chains with the A chain or the B chain natural insulin, but is not limited to this, and those skilled in the art can easily determine this by comparing the sequence constituting the two polypeptide chains with the sequence of the A chain or B chain of natural insulin.

As used herein, the term “homology” is intended to indicate the degree of similarity to the amino acid sequence of a wild-type protein or the nucleotide sequence encoding it and includes sequences having the above percentage or greater homology to the amino acid sequence or nucleotide sequence of the present invention. Homology can be determined by comparing two given sequences with the naked eye, but can be determined using a bioinformatics algorithm that allows homology analysis by aligning the sequences in question for comparison. The homology of two given amino acid sequences can be reported as a percentage. A useful automated algorithm is available for use in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA). The alignment algorithm automated in the above modules includes the Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Other useful sequence alignment and homology determination algorithms are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

Information about the insulin and glucagon sequences used in the present invention and the nucleotide sequences encoding them can be obtained from a publicly available database such as NCBI and the like.

In one particular exemplary embodiment, the insulin analog of the present invention may be a composition comprising an A chain of SEQ ID NO: 48 represented by the following general formula 3 and a B chain of SEQ ID NO: 49 represented by the following general formula 4, but without limitation:

[general formula 3]

Xaa1-Xaa2-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 48), in general formula 3:

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

Xaa2 is alanine or isoleucine;

Xaa5 is 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 4]

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: 49),

and in general formula 4:

Xaa8 is alanine or glycine;

Xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or absent;

Xaa23 is glycine or alanine;

Xaa24 is alanine or phenylalanine;

Xaa25 is alanine, phenylalanine, aspartic acid or glutamic acid or absent;

Xaa27 is threonine or absent; And

Xaa28 is proline, glutamic acid or aspartic acid or absent

(wherein the peptide containing the A chain of SEQ ID NO: 124 and the B chain of SEQ ID NO: 125 is excluded).

More specifically, the insulin analog may be an insulin analog prepared by replacing with alanine one or more amino acids selected from the group consisting of an amino acid at position 8, an amino acid at position 23, an amino acid at position 24, and an amino acid at position 25 of the B chain natural insulin and the amino acid at position 1, the amino acid at position 2 and the amino acid at position 19 of the A chain of natural insulin or substitution with glutamic acid or asparagine of the amino acid at position 14 of the A chain of natural insulin, and in particular the insulin analogue may contain the amino acid sequence , selected from the group consisting of SEQIDNO: 51, 53, 55, 57, 59, 61, 63, 65 and 67, or may consist of, but is not limited to, such an amino acid sequence.

In addition, the insulin analog may be an insulin analog obtained by substituting glutamic acid for the amino acid at position 16 of the natural insulin B chain, deleting an amino acid at position 25 of the natural insulin B chain, or substituting glutamic acid or alanine for the amino acid at position 14 A- natural insulin chains, and in particular the insulin analogue, may contain, but are not limited to, the amino acid sequence of SEQ ID NO: 69 or 71.

In addition, the insulin analog may be an insulin analog obtained by substituting glutamic acid, serine, threonine, or aspartic acid for an amino acid at position 16 of the natural insulin B chain, substituting aspartic acid or glutamic acid for an amino acid at position 25 of the natural insulin B chain , substitution with histidine, lysine, alanine or aspartic acid of the amino acid at position 14 of the A-chain of natural insulin, or substitution with glutamic acid, serine or threonine of the amino acid at position 19 of the A-chain of natural insulin, and, in particular, the insulin analogue may contain the amino acid sequence , selected from the group consisting of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123, or may consist of, but is not limited to, such an amino acid sequence.

Although described in the present invention as "consisting of a particular SEQ ID NO", provided that the protein may have an activity identical to or corresponding to the activity of a polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO, it is possible to add a nonsense sequence above or below the amino acid sequence corresponding to SEQ ID NO, a natural mutation or a silent mutation thereof, and it is understood that variants having such a sequence addition or mutation are also included within the scope of the present invention.

The above description may be applied to, but is not limited to, other specific embodiments or aspects of the present invention.

Meanwhile, the insulin analog of the present invention may be in the form of single chain insulin wherein the C-peptide is not removed, and may be a substance containing an A chain of SEQ ID NO: 48 represented by the general formula 3, and a B-chain a chain of SEQ ID NO: 49, represented by the general formula 4, and having the activity and function of insulin.

In addition, the insulin analog of the present invention can be obtained by removing the C-peptide from proinsulin containing the C-peptide between the A chain and the B chain. Removal of C-peptide can be accomplished by a method known in the art, and specifically, it can be accomplished by, but not limited to, treatment with trypsin and carboxypeptidase B.

Specifically, the insulin analog of the present invention may be composed of an A chain of SEQ ID NO: 48 represented by the general formula 3 and a B chain of SEQ ID NO: 49 represented by the general formula 4, and more specifically, the insulin analog may be is presented in the form of two polypeptide chains, where the A chain and the B chain are linked to each other through two disulfide bonds, but not limited to this.

It is understood that the insulin analog in the form of two polypeptide chains obtained by removing the C-peptide from the insulin analog in the form of proinsulin represented in the present invention by the specific SEQ ID NO is also included within the scope of the present invention.

An insulin analog may include a peptide having one or more than one sequence different from the amino acid sequence of natural insulin, a peptide altered by modifying the sequence of natural insulin, and a mimetic of natural insulin that regulates the blood glucose control function in a living body, similar to natural insulin. In the present invention, an insulin analog can be used interchangeably with an insulin derivative. Such a derivative of natural insulin can perform the function of controlling blood glucose in a living organism.

Specifically, the insulin derivative can be modified by any method of replacing, adding, deleting and modifying a portion of the amino acids of natural insulin or by a combination of these methods.

Specifically, the insulin derivative may have an amino acid sequence that is at least 80% or more homologous to the amino acid sequence of the A chain or B chain of natural insulin, and/or may be presented in a form wherein a portion of a group of one amino acid residue of insulin is chemically substituted (eg , alpha-methylation, alpha-hydroxylation), removed (eg, deamination) or modified (eg, N-methylation), but not limited to.

The natural insulin derivative used in the present invention can be obtained by a combination of various derivatization methods.

In addition, such modification to produce insulin derivatives may include all changes using L-type or D-type amino acids and/or non-native amino acid types and/or native sequence modification or post-translational modification (e.g., methylation, acylation, ubiquitination, formation of intramolecular covalent connections and the like).

In addition, the change may also include all changes adding one or more amino acids to the N- and/or C-terminus of insulin.

When replacing or adding amino acids, not only the 20 amino acids commonly found in human proteins can be used, but also atypical amino acids not found in nature. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptides containing these amino acids and atypical peptide sequences can be synthesized and purchased from commercial suppliers, such as, but not limited to, American Peptide Company, Bachem (USA) or Anygen (Korea).

As used herein, the term “natural insulin, insulin analog, or insulin derivative fragment” refers to a form where one or more amino acids have been removed from the N- and/or C-terminus of the natural insulin, insulin analog, or natural insulin derivative. Such a fragment can perform the function of controlling blood glucose in the body.

In addition, the insulin analog of the present invention can be produced using production methods used in the production of natural insulin derivatives and fragments, independently or in combination.

In addition, the insulin analog of the present invention may contain a change in a certain amino acid residue of the A chain and B chain of natural insulin, and, specifically, may contain a change in a certain amino acid residue of the A chain of natural insulin and/or a change in a certain amino acid residue of the B chain of natural insulin. insulin.

The pharmaceutical composition of the present invention may contain (1) natural insulin, (2) an insulin analog, (3) an insulin derivative, (4) a fragment thereof, or (5) a combination thereof as insulin being one of the active ingredients, provided that They perform the function of insulin in controlling blood glucose.

As used herein, the term "glucagon" refers to a type of hormone secreted by the alpha cells of the pancreas and has the function in the body of stimulating the breakdown of glycogen to increase glucose levels and increase blood glucose. 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: 1).

In the present invention, glucagon may be native glucagon or may be an analogue, derivative or fragment obtained by altering one or more amino acids of native glucagon selected from the group consisting of substitution, addition, deletion, modification and combinations thereof.

As used herein, the term “glucagon analog” refers to a non-natural glucagon other than naturally occurring glucagon.

A glucagon analog includes an analog obtained by modifying a portion of the amino acids of naturally occurring glucagon by addition, deletion, or substitution.

The sequence of the glucagon analog of the present invention may be at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more more than, 92% or more, 93% or more, 94% or more, or 95% or more identical to the sequence of native glucagon and is not limited to a specific sequence, provided that it has the effect of increasing blood glucose. The glucagon analogue of the present invention may refer to Korean Patent Publication Nos. 10-2017-003466, 10-2018-0002544 and 10-2016-0082482.

In one particular aspect, the glucagon analog may be a peptide containing the amino acid sequence of the following general formula 1:

X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-T-X30 ( general formula 1, SEQ ID NO: 46),

in general formula 1:

X1 is histidine, desaminohistidyl, dimethylhistidyl (N-dimethylhistidyl), beta-hydroxyimidazopropionyl, 4-imidazoacetyl, beta-carboxyimidazopropionyl, tryptophan or tyrosine, or none;

X2 is alpha-methylglutamic acid (α-methylglutamic acid), Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine;

X7 is threonine, valine or cysteine;

X10 represents tyrosine or cysteine;

X12 represents lysine or cysteine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 is aspartic acid, glutamic acid or cysteine;

X16 is glutamic acid, aspartic acid, serine, alpha-methylglutamic acid or cysteine or absent;

X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine or valine or none;

X18 is alanine, aspartic acid, glutamine, glutamic acid, arginine, valine or cysteine or absent;

X19 is alanine, arginine, serine, valine or cysteine or absent;

X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, alpha-methylglutamic acid or cysteine or absent;

X21 is aspartic acid, glutamic acid, leucine, valine or cysteine or absent;

X23 is isoleucine, valine or arginine or absent;

X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methylglutamic acid or leucine or none;

X27 is isoleucine, valine, alanine, lysine, methionine, glutamine or arginine or absent;

X28 is glutamine, lysine, asparagine or arginine or absent; And

X30 is cysteine or is absent (provided that when the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1, it is excluded).

Much more specifically, in general formula 1:

X1 is histidine, tryptophan or tyrosine or absent;

X2 represents serie or Aib (aminoisobutyric acid);

X7 is threonine, valine or cysteine;

X10 represents tyrosine or cysteine;

X12 represents lysine or cysteine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid, serine or cysteine;

X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine or valine;

X18 is aspartic acid, glutamic acid, arginine or cysteine;

X19 is alanine or cysteine;

X20 represents glutamine, aspartic acid, lysine or cysteine;

X21 is aspartic acid, glutamic acid, leucine, valine or cysteine;

X23 is isoleucine, valine or arginine;

X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine or leucine;

X27 is isoleucine, valine, alanine, methionine, glutamine or arginine;

X28 is glutamine, lysine, asparagine or arginine; and X30 is cysteine or is absent (provided that, when the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1, it is excluded).

For example, a glucagon analog may contain an amino acid sequence selected from the group consisting of SEQ ID NO: 2-45, and, specifically, may consist (substantially) of an amino acid sequence selected from the group consisting of SEQ ID NO: 2-45 , but not limited to this. In addition, the glucagon analog may have a pi of 6 to 7 and/or may have in vitro activity that is 200% or greater than that of native glucagon. More specifically, the glucagon analog may comprise the amino acid sequence of SEQ ID NO: 37, and, much more specifically, it may consist (substantially) of the amino acid sequence of SEQ ID NO: 37, but is not limited to it.

