Pharmaceutical composition containing insulin and glucagon

Abstract

To provide a drug capable of improving side effects associated with insulin administration while maintaining the efficacy of insulin administration.SOLUTION: The present invention provides a pharmaceutical composition for prevention or treatment of insulin-related diseases, comprising insulin and glucagon.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a composition comprising insulin and glucagon and a combined preparation comprising the same. [Background technology]

[0002] Glucagon is produced and secreted by the pancreas when blood glucose levels drop due to various causes, including drug therapy, disease, hormone or enzyme deficiency, etc. The secreted glucagon acts on the liver to break down glycogen and release glucose, thereby raising blood glucose levels to normal levels.

[0003] Insulin, a hormone produced in the pancreas like glucagon, is a hormone secreted from the pancreas and plays a role in maintaining blood glucose levels at an appropriate level when blood glucose levels rise, and various diseases occur due to insulin deficiency, insufficiency, or impaired function. Therefore, administration of insulin is considered to be the core treatment for insulin-related diseases, including diabetes, which causes high blood glucose levels.

[0004] It is widely known that glucagon and insulin have opposite effects on blood glucose, i.e., antagonistic effects, and have been used as therapeutic agents for different diseases. In particular, Patent Document 1 discloses a method for treating diabetes by blocking glucagon receptors with antagonistic antigen-binding proteins to inhibit glucagon action, while supplementing insulin.

[0005] That is, because insulin and glucagon play opposing roles in the body, no drug therapy has yet been demonstrated in which they are administered together.

[0006] On the other hand, when insulin, which is a therapeutic agent for various insulin-related diseases, is administered to a patient, there is a risk of side effects such as weight gain, overdosage, and hypoglycemia.

[0007] Therefore, there is a need to develop drugs that can improve these side effects while maintaining the pharmacological effects of insulin administration. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 10-2017-0023066 [Patent Document 2] Korean Patent Publication No. 10-2014-0106452 [Patent Document 3] Korean Patent Publication No. 10-2017-0026284 [Patent Document 4] Korean Patent Publication No. 10-2017-0003466 [Patent Document 5] Korean Patent Publication No. 10-2018-0002544 [Patent Document 6] Korean Patent Publication No. 10-2016-0082482 [Patent Document 7] International Publication No. 97 / 034631 [Patent Document 8] International Publication No. 96 / 032478 [Non-patent literature]

[0009] [Non-Patent Document 1] http: / / expasy.org / tools / pi_tool.html; Gasteiger et al.,2003 [Non-Patent Document 2] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a pharmaceutical composition for preventing or treating insulin-related diseases, which comprises insulin and glucagon.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hypoglycemia, which comprises insulin and glucagon.

[0012] A further object of the present invention is to provide a composition for improving insulin side effects, which comprises insulin and glucagon.

[0013] A further object of the present invention is to provide a combination preparation containing insulin and glucagon for improving hypoglycemia in patients with insulin-related diseases.

[0014] A further object of the present invention is to provide a use of the composition in the manufacture of a medicament.

[0015] Furthermore, the present invention aims to provide a method for preventing or treating an insulin-related disease, comprising the step of administering the composition or combined preparation to an individual.

[0016] A further object of the present invention is to provide a method for improving insulin side effects, comprising the step of administering the composition or combined preparation to an individual. [Means for solving the problem]

[0017] One aspect of the present invention is a pharmaceutical composition for preventing or treating an insulin-related disease, comprising insulin and glucagon.

[0018] In one embodiment of the composition, the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its half-life in vivo is bound.

[0019] In the composition according to the above embodiment, the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

[0020] As a composition according to any of the above-mentioned specific examples, the insulin is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life, and the glucagon is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life.

[0021] The composition according to any of the above embodiments, wherein the insulin-related disorder is selected from the group consisting of insulin resistance disorders, diabetes, hyperglycemia and obesity.

[0022] The composition according to any of the above-mentioned embodiments is characterized in that the composition alleviates hypoglycemia, a side effect of insulin, and inhibits weight gain.

[0023] The composition according to any of the above-mentioned specific examples is characterized in that the composition contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a weight ratio of 0.1:1 to 100:1.

[0024] In a composition according to any of the above-mentioned embodiments, the glucagon is characterized in that it is native glucagon or a glucagon analogue in which at least one amino acid of native glucagon has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification and combinations thereof.