Specifically, in general formula 1:

X1 is histidine, tryptophan or tyrosine;

X2 represents serie or Aib (aminoisobutyric acid);

X7 is cysteine, threonine or valine;

X10 represents tyrosine or cysteine;

X12 represents lysine or cysteine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid, serine or cysteine;

X17 is glutamic acid, lysine, arginine, cysteine or valine;

X18 is arginine or cysteine;

X19 is alanine or cysteine;

X20 represents glutamine or lysine;

X21 is aspartic acid, glutamic acid, valine or cysteine;

X23 is valine;

X24 is valine or glutamine;

X27 is methionine;

X28 is asparagine or arginine; And

X30 is cysteine or absent.

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11-17, 19-27, 29, 31, 33 and 35-44, and specifically may consist of (substantially) the amino acid a sequence selected from the group consisting of, but not limited to, SEQ ID NOs: 3, 11-17, 19-27, 29, 31, 33 and 35-44.

Specifically, in general formula 1:

X1 is tyrosine;

X2 represents Aib (aminoisobutyric acid);

X7 is cysteine, threonine or valine;

X10 represents tyrosine or cysteine;

X12 represents lysine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid, serine or cysteine;

X17 is lysine, arginine, cysteine or valine;

X18 is arginine or cysteine;

X19 is alanine or cysteine;

X20 represents glutamine or lysine;

X21 is aspartic acid, glutamic acid or cysteine;

X23 is valine;

X24 is glutamine;

X27 is methionine;

X28 is asparagine or arginine; And

X30 is cysteine or is absent (provided that when the amino acid sequence of general formula 1 is the same as SEQ ID NO: l, it is excluded).

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQIDNO: 12, 14, 17, 19-25, 27, 29, 33, 35-38, 40-42 and 44, and, specifically, may consist of essentially) from an amino acid sequence selected from the group consisting of, but not limited to, SEQ ID NOs: 12, 14, 17, 19-25, 27, 29, 33, 35-38, 40-42 and 44.

Specifically, in general formula 1:

X1 is histidine, tryptophan or tyrosine or absent;

X2 represents serie or Aib (aminoisobutyric acid);

X7 is threonine, valine or cysteine;

X10 represents tyrosine or cysteine;

X12 represents lysine or cysteine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid, serine or cysteine;

X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine or valine;

X18 is aspartic acid, glutamic acid, arginine or cysteine;

X19 is alanine or cysteine;

X20 represents glutamine, aspartic acid or lysine;

X21 is aspartic acid or glutamic acid;

X23 is valine;

X24 is valine or glutamine;

X27 is isoleucine or methionine;

X28 is asparagine or arginine; And

X30 is cysteine or is absent (provided that when the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1, it is excluded).

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30 and 32-45, and specifically may consist of (substantially) the amino acid a sequence selected from the group consisting of, but not limited to, SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30 and 32-45.

Specifically, in general formula 1:

X1 is tyrosine;

X2 represents Aib (aminoisobutyric acid);

X7 is threonine;

X10 is tyrosine;

X12 represents lysine;

X13 is tyrosine;

X14 is leucine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid, serine or cysteine;

X17 is lysine or arginine;

X18 is arginine;

X19 is alanine;

X20 is glutamine, cysteine or lysine;

X21 is aspartic acid, cysteine, valine or glutamic acid;

X23 is valine or arginine;

X24 is glutamine or leucine;

X27 is methionine;

X28 is asparagine or arginine; And

X30 is missing.

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25 and 31, and specifically may consist (substantially) of an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 19, 25 and 31, but not limited to.

More specifically, the peptide may be a peptide containing the amino acid sequence of the following general formula 2:

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

in general formula 2:

X7 is threonine, valine or cysteine;

X10 represents tyrosine or cysteine;

X12 represents lysine or cysteine;

X15 represents aspartic acid or cysteine;

X16 represents glutamic acid or series;

X17 is lysine or arginine;

X20 represents glutamine or lysine;

X21 is aspartic acid or glutamic acid;

X24 is valine or glutamine; And

X30 is cysteine or absent.

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15 and 36-44, and specifically may consist (substantially) of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 15 and 36-44, but not limited to these. More specifically, the peptide may contain the amino acid sequence of SEQ ID NO: 12, 20 or 37 or may consist of (substantially) the corresponding amino acid sequence, but is not limited to this.

Specifically, in general formula 2:

X7 is threonine, valine or cysteine;

X10 represents tyrosine or cysteine;

X12 represents lysine;

X15 is aspartic acid;

X16 represents glutamic acid or series;

X17 is lysine or arginine;

X20 represents glutamine or lysine;

X21 is aspartic acid or glutamic acid;

X24 is glutamine; And

X30 is, but not limited to, cysteine.

For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 36-38, 40-42 and 44, and, specifically, may consist (substantially) of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 36-38, 40-42 and 44, but not limited to.

The glucagon analog described above may contain an intramolecular linkage (eg, covalent cross-link or non-covalent cross-link) and, specifically, it may be presented in a form containing a ring. For example, the glucagon analog may be presented in a form where a ring is formed between the amino acids at positions 16 and 20 of the glucagon derivative, but is not limited to this.

Non-limiting examples of the ring may include a lactam cross-link (or lactam ring).

In addition, a glucagon analog includes all glucagon analogs modified to include a ring-forming amino acid at the desired site for ring formation.

The ring may be formed between the amino acid side chains of a glucagon analogue, 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.

For example, a peptide containing an amino acid sequence of general formula 1 or 2 may be a peptide wherein each amino acid in each amino acid pair of amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 and X24 and X28 of general formula 1 or 2 may be replaced by glutamic acid or lysine, respectively, but without limitation. In Xn (n is a natural number), n refers to the amino acid position at the N-terminus of the represented amino acid sequence.

In addition, the peptide containing the amino acid sequence of the general formula 1 or 2 may be a peptide wherein each amino acid in each of the pair of amino acids X12 and X16, or the pair of amino acids X16 and X20, or the pair of amino acids X17 and X21 is replaced by glutamic acid or lysine , respectively, which are capable of forming a ring, but are not limited thereto.

In addition, in the general formula 1 or 2, the peptide may be a peptide in which between the corresponding amino acids in at least one pair of amino acids from the pairs of amino acids X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28, a ring is formed (eg, a lactam ring), but is not limited to this.

Additionally, in the general formula 1 or 2, X16 may be glutamic acid, X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but are not limited to this.

As used herein, the term "glucagon derivative" includes a peptide whose amino acid sequence has one or more differences compared to native glucagon, a peptide where the sequence of native glucagon is altered by modification, and a native glucagon mimetic capable of activating glucagon receptors like native glucagon . Such a derivative of native glucagon can perform the function of controlling blood glucose in a living organism. In the present invention, a glucagon derivative can be used interchangeably with a glucagon analogue.

The glucagon analogue or glucagon derivative of the present invention may have improved physical properties by having an altered pI compared to native glucagon. In addition, a glucagon analogue or glucagon derivative may have improved solubility while having, but not limited to, glucagon receptor activating activity.

In addition, the glucagon analogue or glucagon derivative may be a glucagon that does not occur in nature.

As used herein, the term "isoelectric point" or "pI" refers to the pH value at which the net charge of a molecule, such as a polypeptide or peptide, is zero (0). In the case of a polypeptide with different charged functional groups, at pI the sum of their charges is zero. At a pH above pI, the net charge of the polypeptide will be negative, and at a pH below pI, the net charge of the polypeptide will be positive.

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

As used herein, the term “altered pI” refers to a pI that differs from the pI of native glucagon due to the replacement of part of the amino acid sequence of native glucagon with an amino acid residue having a negative charge or a positive charge, that is, a reduced or increased pI value. A peptide with such an altered pI may exhibit improved solubility and/or high stability at neutral pH, but is not limited to this.

More specifically, a glucagon analogue or derivative may have an altered pI value rather than the pI value (6.8) of native glucagon, and, even more specifically, a pI value less than 6.8, more specifically 6.7, or less, more specifically 6.5 or less, and further, a pI value greater than 6, 8, 7 or more, more particularly 7.5 or greater, but without limitation, and the scope of the present invention includes any pI value other than the pI value of native glucagon. In particular, the scope of the present invention includes a glucagon analogue or derivative having a pI value different from the pI value of native glucagon, and thus demonstrating improved solubility at neutral pH compared to the solubility of native glucagon and, therefore, a low level of aggregation.

More specifically, the glucagon analog or derivative may have a pI value of from 4 to 6.5 and/or from 7 to 9.5, specifically from 7.5 to 9.5 and more particularly from 8.0 to 9.3. but the value of pI is not limited to this. In this case, by virtue of having a pI value higher or lower than the pI value of native glucagon, the glucagon analogue or derivative may exhibit improved solubility and high stability at neutral pH compared to, but not limited to, native glucagon.

Specifically, the glucagon derivative may be modified by any method of replacing, adding, deleting, or modifying a portion of the amino acids of native glucagon, or a combination of these methods. Amino acid substitutions can generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Conservative substitutions with amino acids having similar properties can be expected to exhibit the same or similar activity.

Examples of a glucagon derivative prepared by a combination of the above methods include a peptide having a glucagon receptor activating function, the amino acid sequence of which has one or more than one difference from the amino acid sequence of native glucagon, and wherein the N-terminal amino acid residue is deaminated, but is not limited to, and The native glucagon derivatives used in the present invention can be prepared by a combination of various derivatization methods.

In addition, such modification to produce native glucagon derivatives may include all modifications using L-type or D-type amino acids and/or non-native amino acids and/or modification of the native sequence, e.g. modification of a side chain functional group, formation of intramolecular covalent bonds, e.g. rings between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation and so on. In addition, the change may also include substitutions in non-native compounds.

In addition, the change may also include all changes where one or more than one amino acid is attached to the N- and/or C-terminus of the native glucagon.

Not only the 20 amino acids commonly found in human proteins, but also atypical amino acids or amino acids not found in nature can be used as amino acids to be replaced or added. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptides containing these amino acids and conventional peptide sequences can be synthesized and purchased from commercial peptide suppliers such as American Peptide Company, Bachem (USA) and Anygen (Korea).

In addition, amino acid derivatives can be prepared in the same way. For example, 4-imidazoleacetic acid and so on can be used.

As used herein, the term "native glucagon, glucagon analogue or glucagon derivative fragment" refers to a form wherein one or more amino acids are removed from the N- and/or C-terminus of the native glucagon, glucagon analogue or natural glucagon derivative, while which has the effect of increasing blood glucose in the body.

In addition, the glucagon analogue of the present invention can be prepared using production methods used in the production of natural glucagon derivatives and fragments, independently or in combination.

The pharmaceutical composition of the present invention may contain (1) natural glucagon, (2) a glucagon analog, (3) a glucagon derivative, (4) a fragment thereof, or (5) a combination thereof as glucagon being one of the active ingredients, provided that They perform the function of glucagon in controlling blood glucose.

In addition, the peptides of the present invention (eg, insulin, insulin analog, glucagon, glucagon analog, etc.) may include peptides in which the N-terminus and/or C-terminus are not modified. However, the peptides of the present invention also include modified peptides where the N-terminus and/or C-terminus are chemically modified(s) or protected with organic groups, or an amino acid is attached to the end of the peptide, and so on. When the C-terminus is not modified, this end of the peptide of the present invention may have a carboxyl group, but is not particularly limited to this.

In particular, in the case of a peptide that is chemically synthesized, its N- and C-termini are charged, and N-terminal acetylation and/or C-terminal amidation can be performed to remove this charge, but is not particularly limited to this.

Specifically, the N- and C-termini of insulin or an analog thereof or glucagon or an analogue thereof of the present invention may have an amino group (-NH ₂ ) or a carboxyl group (-COOH), and the C-terminus of glucagon or an analogue thereof may have an amino group, but they are not limited to this.

Unless otherwise indicated in the present invention, the descriptions given in the detailed description or claims relating to a “peptide” of the present invention or a “conjugate” where such a peptide is covalently linked to a biologically compatible substance may also apply to forms containing only the corresponding peptide or conjugate, but also salts of the corresponding peptide or conjugate (eg, pharmaceutically acceptable salts thereof) or solvates thereof. Accordingly, even in the case where a “peptide” or a “conjugate” is described in the present invention, its description can also be equally applied to its specific salt, its specific solvate, and its specific solvate. These salts may, for example, be presented in the form of any pharmaceutically acceptable salts. There are no specific restrictions on the type of salt. However, the salt is preferably, but not particularly limited to, a salt that is safe and effective for use in an individual, such as a mammal.

The term "pharmaceutically acceptable" refers to a substance that can be effectively used for its intended use in a pharmacomedical solution without inducing excessive toxicity, irritation, allergic responses, and the like.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, 2-naphthalenesulfonic acid, benzenesulfonic acid and so on. Examples of salts derived from suitable bases may contain alkali metals such as sodium, potassium and so on, alkaline earth metals such as magnesium, ammonium and so on.

Additionally, as used herein, the term “solvate” refers to a complex formed by a peptide, conjugate, or salt thereof of the present invention and a solvent molecule.

In addition, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, using an automatic peptide synthesizer, and can also be obtained using genetic engineering techniques.

In particular, the peptide of the present invention can be produced by a standard synthesis method, a recombinant expression system, or any other method known in the art. Accordingly, the glucagon derivative of the present invention can be synthesized by various methods, including, for example, the methods below:

(a) a method for solid-phase or liquid-phase synthesis of a peptide, stepwise or by fragment assembly, followed by isolation and purification of the final peptide product;

(b) a method for expressing a nucleic acid construct encoding a peptide in a host cell and isolating the expression product from a host cell culture; or

(c) a method for performing cell-free expression of a nucleic acid construct encoding a peptide in vitro and isolating the expression product; or

a method for producing peptide fragments by any combination of methods (a), (b) and (c), with the production of a peptide by linking said peptide fragments and subsequent isolation of the peptide.

As a more specific example, the desired peptide can be obtained by genetic manipulation by obtaining a fusion gene encoding a fusion protein containing a fusion partner and insulin, glucagon or an analog thereof, transforming a host cell with said fusion gene, expressing said fusion gene in the form of the fusion protein , and cleaving and releasing insulin, glucagon or an analogue thereof from the fusion protein using a protease or compound. To do this, for example, a DNA sequence encoding amino acid residues that can be cleaved by a protease such as factor Xa or enterokinase, CNBr or a compound such as hydroxylamine can be introduced between the fusion partner and a polynucleotide encoding insulin, glucagon or an analog thereof.

In one specific embodiment, the insulin or glucagon of the present invention can be presented in the form of a long-acting conjugate, where it is associated with a biocompatible substance capable of increasing its half-life in vivo.

As used herein, a "long-acting conjugate" or "conjugate" of insulin or glucagon has a structure wherein the insulin or glucagon is bound to a biocompatible substance and may exhibit an increased duration of effectiveness compared to insulin or glucagon not bound to such a biocompatible substance. In a long-acting conjugate, the biocompatible substance may be linked to insulin or glucagon through a covalent bond, without limitation. In the present invention, the insulin or glucagon that is one of the components of the conjugate may be insulin or glucagon having a native sequence, or may be an insulin or glucagon analog or derivative or fragment thereof with the substitution, addition, deletion, modification of one or more amino acids of the native sequence or a combination thereof. However, provided that it has the effect of natural insulin or glucagon in lowering/increasing blood glucose, it can be used as one of the components of the conjugate of the present invention without limitation.

As used herein, the term "biocompatible substance" refers to a substance associated with a physiologically active substance (for example, a glucagon derivative, insulinotropic peptide, etc.), thereby increasing the duration of its effectiveness compared to a physiologically active substance not related to the biocompatible group. substance or carrier. The biologically compatible substance may be linked to the physiologically active substance covalently, without being particularly limited thereto.

Specifically, the conjugate is a conjugate represented by the following chemical formula 1:

[chemical formula 1]

XL _a -F,

where: X represents insulin or glucagon;

L represents a linker;

a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent; And

F is a substance capable of increasing the half-life of X in vivo.

The composition of the present invention may contain (1) insulin and glucagon, (2) a long-acting insulin-glucagon conjugate, (3) insulin and a long-acting glucagon conjugate, or (4) a long-acting insulin conjugate and a long-acting glucagon conjugate. Insulin or glucagon in the form of a long-acting conjugate may reduce the side effects of insulin while providing superior blood glucose control benefits based on improved duration of effectiveness.

In the conjugate, F is a substance capable of increasing the half-life of X, that is, insulin or glucagon, and corresponds to one of the structures of the moieties constituting the conjugate of the present invention.

F and X may be linked to each other via a covalent chemical bond or a non-covalent chemical bond, and F and X may be linked to each other via L via 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, for example, may be selected from the group consisting of a high molecular weight polymer, a fatty acid, cholesterol, albumin and a fragment thereof, an albumin-binding substance, a polymer of repeating units with a specific amino acid sequence, antibody, antibody fragment, FcRn binding substance, in vivo connective tissue, nucleotide, fibronectin, transferrin, saccharide, heparin and elastin, but not limited to.

Elastin may be human tropoelastin, which is a water-soluble precursor, and may be a polymer of some sequences or some repeat units, for example, including but not limited to all elastin-like polypeptides.

Examples of the high molecular weight polymer may include polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotides, and combinations thereof, and the polysaccharides may be dextran, but are not limited to this.

Polyethylene glycol is a general term including, but not specifically limited to, all forms of ethylene glycol homopolymers, PEG copolymers (polyethylene glycol, PEG) and monomethyl-substituted PEG polymers (mPEG).

In addition, the biocompatible material may include, but is not limited to, polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid.

In addition, the fatty acid may have binding affinity for albumin in vivo, but is not limited to this.

As a more specific example, the FcRn-binding substance may be an immunoglobulin Fc region and, more particularly, an IgG Fc region, but is not particularly limited thereto.

A side chain of one or more amino acids of a peptide of the present invention may be coupled to a biocompatible substance to increase its solubility and/or half-life in vivo and/or increase its bioavailability. These modifications may reduce the clearance of therapeutic proteins and peptides.

The biocompatible substance described above may be soluble (amphipathic or hydrophilic) and/or non-toxic and/or pharmaceutically acceptable.

F may be linked to X directly (i.e., in chemical formula 1, a represents 0), or F may be linked to X via a linker (L).

As used herein, "immunoglobulin Fc region" refers to the region comprising heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions of an immunoglobulin. The Fc region of an immunoglobulin may be one of the structures constituting the conjugate moiety of the present invention.

The Fc region of an immunoglobulin may comprise, but is not limited to, a heavy chain constant region hinge region. In addition, the Fc region of the immunoglobulin of the present invention may be an extended Fc region containing part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1), excluding the heavy chain and light chain variable regions immunoglobulin, provided that the Fc region of the immunoglobulin produces an effect substantially identical to or superior to that of the native type. In addition, the Fc region of the immunoglobulin of the present invention may be a region from which an extended portion of the amino acid sequence corresponding to CH2 and/or CH3 has been deleted.

For example, the Fc region of the immunoglobulin of the present invention may be: (1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (2) CH1 domain and CH2 domain; (3) CH1 domain and CH3 domain; (4) CH2 domain and CH3 domain; (5) a combination of one or two or more domains of a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain and an immunoglobulin hinge region (or part of an immunoglobulin hinge region); and (6) a dimer of each heavy chain constant region and light chain constant region domain; but without limiting it.

Additionally, in one particular embodiment, the immunoglobulin Fc region may be in dimeric form, and one X molecule may be covalently linked to the dimeric form Fc region, wherein the immunoglobulin Fc and X may be linked to each other through a non-peptide polymer. Moreover, two molecules and X can also be conjugated in a symmetrical manner to one Fc region of the dimeric form. In this regard, the immunoglobulin Fc and X may be linked to each other through a non-peptide linker, but without being limited to the embodiment described above.

In addition, the Fc region of the immunoglobulin of the present invention includes not only the native amino acid sequence, but also a derivative of this sequence. An amino acid sequence derivative refers to an amino acid sequence that differs from the native amino acid sequence by one or more amino acid residues due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

For example, amino acid residues at positions 214-238, 297-299, 318-322 or 327-331, which are known to be important for IgG Fc binding, can be used as suitable sites for modification.

In addition, various other derivatives are possible, including derivatives having a deletion of a region capable of forming a disulfide bond, or a deletion of certain amino acids at the N-terminus of the native Fc, or the addition of a methionine residue at the N-terminus of the native Fc. In addition, deletions in the complement binding site, such as the Clq binding site, and the antibody-dependent cell-mediated cytotoxicity (ADCC) site are possible to eliminate effector functions. Methods for obtaining such derivatives of the immunoglobulin Fc region sequence are disclosed in the publications of international patent applications No. WO 97/34631, WO 96/32478 and so on.

Amino acid substitutions in proteins and peptides are known in the art and generally do not change the activity of the molecule (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions are 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 both directions. If necessary, the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and so on.

The Fc derivatives described above exhibit biological activity identical to that of the Fc region of the present invention, and have improved structural stability under heating, pH changes, and so on.

In addition, this Fc region may be derived from native forms isolated in vivo from humans or animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or may be recombinants thereof or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining the Fc region from native immunoglobulin may be a method of isolating full-length immunoglobulin from a living human or animal body and then treating the isolated immunoglobulin with a protease. When treated with papain, immunoglobulin is split into Fab and Fc regions. When treated with pepsin, immunoglobulin is split into pF'c and F(ab) ₂ . Fc or pF'c can be isolated using size exclusion chromatography and so on. In a more specific embodiment, the Fc region of human origin is a recombinant immunoglobulin Fc region obtained from a microorganism.

In addition, the Fc region of an immunoglobulin may have natural sugar chains or increased or decreased sugar chain content compared to the natural type, or may be present in deglycosylated form. Increasing or decreasing the content of sugar chains or removing sugar chains of immunoglobulin Fc can be achieved by conventional methods such as chemical method, enzyme method and genetic engineering method using a microorganism. Here, the immunoglobulin Fc region obtained by removing sugar chains from the Fc region exhibits a significant reduction in binding affinity to the Clq moiety and a reduction or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and thus does not induce unnecessary immune responses in vivo . In this aspect, a deglycosylated or aglycosylated immunoglobulin Fc region may be a more suitable form for the original purpose of the present invention as a drug carrier.

As used herein, the term "deglycosylation" refers to the enzymatic removal of sugar moieties from the Fc region, and the term "aglycosylation" refers to the non-glycosylated Fc region produced in prokaryotes, more specifically in E. coli.

Meanwhile, the Fc region of an immunoglobulin may be of human or other animal origin, including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. In a more specific incarnation, she has human origins.

In addition, the Fc region of an immunoglobulin may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant proteins in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to increase the half-life of ligand binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4, without limitation.

However, as used herein, “combination” means that polypeptides encoding single-chain Fc regions of immunoglobulins of the same origin are combined with a single-chain polypeptide of a different origin to form a dimer or multimer. That is, it is possible to form a dimer or multimer from two or more fragments selected from the group consisting of an IgG Fc fragment, an IgA Fc fragment, an IgM Fc fragment, an IgD Fc fragment and an IgE Fc fragment.

In addition, the conjugate described above may have an increased duration of effectiveness compared to natural insulin or glucagon or compared to X not modified with F, and the conjugate includes not only the form described above, but also a form where the conjugate is included in biodegradable nanoparticles.

In addition, L is a peptidyl linker or a non-peptidyl linker.