[0025] The composition according to any of the above embodiments, wherein the glucagon analogue comprises an amino acid sequence of general formula 1:

[0026] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number 46)

[0027] In the above formula, X1 is histidine (H), desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan (W), tyrosine (Y), or absent, and X2 is α-methyl-glutamic acid, Aib (aminoisobutyric 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), and X14 is leucine. X15 is aspartic acid (D), glutamic acid (E) or cysteine ​​(C); X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid or cysteine ​​(C) or is absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), arginine (R), selenium (C), or arginine (L). X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), cysteine ​​(C), or absent; X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine ​​(C), or absent. X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, cysteine ​​(C), or is absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), cysteine ​​(C), or is absent; X23 is isoleucine (I),X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid or leucine (L) or is not present, X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q) or arginine (R) or is not present, X28 is glutamine (Q), lysine (K), asparagine (N) or arginine (R) or is not present, and X30 is cysteine ​​(C) or is not present (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1).

[0028] As a composition according to any of the above-mentioned specific examples, in general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y), or is absent, X2 is 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), and 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), and 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), and X24 is valine (V), arginine (R), alanine (A), glutamine 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.

[0029] As a composition according to any of the above-mentioned specific examples, in general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y), X2 is 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), and 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 is methionine (M), X28 is asparagine (N) or arginine (R), and X30 is cysteine ​​(C) or absent.

[0030] As a composition according to any of the above-mentioned specific examples, in general formula 1, X1 is tyrosine (Y), X2 is Aib (aminoisobutyric acid), X7 is cysteine ​​(C), threonine (T) or valine (V), X10 is tyrosine (Y) or cysteine ​​(C), X12 is 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), and 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 is methionine (M), X28 is asparagine (N) or arginine (R), and X30 is cysteine ​​(C) or absent.

[0031] As a composition according to any of the above-mentioned embodiments, in general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y), or is absent, and X2 is 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), and 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.

[0032] As a composition according to any of the above-mentioned specific examples, in general formula 1, X1 is tyrosine (Y), X2 is Aib (aminoisobutyric acid), X7 is threonine (T), X10 is tyrosine (Y), X12 is 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 is arginine (R), and X19 is alanine ( A), in which 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 is methionine (M), X28 is asparagine (N) or arginine (R), and X30 is absent.

[0033] The composition according to any of the above embodiments, wherein the glucagon analogue comprises an amino acid sequence of general formula 2:

[0034] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula 2, sequence number 47)

[0035] 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 is 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.

[0036] The composition according to any of the above-mentioned specific examples is characterized in that a ring is formed between each amino acid in at least one of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1.

[0037] The composition according to any of the above-mentioned specific examples is characterized in that each amino acid of at least one of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 is substituted with glutamic acid or lysine to form a ring.

[0038] The liquid formulation according to any one of the above-mentioned specific examples is characterized in that the glucagon analog in the long-acting conjugate of the glucagon analog has a pI of 6 to 7 and a relative in vitro activity of 200% or more compared to native glucagon.The composition according to any one of the above-mentioned specific examples is characterized in that the glucagon analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45.

[0039] The composition according to any of the previous embodiments, wherein the glucagon analogue comprises the amino acid sequence of SEQ ID NO:37.

[0040] The composition according to any of the above-mentioned embodiments is characterized in that the insulin is a natural insulin or an insulin analogue in which at least one amino acid of natural insulin has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification and combinations thereof.

[0041] In the composition according to any of the above-mentioned embodiments, the insulin analogue is characterized in that at least one amino acid selected from the group consisting of the 1st, 2nd, 3rd, 5th, 8th, 10th, 12th, 16th, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, and 30th amino acids of the B chain and the 1st, 2nd, 5th, 8th, 10th, 12th, 14th, 16th, 17th, 18th, 19th, and 21st amino acids of the A chain of native insulin is substituted with another amino acid or deleted.

[0042] A composition according to any of the above-mentioned specific examples, characterized in that the insulin analogue comprises an A chain of SEQ ID NO: 48, represented by general formula 3, and a B chain of SEQ ID NO: 49, represented by general formula 4.

[0043] [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)

[0044] 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.

[0045] [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)

[0046] In general formula 4, Xaa8 is alanine or glycine, Xaa16 is tyrosine, glutamic acid, serine, threonine, aspartic acid, or absent, Xaa23 is glycine or alanine, Xaa24 is alanine or phenylalanine, Xaa25 is alanine, phenylalanine, aspartic acid, glutamic acid, or absent, Xaa27 is threonine or absent, and Xaa28 is proline, glutamic acid, aspartic acid, or absent (excluding peptides comprising chain A of SEQ ID NO: 48 and chain B of SEQ ID NO: 49).

[0047] In a composition according to any of the above-mentioned embodiments, the insulin analogue is characterized in that one amino acid selected from the group consisting of the 8th, 23rd, 24th and 25th amino acids of the B chain and the 1st, 2nd and 19th amino acids of the A chain of natural insulin is substituted with alanine, or the 14th amino acid of the A chain is substituted with glutamic acid or asparagine.