When L is a peptidyl linker, it may contain one or more amino acids, for example from 1 amino acid to 1000 amino acids, but not particularly limited thereto. In the present invention, a variety of known peptidyl linkers can be used to link F and X, and examples thereof may include [GS] _x linker, [GGGS] _x linker and [GGGGS] _x linker, where x can be a natural number being 1 or more, but binding is not limited to this.

As used herein, a “non-peptidyl linker” includes a biocompatible polymer where two or more than two repeating units are linked. The repeating units are linked to each other by any covalent bond other than a peptide bond. The non-peptidyl linker may be one of the structures constituting the conjugate moiety of the present invention and corresponds to L in Chemical Formula 1. In the present invention, the non-peptidyl linker may be used interchangeably with “non-peptidyl polymer”.

In the present invention, the non-peptidyl linker contains reactive groups at its ends, and thus a conjugate can be formed by reaction with other components constituting the conjugate. When a non-peptidyl polymer having reactive functional groups at both ends can be linked to X and F in Chemical Formula 1 through these reactive groups to form a conjugate, the non-peptidyl linker or non-peptidyl polymer can be called a non-peptidyl polymer linker moiety or a non-peptidyl linker moiety .

In L _a a can be 1 or more and, when a is 2 or more, each L can be independent.

Additionally, in one specific embodiment, the conjugate may be a conjugate wherein F, specifically the Fc region of an immunoglobulin, and X, specifically a peptide drug, can be covalently linked to each other via a non-peptide linker containing reactive groups capable of reacting with X and F.

Specifically, the non-peptidyl linker may be selected from the group consisting of fatty acids, polysaccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

In the present invention, the high molecular weight polymer may have a molecular weight ranging from greater than 0 kDa to about 100 kDa, specifically from about 1 kDa to about 100 kDa, and more particularly from about 1 kDa to about 20 kDa, without limitation.

As used herein, the term “about” refers to a range including all of plus/minus 0.5, plus/minus 0.4, plus/minus 0.3, plus/minus 0.2, plus/minus 0.1, etc. hereinafter and includes all values equivalent to, but not limited to, the values indicated immediately following the term “about” or values within a similar range.

The polymer may include, but is not limited to, polymers selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotides, and combinations thereof , and the polysaccharides may be dextran. In a more specific embodiment, L may be polyethylene glycol, but is not limited to this. Moreover, the scope of the present invention may also include derivatives thereof already known in the art and derivatives that can be easily obtained at the technological level of the art.

The non-peptidyl linker that can be used in the present invention can be used without limitation, provided that it is a polymer that is resistant to proteases in vivo. The molecular weight of the non-peptidyl polymer can range from, but is not limited to, about 1 kDa to about 100 kDa, and specifically from about 1 kDa to about 20 kDa. In addition, the non-peptidyl linker of the present invention associated with a polypeptide corresponding to F may include not only one type of polymer, but also a combination of different types of polymers.

As used herein, the term “about” refers to a range including all of plus/minus 0.5, plus/minus 0.4, plus/minus 0.3, plus/minus 0.2, plus/minus 0.1, etc. hereinafter and includes all values equivalent to, but not limited to, the values indicated immediately following the term “about” or values within a similar range.

In one particular embodiment, both ends of the non-peptidyl linker may be linked to an amino group or thiol group of F, that is, the Fc region of an immunoglobulin, and to an amino group or thiol group of X.

Specifically, the non-peptidyl polymer may contain reactive groups at both ends capable of binding to F (i.e., the Fc region of an immunoglobulin) and X, respectively, and specifically reactive groups capable of binding to an amino group located at the N-terminus or lysine, or with a thiol group of cysteine X or F (ie, the Fc region of an immunoglobulin), but not limited to this.

In addition, the reactive groups of the non-peptidyl polymer capable of binding to F, such as the Fc region of an immunoglobulin, and X may be selected from the group consisting of, but not limited to, an aldehyde group, a maleimide group, and a succinimide derivative.

In the above, examples of the aldehyde group may include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

In the above, examples of the succinimidyl derivative may include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanonate, succinimidyl methyl propionate, succinimidyl butanonate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate.

The non-peptidyl linker may be linked to X and F through these reactive groups, but is not limited to this.

In addition, the final product obtained by reductive amination via an aldehyde bond is more stable than the product linked by an amide bond. The aldehyde reactive group selectively interacts with the N-terminus at low pH, while at high pH, such as pH 9.0, it can form a covalent bond with the lysine residue.

In addition, the reactive groups at both ends of the non-peptidyl linker may be the same or different from each other; for example, a maleimide group may be present at one end, and an aldehyde group, propionaldehyde group, or butyraldehyde group may be present at the other end. However, if F, specifically the Fc region of an immunoglobulin, and X can be conjugated to each end of a non-peptidyl linker, there are no particular restrictions regarding the reactive groups.

For example, the non-peptidyl polymer may contain, as reactive groups, a maleimide group at one end and an aldehyde group, propionaldehyde group or butyraldehyde group at the other end.

When using polyethylene glycol having a reactive hydroxy group at both ends thereof as a non-peptidyl polymer, the hydroxy group can be activated to form various reactive groups through known chemical reactions, or polyethylene glycol with a commercially available modified reactive group can be used to obtain a long-acting protein conjugate of the present invention. group.

In one particular embodiment, the non-peptidyl polymer may be linked to a cysteine residue X and, more specifically, to a cysteine SH group, but is not limited to this.

For example, a non-peptidyl polymer may be linked to a cysteine residue at position 10, a cysteine residue at position 13, a cysteine residue at position 15, a cysteine residue at position 17, a cysteine residue at position 19, a cysteine residue at position 21, a cysteine residue at position 24, a cysteine residue at position 28, a cysteine residue at position 29, a cysteine residue at position 30, a cysteine residue at position 31, a cysteine residue at position 40, or a cysteine residue at position 41 of the peptide corresponding to X, but not specifically limited to this.

Specifically, the reactive group of the non-peptidyl polymer may be bonded to the -SH group of the cysteine residue, and the above also applies to the reactive group. When using a maleimide-PEG aldehyde, the maleimide group may be linked to the SH X group by a thioether linkage, and the aldehyde group may be linked to the -NH ₂ F, specifically the Fc region of an immunoglobulin, by, but not limited to, reductive amination, and the above is only a typical embodiment.

In addition, in the conjugate described above, the reactive group of the non-peptidyl polymer may be linked to the -NH ₂ located at the N-terminus of the Fc region of the immunoglobulin, but this is only a typical embodiment.

As used herein, the term "prevention" refers to all types of activities associated with inhibiting or delaying the onset of insulin-related diseases through the administration of a composition, and the term "treatment" refers to all types of activities associated with improving or beneficially changing the symptoms of insulin-related diseases. with insulin, due to the administration of the composition.

As used herein, the term "administration" refers to the administration of a specific substance to a patient in a suitable manner. The composition can be administered in a conventional manner allowing delivery of the composition to the target tissue in vivo, such as, but not particularly limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and intrarectal administration.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" refers to the property of being present in sufficient quantity to produce a therapeutic effect without undesirable effects and can be readily determined by one skilled in the art based on factors well known in the medical field, such as disease type, age, body weight , health status, gender of the patient, his sensitivity to drugs, route of administration, mode of administration, frequency of administration, duration of treatment, drug to be mixed or administered simultaneously in combination and so on.

A pharmaceutically acceptable carrier may include: for oral administration, a binding agent, a glidant, a disintegrating agent, an excipient, a solubilizing agent, a dispersing agent, a stabilizing agent, a suspending agent, a coloring agent, a flavoring agent, and the like; for injection - a buffering agent, preservative, analgesic, solubilizing agent, isotonic agent, stabilizing agent and so on, which can be used in combination; and, for topical administration, a base, excipient, lubricant, preservative, and so on. The pharmaceutical composition of the present invention can be prepared in various ways by combination with a pharmaceutically acceptable carrier as described above. For example, for oral administration, the composition may be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. For injection, the composition may be formulated in single-dose ampoules or multi-dose containers. The composition may also be formulated as solutions, suspensions, tablets, pills, capsules, sustained release formulations, and the like.

Meanwhile, examples of carriers, excipients and diluents suitable for the composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and so on. In addition, the composition may further contain a filler, an anticoagulant, a lubricant, a humectant, a flavoring agent, an emulsifier, a preservative, and so on.

Moreover, the pharmaceutical composition of the present invention can be formulated in any form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents , lyophilized compositions and suppositories.

In addition, the conjugate can be used by mixing with various pharmaceutically acceptable carriers such as saline or organic solvents. To enhance stability or absorption, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used.

The administered dose and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of active ingredient(s) and various factors such as the disease being treated, the route of administration, the age, sex and weight of the patient and the severity of the disease.

The total effective dose of the composition of the present invention can be administered to a patient in a single dose or can be administered over an extended period of time in multiple doses according to a fractionated administration protocol. The content of active ingredient(s) in the pharmaceutical composition of the present invention may vary depending on the severity of the disease. Specifically, the preferred total daily dose of the conjugate of the present invention may be from about 0.0001 mg to 500 mg per 1 kg of body weight of the patient. However, the effective dose of the conjugate is determined by taking into account various factors, including the age, body weight, health status and gender of the patient, severity of the disease, nutrition and elimination rate, along with the route and frequency of administration of the pharmaceutical composition. In this regard, those skilled in the art can readily determine an effective dose suitable for a particular use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in terms of composition and route of administration as long as it produces the effects of the present invention.

In connection with the pharmaceutical composition of the present invention, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof can be administered simultaneously, sequentially or in reverse order, but the administration is not limited thereto.

As described above, combination administration may include:

(a) administering (1) insulin or a long-acting conjugate thereof and (2) glucagon or a long-acting conjugate thereof in one mixture; or

(b) administering each of (1) insulin or a long-acting conjugate thereof and (2) glucagon or a long-acting conjugate thereof in separate form, but without limitation.

When each of insulin or its long-acting conjugate and glucagon or its long-acting conjugate is administered in separate form, insulin or its long-acting conjugate and glucagon or its long-acting conjugate are included in separate preparations, which are then administered simultaneously, separately, sequentially or in reverse ok.

To achieve the present invention, according to another aspect, there is provided a pharmaceutical composition for preventing or treating hypoglycemia containing insulin and glucagon.

Specifically, the composition may contain:

(1) insulin and glucagon;

(2) a long-acting insulin conjugate, wherein insulin and a biocompatible substance capable of increasing its half-life in vivo are associated with each other, and glucagon;

(3) insulin and a long-acting glucagon conjugate, wherein the glucagon and a biocompatible substance capable of increasing its half-life in vivo are linked to each other; or

(4) long-acting insulin conjugate and long-acting glucagon conjugate.

The insulin or glucagon of the present invention may be native sequence insulin or glucagon, or may be an analogue, derivative or fragment thereof obtained by substitution, addition, deletion, modification of one or more amino acids or combinations thereof, as described above .

The pharmaceutical composition for preventing or treating hypoglycemia of the present invention may contain insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof and thus may have the effect of alleviating hypoglycemia occurring as a side effect caused by the administration of insulin while providing effect of suppressing body weight gain.

In one exemplary embodiment of the present invention, the effects of lowering blood glucose, suppressing body weight gain, and alleviating hypoglycemia were confirmed in db/db mice that were administered a composition containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and thus Thus, it has been demonstrated that the pharmaceutical composition of the present invention can maintain the effectiveness of insulin while reducing its side effects.

In connection with the pharmaceutical composition of the present invention, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be administered in combination simultaneously, sequentially, or in reverse order, but without limitation. Combined administration is the same as described above.

To achieve the present invention, according to another aspect, there is provided a composition for reducing the side effects of insulin containing insulin and glucagon.

Specifically, the composition may contain:

(1) insulin and glucagon;

(2) a long-acting insulin conjugate, wherein insulin and a biocompatible substance capable of increasing its half-life in vivo are associated with each other, and glucagon;

(3) insulin and a long-acting glucagon conjugate, wherein the glucagon and a biocompatible substance capable of increasing its half-life in vivo are linked to each other; or

(4) long-acting insulin conjugate and long-acting glucagon conjugate.

The insulin or glucagon of the present invention may be native sequence insulin or glucagon, or may be an analogue, derivative or fragment thereof obtained by substitution, addition, deletion, modification of one or more amino acids or combinations thereof, as described above .

The composition for reducing the side effects of insulin of the present invention may contain insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof and thus may have the effect of reducing the side effects caused by the administration of insulin (eg, hypoglycemia or weight gain).

In connection with the pharmaceutical composition of the present invention, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be administered in combination simultaneously, sequentially, or in reverse order, but without limitation. Combined administration is the same as described above.

To achieve the present invention, according to another aspect, there is provided a complex composition for reducing hypoglycemia in a patient with an insulin-related disease, comprising:

(1) insulin and glucagon;

(2) long-acting insulin-glucagon conjugate;

(3) insulin and long-acting glucagon conjugate; or

(4) long-acting insulin conjugate and long-acting glucagon conjugate.

In one specific embodiment, the complex composition of the present invention may contain a pharmaceutically acceptable carrier.

As used herein, the terms “pharmaceutically acceptable” and “pharmaceutically acceptable carrier” are the same as described above.

The complex composition of the present invention can be a complex composition for treating an insulin-related disease, and can have the effect of alleviating side effects caused by insulin administration (eg, hypoglycemia or weight gain). Specifically, the complex composition can reduce hypoglycemia, which is a side effect of insulin, and can also suppress weight gain.

The complex composition of the present invention may contain insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a mass ratio from 0.1:1 to 100:1 or in a molar ratio from 1:1 to 30:1, from 1:1 up to 20:1 or from 1:1 to 16:1, but not limited to this.

To achieve the present invention, according to another aspect, there is provided a method of preventing or treating an insulin-related disease, comprising the step of administering (1) insulin and glucagon, (2) a long-acting insulin-glucagon conjugate, (3) insulin and a long-acting glucagon conjugate or ( 4) a long-acting insulin conjugate and a long-acting glucagon conjugate to an individual at risk of developing an insulin-related disease or an insulin-related disease.

The administration step can be carried out by combined administration of (1) insulin and glucagon, (2) a long-acting insulin-glucagon conjugate, (3) insulin and a long-acting glucagon conjugate, or (4) a long-acting insulin conjugate and a long-acting glucagon conjugate. The substances may be administered simultaneously, sequentially, or in reverse order, and may be administered together in a combination of suitable effective amounts, without limitation to a particular route or order of administration. In addition, the prevention or treatment method of the present invention may be to administer to an individual a composition or complex composition containing any of (1) to (4), without limitation. Insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be separate compositions or a complex composition.

The composition or complex composition containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof of the present invention can complement the activity or function of insulin to significantly lower blood glucose while suppressing the side effects of insulin such as weight gain and hypoglycemia, thereby providing excellent therapeutic effects on insulin-related diseases.

In addition, the composition or complex composition can be formulated in a single-dose form suitable for the patient's body, and, specifically, in the form of a preparation useful for administering protein drugs, in a manner conventional in the pharmaceutical field, and can be administered using a method of administration, commonly used in the art, by oral or parenteral routes, such as, but not limited to, percutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal this.

The method of the present invention may include administering a pharmaceutical composition containing the peptide in a pharmaceutically effective amount. The total daily dose must be determined by a physician within the framework of proper medical logic and administered at one time or several times in divided doses. In connection with the purposes of the present invention, in each individual patient, the specific therapeutically effective dose may preferably be administered differently depending on various factors well known in the medical field, including the type and extent of response to be achieved, the specific compositions, including periodic co-administration use of other agents, age, body weight, general health, sex and nutrition of the patient, time and route of administration, rate of elimination of the composition, duration of treatment, other drugs used in combination or concurrently with a particular composition, and similar factors well known in the art medical field.

To achieve the present invention, according to yet another aspect, there is provided a method of reducing the side effects of insulin, comprising the step of administering (1) insulin and glucagon, (2) a long-acting insulin-glucagon conjugate, (3) insulin and a long-acting glucagon conjugate, or (4) a long-acting glucagon conjugate. an insulin conjugate and a long-acting glucagon conjugate to an individual at risk of developing an insulin-related disease or an insulin-related disease.

According to yet another aspect of the present invention, there is provided the use of a composition or complex composition in the manufacture of a medicament for the treatment of an insulin-related disease.

OPTION FOR IMPLEMENTATION OF THE INVENTION

The present invention will now be described in more detail with reference to the following Examples. However, the following Examples are provided for illustrative purposes only, and the scope of the present invention is not limited to the Examples provided.

Example 1: Preparation of glucagon analogues

It was the intention of the present inventors to confirm the therapeutic effects on diabetes of combined administration of insulin and glucagon, and to determine whether the risk of hypoglycemia, a side effect of insulin, could be reduced by such combination administration.

Accordingly, native glucagon and glucagon analogues having activity equivalent to that of native glucagon were prepared as described in Table 1 below. Glucagon analogues were synthesized as described in Korean Patent Publications Nos. 10-2016-0082482, 10-2017-003466 and 10-2018-0002544, and their pI and in vitro activity were measured.

In the sequences described in Table 1, the amino acid represented by X represents the non-native amino acid, aminoisobutyric acid (Aib), the underlined amino acid residues correspond to the formation of a lactam ring between the side chains of the corresponding underlined amino acids and "-" indicates the absence of an amino acid residue at the corresponding position . Additionally, in the column related to ring formation, “−” indicates no ring formation in the corresponding sequences.

Example 2: Preparation of insulin analogues

The authors of the present invention intended to produce insulin analogues for combined administration with glucagon or its long-acting conjugate. Specifically, the analogs shown in Table 2 below were obtained as described in Korean Patent Publication No. 10-2014-0106452.

The amino acid sequence variation of the A chain or B chain and the names of the analogues are shown in Table 2 below. That is, analog 1 means that the glycine at position 1 of the A chain has been replaced by alanine, and analog 4 means that the glycine at position 8 of the B chain has been replaced by alanine.

The DNA sequences and protein sequences of each of the insulin analogues 1-9 are shown in Table 3 below.

In addition, the inventors of the present invention obtained the insulin analogues shown in Table 4 below, as described in Korean Patent Publication No. 10-2017-0026284.

The DNA sequences and protein sequences of each of the insulin analogues 10 and 11 are shown in Table 5 below.

In addition, the inventors of the present invention have prepared insulin analogues shown in Table 6 below. In detail, forward and reverse oligonucleotides were synthesized (Table 7) to produce insulin analogues with one modified amino acid A chain or B chain using a natural insulin expression vector as a template and PCR (polymerase chain reaction) was performed to amplify the genes of each analogs.

Primers for amplification of insulin analogues are shown in Table 7 below.

To amplify insulin analogs, PCR was performed under conditions of 18 cycles at 95°C for 30 sec, 55°C for 30 sec, and 68°C for 6 min. Fragments of insulin analogues obtained under the conditions described above were introduced into the pET22b vector for expression in the form of intracellular inclusion bodies, and the resulting expression vectors were named pET22b-insulin analogues 12-24. The expression vectors contained nucleotides encoding the amino acid sequences of each of the insulin analogues 12-24, under the control of the T7 promoter. The protein of each insulin analogue was expressed in the form of inclusion bodies in host cells containing the expression vector.

The DNA sequences and protein sequences of each of the insulin analogues 12-24 are shown in Table 8 below.

Each sequence change was analyzed by DNA sequencing and it was confirmed that the sequence of each of the insulin analogues was changed in the expected manner.

Example 3: Expression of Recombinant Insulin Analog Fusion Peptide

Expression of recombinant insulin analogues was carried out under the control of the T7 promoter. E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λDE3; Novagen) was transformed with each of the recombinant insulin analogue expression vectors. The transformation procedure was carried out in accordance with Novagen recommendations. Each of the individual colonies transformed with each of the recombinant expression vectors was collected, plated in 2X liquid Luria Broth (LB) medium containing ampicillin (50 μg/ml), and incubated at 37°C for 15 hours. The culture liquid of the recombinant strain was mixed with 2X LB medium containing 30% glycerol in a ratio of 1:1 (v/v), and each 1 ml of this mixture was added to cryovials and stored at 140°C, then using them as initial cells to produce recombinant fusion proteins.

To express recombinant insulin analogs, 1 tube of each source cell was thawed, added to 500 ml of 2X liquid Luria (LB) medium, and then incubated with shaking at 37°C for 14-16 hours. When the OD600 value (optical density at 600 im) reached 5.0 or more, the cultivation was stopped and the culture liquid was used as a seed culture. The seed culture was sown in 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, B.E. MARUBISHI, Japan) and the first batch fermentation was started. The following culture conditions were maintained: temperature 37°C, air flow rate 20 L/min (1 rpm), rocking speed 500 rpm and pH 6.70 using 30% ammonia solution. Fermentation was carried out in fed-batch mode, adding a feeding solution as the nutrient content in the culture liquid decreased. Strain growth was monitored by OD value. When the OD value was more than 100, IPTG was added at a final concentration of 500 μM. After application, cultivation was carried out for another 23-25 hours. After completion of cultivation, the recombinant strains were collected using a centrifuge and stored at -80°C until use.

Example 4: Isolation and Refolding of Recombinant Insulin Analogues

To convert the recombinant insulin analogues expressed in Example 3 into soluble forms, cell destruction was carried out followed by refolding. 100 g (wet weight) of cell pellet was resuspended in 1 L of lysis buffer (50 mM Tris-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton X- 100). Cells were disrupted using a microfluidizer (Microfluidic Corp., model M-110EN-30) at an operating pressure of 15,000 psi (103.4 MPa). The lysate of cells destroyed in this way was centrifuged at 7000 rpm and 4-8°C for 20 minutes. The supernatant was removed, and the pellet was resuspended in 3 L of wash buffer (0.5% Triton X-100 and 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA). After centrifugation at 7000 rpm and 4-8°C for 20 minutes, the cell pellet was resuspended in distilled water, followed by centrifugation in the same way. The pellet was collected and resuspended in buffer (1 M L-glycine, 3.78 g L-cysteine-HCl, pH 10.6) and stirred at room temperature for 1 hour. To isolate the recombinant insulin analog resuspended in this way, 8 M urea was added, followed by stirring for 3-5 hours. To refold solubilized recombinant insulin analogues, centrifugation was performed at 7000 rpm and 4-8°C for 30 minutes, the supernatant was obtained and treated with a reducing agent (15 mM L-cysteine-HCl) for 1 hour. A predetermined multiple equivalent of distilled water was added to it using a peristaltic pump with stirring at 4-8°C for 12 hours or more.

Example 5: Purification by Cation Exchange Chromatography

The refolded sample was loaded onto an SP FF column (GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol, and insulin analog proteins were then eluted onto 10 column volumes with a linear gradient from 0% to 100% using 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol.

Example 6: Treatment with trypsin and carboxypeptidase B

The eluted samples were desalted using an ultrafiltration membrane and the buffer was replaced with another buffer (10 mM Tris-HCl, pH 8.0). Trypsin was added to the resulting protein sample at a molar ratio of about 30,000 and carboxypeptidase B at a molar ratio of about 30,000, and then it was stirred at 4-8°C for 16 hours or more.

Example 7: Purification by Cation Exchange Chromatography

The sample resulting from this reaction was loaded onto an SP HP column (GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol, and insulin analogue proteins were then eluted into 10 column volumes with a linear gradient from 0% to 100% using 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol.