[0048] The composition according to any of the previous embodiments, wherein the insulin analogue comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67.

[0049] A composition according to any of the above embodiments, wherein the insulin analogue is characterized in that the 16th amino acid of the B chain of natural insulin is substituted with glutamic acid, the 25th amino acid is deleted, or the 14th amino acid of the A chain of natural insulin is substituted with glutamic acid or alanine.

[0050] The composition according to any of the above embodiments, wherein the insulin analogue comprises the amino acid sequence of SEQ ID NO: 69 or 71.

[0051] In a composition according to any of the above-mentioned embodiments, the insulin analogue is characterized in that the 16th amino acid of the B chain of natural insulin is replaced by glutamic acid, serine, threonine or aspartic acid, the 25th amino acid is replaced by aspartic acid or glutamic acid, the 14th amino acid of the A chain of natural insulin is replaced by histidine, lysine, alanine or aspartic acid, or the 19th amino acid is replaced by glutamic acid, serine or threonine.

[0052] The composition according to any of the previous embodiments, wherein the insulin analogue comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123.

[0053] A composition according to any of the above-mentioned specific examples, wherein the insulin analogue is in the form of a double chain consisting of an A chain of SEQ ID NO: 48, represented by general formula 3, and a B chain of SEQ ID NO: 49, represented by general formula 4.

[0054] The composition according to any of the above embodiments, wherein the A and B chains are linked by a disulfide bond.

[0055] In a composition according to any of the above-mentioned embodiments, the conjugate is characterized in that it is represented by Chemical Formula 1:

[0056] [Chemical formula 1] X-La-F

[0057] Here, X is insulin or glucagon, L is a linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others, F is a substance capable of extending the in vivo half-life of X, and the "-" is a covalent or non-covalent bond.

[0058] In the composition according to any of the above-mentioned specific examples, F is characterized in that it is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

[0059] In the composition according to any of the above-mentioned specific examples, the polymer is characterized in that it 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.

[0060] The composition according to any of the above embodiments, wherein F is an immunoglobulin Fc region.

[0061] The composition according to any of the above embodiments, wherein F is an IgG Fc region.

[0062] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region is aglycosylated.

[0063] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it is selected from the group consisting of: (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region; and (f) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0064] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it has a disulfide bond-forming site removed, some amino acids at the N-terminus of a native Fc are deleted, a methionine residue is added to the N-terminus of a native Fc, a complement binding site removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site removed.

[0065] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.

[0066] The composition according to any of the above-mentioned specific examples is characterized in that the immunoglobulin Fc region is a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

[0067] In the composition according to any of the above-mentioned embodiments, L is selected from the group consisting of peptides, fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0068] In the composition according to any of the above-mentioned specific examples, the polymer is characterized in that it 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.

[0069] The composition according to any of the above-mentioned embodiments, wherein L is polyethylene glycol.

[0070] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating hypoglycemia, comprising insulin and glucagon.

[0071] In one embodiment of the composition, the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

[0072] In the composition according to the above embodiment, the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its half-life in vivo is bound.

[0073] As a composition according to any of the above-mentioned specific examples, the insulin is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life, and the glucagon is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life.

[0074] Yet another aspect of the present invention provides a composition for improving insulin side effects, comprising insulin and glucagon.

[0075] In one embodiment of the composition, the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

[0076] In the composition according to the above embodiment, the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its half-life in vivo is bound.

[0077] As a composition according to any of the above-mentioned specific examples, the insulin is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life, and the glucagon is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life.

[0078] Yet another aspect of the present invention provides a combination preparation for improving hypoglycemia in a patient with an insulin-related disease, comprising insulin and glucagon.

[0079] In one embodiment of the combined preparation, the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its half-life in vivo is bound.

[0080] In the above-mentioned embodiment of the combined preparation, the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its half-life in vivo is bound.

[0081] In the combined preparation according to any of the above-mentioned specific examples, the insulin is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life, and the glucagon is in the form of a long-acting conjugate bound to a biocompatible substance capable of extending its in vivo half-life.

[0082] The composition according to any of the previous embodiments, wherein said hypoglycemia is a side effect of insulin.

[0083] The composition according to any of the above-mentioned embodiments, wherein the combination preparation is characterized in that it inhibits weight gain.