Example 8: Purification by Reverse Phase Chromatography To isolate the pure insulin analogue from the sample obtained in Example 7, the sample was loaded onto a Source 30RPC reverse phase chromatography (GE Healthcare, USA) equilibrated with a buffer containing sodium phosphate and isopropanol, and then the proteins insulin analogues were eluted with a linear gradient using a buffer containing sodium phosphate and isopropanol.

Example 9: Preparation of long-acting insulin-glucagon conjugates

Long-acting insulin-glucagon conjugates were prepared by coupling insulin and the glucagon analogue prepared in the Examples to the Fc region of an immunoglobulin using polyethylene needles as a linker, respectively. The glucagon used in the preparation of the long-acting glucagon conjugate was an analog having a pI of 6 to 7 and an in vitro activity of 200% or more of that of native glucagon. The insulin used in the preparation of the long-acting insulin conjugate was natural insulin or an analogue obtained by replacing one amino acid of the A chain of natural insulin.

In detail, for PEGylation of the N-terminus of the insulin B chain (Biocon, India) using 3.4 K propion-ALD(2) PEG (PEG with a molecular weight of 3.4 kDa, having one propionaldehyde group at both ends, NOF, USA ) reacted insulin and PEG in a molar ratio of 1:4 at an insulin concentration of 5 mg/ml and 4°C for approximately 2 hours. In this case, the reaction was carried out in a mixed solvent of 50 mM sodium citrate (pH 5.0) and 45% isopropanol with the addition of 3 mM sodium cyanoborohydride (NaCNBH ₃ ) as a reducing agent. The reaction solution was purified on an SP-HP column (GE Healthcare) using a buffer containing sodium citrate (pH 3.0) and 45% EtOH and a KCl concentration gradient.

Then, to bind insulin-PEG to the N-terminus of the Fc fragment of immunoglobulin, purified mono-PEGylated insulin was reacted with the Fc fragment of immunoglobulin in a molar ratio of 1:1.2 at 25°C for 15 hours at a total protein concentration of 20 mg/ml. In this case, 100 mM HEPES buffer (pH 8.2) with sodium chloride and 20 mM sodium cyanoborohydride as a reducing agent was added to the reaction solution.

After completion of the reaction, the reaction solution was loaded onto a Q HP column (GE, USA) with a concentration gradient of buffer with Tris-HCl (pH 7.5) and NaCl and loaded onto a Source 15ISO (GE, USA) with a concentration gradient of ammonium sulfate and Tris-HCl (pH 7.5) for purification of the conjugate “insulin - 3.4K PEG - immunoglobulin Fc”.

Next, to obtain a long-acting glucagon conjugate, PEGylate the cysteine residue of a glucagon analog using a 10 kDa PEG having a maleimide group and an aldehyde group at both ends, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the interaction was carried out at a molar ratio of glucagon analogue to maleimide-PEG-aldehyde from 1:1 to 1:5 and a peptide concentration from 3 mg/ml to 10 mg/ml for 1-3 hours at low temperature. In this case, the interaction was carried out in a medium to which 50 mM Tris buffer (pH 7.5) and 20% to 60% isopropanol were added. After completion of the reaction, the reaction solution was loaded onto SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative monopegylated at cysteine.

The thus purified mono-PEGylated analogue of glucagon and immunoglobulin Fc was reacted in a molar ratio of 1:2 to 1:10 at a protein concentration of 10 mg/ml to 50 mg/ml and 4-8°C for 12-18 hours. The interaction was carried out in a medium in which 10 mM to 50 mM sodium cyanoborohydride as a reducing agent and 10% to 20% isopropanol were added to a buffer with 100 mM potassium phosphate (pH 6.0). After completion of the reaction, the reaction solution was loaded onto a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing a glucagon analog and immunoglobulin Fc.

Example 10: Study of the effect of lowering blood glucose with combined administration of glucagon and insulin to DIO/STZ rats

The long-acting conjugates of glucagon and natural insulin obtained in Example 9 were administered to DIO/STZ rats, a well-known model of obesity and type 2 diabetes. To study the effect of lowering blood glucose with the combined administration of long-acting conjugates of glucagon and natural insulin, the study groups were divided into control with a vehicle, administration of a long-acting insulin conjugate by itself (53.9 nmol/kg (2997 μg/kg), once 3 days (Q3D)) and combined administration of long-acting insulin conjugate (53.9 nmol/kg, Q3D) and long-acting glucagon conjugate (3.4 nmol/kg (182 μg/kg), Q3D), and each of the corresponding substances were injected repeatedly subcutaneously over 2 weeks. When administered repeatedly over 2 weeks, blood was collected from the tail vein of each rat and then changes in blood glucose were measured using a glucometer (OneTouch® Ultra®).

As a result of repeated administration of a long-acting insulin conjugate over 2 weeks, the high blood glucose levels observed in the vehicle control group were reduced to normal and hypoglycemic levels (70 mg/dL or less) (FIG. 1). These results indicate that high doses of insulin had a blood glucose-lowering effect but could lead to hypoglycemia. In contrast, with the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate, blood glucose normalized without persistent hypoglycemia.

In addition, changes in blood glucose during the administration period were quantified by area under the curve (AUC) and analyzed as shown in FIG. 2. As a result, with the combined administration of long-acting insulin conjugate and long-acting glucagon conjugate, the combined administration of long-acting insulin conjugate and long-acting glucagon conjugate did not significantly affect the overall effect of long-acting insulin conjugate in lowering blood glucose while preventing hypoglycemia, which is a side effect of the administration of a long-acting insulin conjugate (FIG. 2). These results indicate that combined administration of long-acting insulin conjugate and long-acting glucagon conjugate can maintain blood glucose-lowering efficacy while reducing the risk of hypoglycemia caused by long-acting insulin conjugate.

Example 11: Effect of Reducing Side Effects of Insulin by Combined Administration of Glucagon and Insulin to DIO/STZ Rats

The long-acting natural insulin conjugate used in Example 10 also has the side effect of weight gain, along with hypoglycemia, like other long-acting insulins.

Indeed, as shown in FIG. 3, when DIO/STZ mice were repeatedly administered a long-acting insulin conjugate for 2 weeks, a significant increase in body weight was observed compared to the vehicle control group. However, with the combined administration of long-acting glucagon conjugate and long-acting insulin conjugate, the weight gain was neutralized and a decrease in body weight was noted compared with the vehicle control group. These results indicate that combined administration of long-acting insulin conjugate and long-acting glucagon conjugate can alleviate hypoglycemia and weight gain, which are side effects of administration of long-acting insulin conjugate.

The exemplary embodiments described above have confirmed that combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate can suppress the side effects (hypoglycemia and weight gain) caused by insulin administration while providing a blood glucose-lowering effect. Thus, it has been confirmed that the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate of the present invention can not only have a therapeutic effect on insulin-related diseases, but also reduce hypoglycemia and the side effects of insulin.

Based on the above description, it will be clear to those skilled in the art that the present invention can be embodied in various specific forms without changing its technical concept or essential features. In this regard, it should be understood that, in all respects, the embodiments described above are for illustrative purposes and are not limiting. The scope of the invention is defined by the appended claims, but not by the foregoing description, and is therefore intended to include all changes and modifications within the scope of the claims or the equivalent of such boundaries and limits.

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<110> HANMI PHARM. CO., LTD.

<120> A pharmaceutical composition containing insulin and glucagon

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<223> Xaa is aminoisobutyric acid (Aib)

<400> 9

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Cys Asp Ala Gln Asp Phe Val Val Trp Leu Ile Asn Thr

20 25

<210> 10

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 10

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Cys Asp Ala Asp Asp Phe Val Val Trp Leu Ile Asn Thr

20 25

<210> 11

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 11

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Cys Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 12

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 12

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210> 13

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 13

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Cys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 14

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 14

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 15

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 15

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Cys Glu

1 5 10 15

Lys Arg Ala Gln Asp Phe Val Val Trp Leu Met Asn Thr

20 25

<210> 16

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 16

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys

1 5 10 15

Arg Arg Ala Gln Val Phe Val Gln Trp Leu Met Arg Thr

20 25

<210> 17

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 17

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys

1 5 10 15

Val Arg Ala Gln Asp Phe Val Gln Trp Leu Met Arg Thr

20 25

<210> 18

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 18

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Cys Asp Phe Arg Leu Trp Leu Met Asn Thr

20 25

<210> 19

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 19

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Cys Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 20

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 20

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 21

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 21

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Cys Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 22

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 22

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Cys Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 23

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 23

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 24

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 24

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 25

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 25

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Cys Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 26

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 26

Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 27

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 27

Tyr Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 28

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 28

Trp Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Cys Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 29

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 29

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 30

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 30

Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 31

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (17)..(21)

<223> amino acids at positions 17 and 21 form a ring

<400> 31

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Cys

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 32

<211> 28

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (15)..(19)

<223> amino acids at positions 15 and 19 form a ring

<400> 32

Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Cys

1 5 10 15

Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 33

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 33

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 34

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 34

Trp Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu

1 5 10 15

Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 35

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 35

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Cys Asp Glu

1 5 10 15

Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 36

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 36

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 37

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 37

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210> 38

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 38

Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 39

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 39

Tyr Xaa Gln Gly Thr Phe Val Ser Asp Cys Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 40

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 40

Tyr Xaa Gln Gly Thr Phe Val Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210> 41

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (16)..(20)

<223> amino acids at positions 16 and 20 form a ring

<400> 41

Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Arg Arg Ala Lys Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 42

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 42

Tyr Xaa Gln Gly Thr Phe Cys Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 43

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 43

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 44

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 44

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210> 45

<211> 29

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<400> 45

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Cys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210> 46

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (1)

<223> Xaa is histidine, desaminohistidyl,

N-dimethylhistidyl, beta-hydroxyimidazopropionyl,

4-imidazoacetyl, beta-carboxyimidazopropionyl, tryptophan or

tyrosine or absent

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is alpha-methylglutamic acid,

aminoisobutyric acid (Aib), D-alanine, glycine,

Sar (N-methylglycine), serine or D-serine

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa is threonine, valine or cysteine

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa is tyrosine or cysteine

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa is lysine or cysteine

<220>

<221> MISC_FEATURE

<222> (13)

<223> Xaa is tyrosine or cysteine

<220>

<221> MISC_FEATURE

<222> (14)

<223> Xaa is leucine or cysteine

<220>

<221> MISC_FEATURE

<222> (15)

<223> Xaa is aspartic acid, glutamic

acid or cysteine

<220>

<221> MISC_FEATURE

<222> (16)

<223> Xaa is glutamic acid, aspartic

acid, serine, alpha-methylglutamic acid or cysteine or

absent

<220>

<221> MISC_FEATURE

<222> (17)

<223> Xaa is aspartic acid, glutamine,

glutamic acid, lysine, arginine, serine, cysteine or

valine or absent

<220>

<221> MISC_FEATURE

<222> (18)

<223> Xaa is alanine, aspartic acid, glutamine,

glutamic acid, arginine, valine or cysteine or absent

<220>

<221> MISC_FEATURE

<222> (19)

<223> Xaa is alanine, arginine, serine, valine or cysteine

or missing

<220>

<221> MISC_FEATURE

<222> (20)

<223> Xaa is lysine, histidine, glutamic acid,

glutamine, aspartic acid, arginine,

alpha-methylglutamic acid or cysteine or absent

<220>

<221> MISC_FEATURE

<222> (21)

<223> Xaa is aspartic acid, glutamic

acid, leucine, valine or cysteine or none

<220>

<221> MISC_FEATURE

<222> (23)

<223> Xaa is isoleucine, valine or arginine or

absent

<220>

<221> MISC_FEATURE

<222> (24)

<223> Xaa is valine, arginine, alanine, cysteine,

glutamic acid, lysine, glutamine, alpha-methylglutamic

acid or leucine or absent

<220>

<221> MISC_FEATURE

<222> (27)

<223> Xaa is isoleucine, valine, alanine, lysine,

methionine, glutamine or arginine or absent

<220>

<221> MISC_FEATURE

<222> (28)

<223> Xaa is glutamine, lysine, asparagine or arginine

or missing

<220>

<221> MISC_FEATURE

<222> (30)

<223> Xaa is cysteine or absent

<400> 46

Xaa Xaa Gln Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Xaa Xaa Thr Xaa

20 25 30

<210> 47

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> glucagon derivative

<220>

<221> MISC_FEATURE

<222> (2)

<223> Xaa is aminoisobutyric acid (Aib)

<220>

<221> MISC_FEATURE

<222> (7)

<223> Xaa is threonine, valine or cysteine

<220>

<221> MISC_FEATURE

<222> (10)

<223> Xaa is tyrosine or cysteine

<220>

<221> MISC_FEATURE

<222> (12)

<223> Xaa is lysine or cysteine

<220>

<221> MISC_FEATURE

<222> (15)

<223> Xaa is aspartic acid or cysteine

<220>

<221> MISC_FEATURE

<222> (16)

<223> Xaa is glutamic acid or serine

<220>

<221> MISC_FEATURE

<222> (17)

<223> Xaa is lysine or arginine

<220>

<221> MISC_FEATURE

<222> (20)

<223> Xaa is glutamine or lysine

<220>

<221> MISC_FEATURE

<222> (21)

<223> Xaa is aspartic acid or glutamic acid

acid

<220>

<221> MISC_FEATURE

<222> (24)

<223> Xaa is valine or glutamine

<220>

<221> MISC_FEATURE

<222> (30)

<223> Xaa is cysteine or absent

<400> 47

Tyr Xaa Gln Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Tyr Leu Xaa Xaa

1 5 10 15

Xaa Arg Ala Xaa Xaa Phe Val Xaa Trp Leu Met Asn Thr Xaa

20 25 30

<210> 48

<211> 21

<212>PRT

<213> Artificial Sequence

<220>

<223> Insulin analogue, 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> 48

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> 49

<211> 30

<212>PRT

<213> Artificial Sequence

<220>

<223> Insulin analogue, 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, serine,

threonine or aspartic acid or 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 absent

<220>

<221> MISC_FEATURE

<222> (27)

<223> Xaa is threonine or absent

<220>

<221> MISC_FEATURE

<222> (28)

<223> Xaa is proline, glutamic acid or

aspartic acid or absent

<400> 49

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> 50

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 1

<400> 50

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtgcgat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 51

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 1

<400> 51

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> 52

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 2

<400> 52

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcgc ggtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 53

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 2

<400> 53

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> 54

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 3

<400> 54

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaacg cgtgcaac 258

<210> 55

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 3

<400> 55

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> 56

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 4

<400> 56

ttcgttaacc aacacttgtg tgcgtcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 57

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 4

<400> 57

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> 58

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 5

<400> 58

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgagcgt tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 59

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 5

<400> 59

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> 60

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analog 6

<400> 60

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggcg cgttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 61

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 6

<400> 61

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> 62

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 7

<400> 62

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgcgtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 63

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 7

<400> 63

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> 64

<211> 261

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 8

<400> 64

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaacag 240

ctggagaact actgcaactg a 261

<210> 65

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 8

<400> 65

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> 66

<211> 261

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 9

<400> 66

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaaccag 240

ctggagaact actgcaactg a 261

<210> 67

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 9

<400> 67

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> 68

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 10

<400> 68

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tctacacacc caagacccgc cgggaggcag aggacctgca ggtggggcag 120

gtggagctgg gcggggggccc tggtgcaggc agcctgcagc ccttggccct ggaggggtcc 180

ctgcagaagc gtggcattgt ggaacaatgc tgtaccagca tctgctccct cgaacagctg 240

gagaactact gcaactga 258

<210> 69

<211> 85

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 10

<400> 69

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> 70

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 11

<400> 70

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgagct agtgtgcggg 60

gaacgaggct tctacacacc caagacccgc cgggaggcag aggacctgca ggtggggcag 120

gtggagctgg gcggggggccc tggtgcaggc agcctgcagc ccttggccct ggaggggtcc 180

ctgcagaagc gtggcattgt ggaacaatgc tgtaccagca tctgctccct cgcccagctg 240

gagaactact gcaactga 258

<210> 71

<211> 85

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 11

<400> 71

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> 72

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 72

cagcatctgc tccctccatc agctggagaa ctac 34

<210> 73

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 73

gtagttctcc agctgatgga gggagcagat gctg 34

<210> 74

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 74

cagcatctgc tccctcaagc agctggagaa ctac 34

<210> 75

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 75

gtagttctcc agctgcttga gggagcagat gctg 34

<210> 76

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 76

ctaccagctg gagaacgagt gcaactgagg atcc 34

<210> 77

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 77

ggatcctcag ttgcactcgt tctccagctg gtag 34

<210> 78

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 78

ctaccagctg gagaactcct gcaactgagg atcc 34

<210> 79

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 79

ggatcctcag ttgcaggagt tctccagctg gtag 34

<210> 80

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 80

ctaccagctg gagaacacct gcaactgagg atcc 34

<210> 81

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 81

ggatcctcag ttgcaggtgt tctccagctg gtag 34

<210> 82

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 82

ctggtggaag ctctcgagct agtgtgcggg gaac 34

<210> 83

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 83

gttccccgca cactagctcg agagcttcca ccag 34

<210> 84

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 84

ctggtggaag ctctctccct agtgtgcggg gaac 34

<210> 85

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 85

gttccccgca cactagggag agagcttcca ccag 34

<210> 86

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 86

ctggtggaag ctctcaccct agtgtgcggg gaac 34

<210> 87

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 87

gttccccgca cactagggtg agagcttcca ccag 34

<210> 88

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 88

cagcatctgc tccctcgccc agctggagaa ctac 34

<210> 89

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 89

gtagttctcc agctgggcga gggagcagat gctg 34

<210> 90

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 90

cagcatctgc tccctcgacc agctggagaa ctac 34

<210> 91

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 91

gtagttctcc agctggtcga gggagcagat gctg 34

<210> 92

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 92

ctggtggaag ctctcgacct agtgtgcggg gaac 34

<210> 93

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 93

gttccccgca cactaggtcg agagcttcca ccag 34

<210> 94

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 94

ggggaacgag gcttcgacta cacaccaag acc 33

<210> 95

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 95

ggtcttgggt gtgtagtcga agcctcgttc ccc 33

<210> 96

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 96

ggggaacgag gcttcgagta cacaccaag acc 33

<210> 97

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 97

ggtcttgggt gtgtactcga agcctcgttc ccc 33

<210> 98

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 12

<400> 98

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctccatcag 240

ctggagaact actgcaac 258

<210> 99

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 12

<400> 99

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> 100

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 13

<400> 100

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaagcag 240

ctggagaact actgcaac 258

<210> 101

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 13

<400> 101

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> 102

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 14

<400> 102

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaacg agtgcaac 258

<210> 103

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 14

<400> 103

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> 104

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 15

<400> 104

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact cctgcaac 258

<210> 105

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 15

<400> 105

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> 106

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 16

<400> 106

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaaca cctgcaac 258

<210> 107

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 16

<400> 107

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> 108

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 17

<400> 108

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgagct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 109

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 17

<400> 109

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> 110

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 18

<400> 110

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctccct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 111

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 18

<400> 111

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> 112

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 19

<400> 112

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcaccct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 113

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 19

<400> 113

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> 114

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 20

<400> 114

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgcccag 240

ctggagaact actgcaac 258

<210> 115

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 20

<400> 115

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> 116

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 21

<400> 116

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaccag 240

ctggagaact actgcaac 258

<210> 117

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 21

<400> 117

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> 118

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 22

<400> 118

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctcgacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 119

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 22

<400> 119

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> 120

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 23

<400> 120

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgactacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 121

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 23

<400> 121

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> 122

<211> 258

<212> DNA

<213> Artificial Sequence

<220>

<223> Analogue 24

<400> 122

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgagtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcgggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210> 123

<211> 86

<212>PRT

<213> Artificial Sequence

<220>

<223> Analogue 24

<400> 123

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

<210> 124

<211> 21

<212>PRT

<213> Homo sapiens

<400> 124

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> 125

<211> 30

<212>PRT

<213> Homo sapiens

<400> 125

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

<---

Claims (232)

1. A pharmaceutical combination for the prevention or treatment of an insulin-related disease, containing insulin and glucagon, wherein insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by the following chemical formula 1: [chemical formula 1] XL _a -F, where in chemical formula 1: X represents insulin or glucagon; L represents polyethylene glycol; a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent; F is an immunoglobulin Fc region capable of increasing the half-life of X in vivo; And "-" represents a covalent or non-covalent bond, and where the specified combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a mass ratio of 0.1:1 to 100:1. 2. The pharmaceutical combination according to claim 1, wherein the insulin-related disease is selected from the group consisting of insulin-resistant disease, diabetes, hyperglycemia and obesity. 3. The pharmaceutical combination according to claim 1, wherein the glucagon is native glucagon or glucagon obtained by altering one or more amino acids of native glucagon selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof. 4. Pharmaceutical combination according to claim 3, where glucagon contains the amino acid sequence of the following general formula 1: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-T-X30 ( general formula 1, SEQ ID NO: 46), where in general formula 1: X1 is tyrosine (Y); X2 is alpha-methylglutamic acid (α-methylglutamic acid), Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine; X7 is threonine (T), valine (V), or cysteine (C); X10 is tyrosine (Y) or cysteine (C); X12 is lysine (K) or cysteine (C); X13 is tyrosine (Y) or cysteine (C); X14 is leucine (L) or cysteine (C); X15 is aspartic acid (D), glutamic acid (E) or cysteine (C); X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methylglutamic acid or cysteine (C) or absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C) or valine (V) or absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V) or cysteine (C), or none; X19 is alanine (A), arginine (R), serine (S), valine (V) or cysteine (C) or absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid or cysteine (C), or none; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V) or cysteine (C) or absent; X23 is isoleucine (I), valine (V) or arginine (R) or absent; X24 is valine (V), arginine (R), alanine (A), cysteine (C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid or leucine (L) or none ; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q) or arginine (R) or absent; X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R) or absent; And X30 represents cysteine (C) or is absent (provided that, when the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1 or 12, it is excluded). 5. Pharmaceutical combination according to claim 4, where in general formula 1: X1 is tyrosine (Y); X2 represents serine (S) or Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine (C); X10 is tyrosine (Y) or cysteine (C); X12 is lysine (K) or cysteine (C); X13 is tyrosine (Y) or cysteine (C); X14 is leucine (L) or cysteine (C); X15 is aspartic acid (D) or cysteine (C); X16 is glutamic acid (E), serine (S) or cysteine (C); X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C) or valine (V); X18 is aspartic acid (D), glutamic acid (E), arginine (R) or cysteine (C); X19 is alanine (A) or cysteine (C); X20 is glutamine (Q), aspartic acid (D), lysine (K) or cysteine (C); X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V) or cysteine (C); X23 is isoleucine (I), valine (V) or arginine (R); X24 is valine (V), arginine (R), alanine (A), glutamic acid (E), lysine (K), glutamine (Q) or leucine (L); X27 is isoleucine (I), valine (V), alanine (A), methionine (M), glutamine (Q) or arginine (R); X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R); And X30 is cysteine (C) or absent. 6. Pharmaceutical combination according to claim 4, where in general formula 1: X1 is tyrosine (Y); X2 represents serine (S) or Aib (aminoisobutyric acid); X7 is cysteine (C), threonine (T), or valine (V); X10 is tyrosine (Y) or cysteine (C); X12 is lysine (K) or cysteine (C); X13 is tyrosine (Y) or cysteine (C); X14 is leucine (L) or cysteine (C); X15 is aspartic acid (D) or cysteine (C); X16 is glutamic acid (E), serine (S) or cysteine (C); X17 is glutamic acid (E), lysine (K), arginine (R), cysteine (C) or valine (V); X18 is arginine (R) or cysteine (C); X19 is alanine (A) or cysteine (C); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D), glutamic acid (E), valine (V) or cysteine (C); X23 is valine (V); X24 is valine (V) or glutamine (Q); X27 represents methionine (M); X28 is asparagine (N) or arginine (R); And X30 is cysteine (C) or absent. 7. Pharmaceutical combination according to claim 4, where in general formula 1: X1 is tyrosine (Y); X2 represents Aib (aminoisobutyric acid); X7 is cysteine (C), threonine (T), or valine (V); X10 is tyrosine (Y) or cysteine (C); X12 represents lysine (K); X13 is tyrosine (Y) or cysteine (C); X14 is leucine (L) or cysteine (C); X15 is aspartic acid (D) or cysteine (C); X16 is glutamic acid (E), serine (S), or cysteine (C); X17 is lysine (K), arginine (R), cysteine (C) or valine (V); X18 is arginine (R) or cysteine (C); X19 is alanine (A) or cysteine (C); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D), glutamic acid (E) or cysteine (C); X23 is valine (V); X24 is glutamine (Q); X27 represents methionine (M); X28 is asparagine (N) or arginine (R); And X30 is cysteine (C) or absent. 8. Pharmaceutical combination according to claim 4, where in general formula 1: X1 is tyrosine (Y); X2 represents serine (S) or Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine (C); X10 is tyrosine (Y) or cysteine (C); X12 is lysine (K) or cysteine (C); X13 is tyrosine (Y) or cysteine (C); X14 is leucine (L) or cysteine (C); X15 is aspartic acid (D) or cysteine (C); X16 is glutamic acid (E), serine (S), or cysteine (C); X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine (C) or valine (V); X18 is aspartic acid (D), glutamic acid (E), arginine (R) or cysteine (C); X19 is alanine (A) or cysteine (C); X20 is glutamine (Q), aspartic acid (D) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X23 is valine (V); X24 is valine (V) or glutamine (Q); X27 is isoleucine (I) or methionine (M); X28 is asparagine (N) or arginine (R); And X30 is cysteine (C) or absent. 9. Pharmaceutical combination according to claim 4, where in general formula 1: X1 is tyrosine (Y); X2 represents Aib (aminoisobutyric acid); X7 is threonine (T); X10 represents tyrosine (Y); X12 represents lysine (K); X13 is tyrosine (Y); X14 is leucine (L); X15 is aspartic acid (D) or cysteine (C); X16 is glutamic acid (E), serine (S) or cysteine (C); X17 is lysine (K) or arginine (R); X18 represents arginine (R); X19 represents alanine (A); X20 is glutamine (Q), cysteine (C) or lysine (K); X21 is aspartic acid (D), cysteine (C), valine (V) or glutamic acid (E); X23 is valine (V) or arginine (R); X24 is glutamine (Q) or leucine (L); X27 represents methionine (M); X28 is asparagine (N) or arginine (R); And X30 is missing. 10. Pharmaceutical combination according to claim 4, where glucagon contains the amino acid sequence of the following general formula 2: Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X30 (general formula 2, SEQ ID NO: 47), where in general formula 2: X7 is threonine (T), valine (V), or cysteine (C); X10 is tyrosine (Y) or cysteine (C); X12 is lysine (K) or cysteine (C); X15 is aspartic acid (D) or cysteine (C); X16 represents glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); And X30 is cysteine (C) or absent, provided that, when the amino acid sequence of general formula 2 is the same as SEQ ID NO: 12, it is excluded. 11. The pharmaceutical combination according to claim 4, where each of one or more than one pair of amino acids from the pairs of amino acids X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 and X24 and X28 of general formula 1 forms a ring between corresponding amino acids. 12. The pharmaceutical combination according to claim 11, where each amino acid of one or more than one pair of amino acids from the pairs of amino acids X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 and X24 and X28 of general formula 1 is replaced by glutamic acid or lysine, which are capable of forming a ring. 13. The pharmaceutical combination according to claim 4, wherein the glucagon contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11, 25, 27, 29, 31, 33 and 35-45. 14. The pharmaceutical combination according to claim 13, where glucagon contains the amino acid sequence SEQ ID NO: 37. 15. The pharmaceutical combination according to claim 1, wherein the insulin is native insulin or insulin obtained by altering one or more amino acids of native insulin selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof. 16. The pharmaceutical combination according to claim 15, wherein the insulin is insulin obtained by substitution with another amino acid or deletion of one or more amino acids selected from the group consisting of an amino acid at position 1, an amino acid at position 2, an amino acid at position 3 , amino acids at position 5, amino acids at position 8, amino acids at position 10, amino acids at position 12, amino acids at position 16, amino acids at position 23, amino acids at position 24, amino acids at position 25, amino acids at position 26, amino acids at position 27 , amino acids at position 28, amino acids at position 29 and amino acids at position 30 of the natural insulin B chain and amino acids at position 1, amino acids at position 2, amino acids at position 5, amino acids at position 8, amino acids at position 10, amino acids at position 12 , amino acids at position 14, amino acids at position 16, amino acids at position 17, amino acids at position 18, amino acids at position 19 and amino acids at position 21 of the natural insulin A chain. 17. The pharmaceutical combination according to claim 15, wherein the insulin contains an A chain of SEQ ID NO: 48 represented by the following general formula 3, and a B chain of SEQ ID NO: 49 represented by the following general formula 4: [general formula 3] Xaa1-Xaa2-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21 (SEQ ID NO: 48), where in general formula 3: Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid or asparagine; Xaa2 is alanine or isoleucine; Xaa5 is 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 4] 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: 49), where in general formula 4: Xaa8 is alanine or glycine; Xaa16 is tyrosine, glutamic acid, serine, threonine or aspartic acid or absent; Xaa23 is glycine or alanine; Xaa24 is alanine or phenylalanine; Xaa25 is alanine, phenylalanine, aspartic acid or glutamic acid or absent; Xaa27 is threonine or absent; And Xaa28 is proline, glutamic acid or aspartic acid or is absent (provided that when the peptide contains the A chain of SEQ ID NO: 124 and the B chain of SEQ ID NO: 125, it is excluded). 18. The pharmaceutical combination according to claim 17, wherein insulin is obtained by replacing with alanine one or more amino acids selected from the group consisting of an amino acid at position 8, an amino acid at position 23, an amino acid at position 24 and an amino acid at position 25 B- chain of natural insulin and amino acid at position 1, amino acid at position 2 and amino acid at position 19 of the A-chain of natural insulin, or substitution of glutamic acid or asparagine for the amino acid at position 14 of the A-chain of natural insulin. 19. The pharmaceutical combination according to claim 18, wherein the insulin contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67. 20. The pharmaceutical combination according to claim 17, where insulin is obtained by replacing the amino acid at position 16 of the B-chain of natural insulin with glutamic acid, deleting the amino acid at position 25 of the B-chain of natural insulin, or replacing the amino acid at position 14 A with glutamic acid or alanine. natural insulin chains. 21. The pharmaceutical combination according to claim 20, wherein insulin contains the amino acid sequence SEQ ID NO: 69 or 71. 22. The pharmaceutical combination according to claim 17, where insulin is obtained by replacing the amino acid at position 16 of the B-chain of natural insulin with glutamic acid, serine, threonine or aspartic acid, replacing the amino acid at position 25 with aspartic acid or glutamic acid in the B-chain of natural insulin , replacement with histidine, lysine, alanine or aspartic acid of the amino acid at position 14 of the A-chain of natural insulin or replacement with glutamic acid, serine or threonine of the amino acid at position 19 of the A-chain of natural insulin. 23. The pharmaceutical combination according to claim 22, wherein insulin contains an amino acid sequence selected from the group consisting of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123 . 24. The pharmaceutical combination according to claim 17, wherein insulin is in the form of two polypeptide chains consisting of an A chain of SEQ ID NO: 48, represented by the general formula 3, and a B chain of SEQ ID NO: 49, represented by the general formula 4 . 25. The pharmaceutical combination according to claim 24, wherein the A chain and the B chain are linked to each other via a disulfide bond. 26. Pharmaceutical combination according to claim 1, where F represents the Fc region of IgG (immunoglobulin G). 27. Pharmaceutical combination according to claim 1, where the Fc region of the immunoglobulin is aglycosylated. 28. The pharmaceutical combination according to claim 1, where the Fc region of the immunoglobulin 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) CH1 domain and CH3 domain; (d) a CH2 domain and a CH3 domain; (e) combinations of one or two or more domains of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region or part of said hinge region; and (e) a dimer of each heavy chain constant region and light chain constant region domain. 29. The pharmaceutical combination according to claim 1, where the Fc region of the immunoglobulin is the Fc region of the immunoglobulin, where the region capable of forming a disulfide bond is removed, where part of the amino acid(s) at the N-terminus of the native Fc is removed, where to the N-terminus of the native Fc is attached to a methionine residue where the complement fixing site is removed or where the antibody-dependent cell-mediated cytotoxicity (ADCC) site is removed. 30. The pharmaceutical combination according to claim 1, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE or IgM. 31. The pharmaceutical combination according to claim 1, wherein the Fc region of the immunoglobulin is a hybrid of a domain having different origins derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM. 32. The pharmaceutical combination according to claim 1, wherein insulin and glucagon are administered in combination simultaneously, sequentially or in reverse order. 33. Pharmaceutical combination for the prevention or treatment of hypoglycemia, containing insulin and glucagon, wherein insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by the following chemical formula 1: [chemical formula 1] XL _a -F, where in chemical formula 1: X represents insulin or glucagon; L represents polyethylene glycol; a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent; F is an immunoglobulin Fc region capable of increasing the half-life of X in vivo; And "-" represents a covalent or non-covalent bond, where the specified pharmaceutical combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a mass ratio of 0.1:1 to 100:1. 34. The pharmaceutical combination according to claim 33, wherein insulin and glucagon are administered in combination simultaneously, sequentially or in reverse order. 35. Combination to reduce the side effects of insulin, containing insulin and glucagon, wherein insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by the following chemical formula 1: [chemical formula 1] XL _a -F, where in chemical formula 1: X represents insulin or glucagon; L represents polyethylene glycol; a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent; F is an immunoglobulin Fc region capable of increasing the half-life of X in vivo; And "-" represents a covalent or non-covalent bond, where the specified combination contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a mass ratio of 0.1:1 to 100:1. 36. The combination of claim 35, wherein insulin and glucagon are administered in combination simultaneously, sequentially or in reverse order. 37. Complex composition for reducing hypoglycemia in a patient with an insulin-related disease, containing insulin and glucagon, wherein insulin is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, and glucagon is associated with a biologically compatible substance capable of increasing its half-life in vivo, in the form of a long-acting conjugate, where the conjugate is represented by the following chemical formula 1: [chemical formula 1] XL _a -F, where in chemical formula 1: X represents insulin or glucagon; L represents polyethylene glycol; a represents 0 or a natural number, with the proviso that when a represents 2 or more, each L is independent; F is an immunoglobulin Fc region capable of increasing the half-life of X in vivo; And "-" represents a covalent or non-covalent bond, where said complex composition contains insulin in the form of a long-acting conjugate and glucagon in the form of a long-acting conjugate in a mass ratio of 0.1:1 to 100:1. 38. The complex composition according to claim 37, wherein hypoglycemia is a side effect of insulin. 39. Complex composition according to claim 37, suppressing weight gain.

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