[0084] As a composition according to any of the above-mentioned specific examples, the composite preparation is characterized in that it contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a weight ratio of 0.1:1 to 100:1. Effect of the Invention

[0085] The composition or composite preparation of the present invention comprising insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof provides a novel combination therapy that exerts a preventive or therapeutic effect on insulin-related diseases, such as diabetes, while at the same time alleviating side effects of insulin administration, such as weight gain and hypoglycemia. [Brief description of the drawings]

[0086] [Figure 1] FIG. 1 shows changes in blood glucose levels following coadministration of a long-acting insulin conjugate and a long-acting glucagon conjugate. [Diagram 2] FIG. 1 shows changes in blood glucose (AUC) following coadministration of a long-acting insulin conjugate and a long-acting glucagon conjugate. [Diagram 3] FIG. 1 shows changes in body weight resulting from combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] The present invention will now be described in more detail.

[0088] In addition, each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the scope of the present invention is not limited to the following specific description.

[0089] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid Asp,D Cysteine ​​Cys,C Glutamic acid Glu,E Glutamine Gln,Q Glycine Gly,G Histidine Isoleucine Ile,I Leucine Leu,L Lysine Lys,K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin,V

[0090] One aspect of the present invention is a pharmaceutical composition for preventing or treating an insulin-related disease, comprising insulin and glucagon.

[0091] Specifically, the composition may be one comprising (i) insulin and glucagon, (ii) a long-acting insulin conjugate in which insulin is conjugated with a biocompatible substance capable of extending its half-life in vivo, and glucagon, (iii) a long-acting insulin conjugate in which glucagon is conjugated with a biocompatible substance capable of extending its half-life in vivo, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate.

[0092] The pharmaceutical composition of the present invention for preventing or treating insulin-related diseases contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and exhibits the pharmacological efficacy of insulin having the preventive or therapeutic effect on insulin-related diseases caused by insulin deficiency, insufficiency or decreased function, while also alleviating side effects caused by insulin administration (e.g., weight gain, hypoglycemia).

[0093] In the examples of the present invention, not only was a blood glucose lowering effect confirmed in db / db mice administered a composition containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, but also weight gain inhibition and hypoglycemia alleviation effects were confirmed, demonstrating that the pharmaceutical composition according to the present invention can improve the side effects of insulin while maintaining its efficacy.

[0094] In one embodiment of the present invention, the composition may provide a therapeutic combination of insulin or a long-acting conjugate thereof with glucagon or a long-acting conjugate thereof.

[0095] The "composition" of the present invention is used in combination with a "combination" containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof. The composition may be provided in the form of a kit.

[0096] In the present invention, the term "combination" refers to the combined administration of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and is synonymous with "combined used". The combination is a) a mixture of (i) insulin or a long-acting conjugate thereof and (ii) glucagon or a long-acting conjugate thereof, which are administered in one mixture, or b) a mixture of (i) insulin or a long-acting conjugate thereof and (ii) glucagon or a long-acting conjugate thereof, which are administered in separate forms, but is not limited thereto.

[0097] When insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are in a separated form, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be formulated into separate preparations and administered simultaneously, separately, sequentially, or in the reverse order.

[0098] In the present invention, the term "combined administration" should be understood to mean not only simultaneous administration, but also an administration form in which insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof act together on an individual, with each substance performing a function equivalent to or higher than its inherent function. Thus, in the present invention, "combined administration" should be understood to mean simultaneous, separate, sequential, or reversed administration. When the administration is sequential, reversed, or separate, the order of administration is not particularly limited, but the interval between administrations of the secondary components must not lose the beneficial effect of the combination.

[0099] The term "composition" in the present invention refers to a combination of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, or a combination containing the combination and having therapeutic applications, but is not limited thereto, such as, but not limited to, those having applications in the prevention or treatment of insulin-related diseases.

[0100] The composition according to the present invention is for co-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 the glucagon or a long-acting conjugate thereof may be formulated into a single preparation or may be formulated separately. Specifically, the insulin or a long-acting conjugate thereof and the glucagon or a long-acting conjugate thereof are administered simultaneously, separately, sequentially, or in reverse order, but are not limited thereto.

[0101] The "kit" of the present invention may include a combination or composition according to the present invention for co-administration of insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof. Specifically, the kit according to the present invention may include a formulation in which insulin or a conjugate thereof and glucagon or a long-acting conjugate thereof are formulated into one preparation, or a formulation in which insulin or a conjugate thereof and glucagon or a conjugate thereof are formulated separately, and may further include substances necessary for co-administration of the two substances, but is not limited thereto.

[0102] The insulin and glucagon include various substances, for example in the form of compounds or peptides, that exhibit a significant level of activity relative to insulin and glucagon.

[0103] In the present invention, the term "insulin-related disease" refers to a disease caused by an abnormality in the blood glucose regulation function inherent to insulin, such as insulin deficiency, insufficiency, or decreased function, and may be any disease that can be prevented or treated by administration of insulin. Specifically, the insulin-related disease is selected from the group consisting of insulin resistance disease, diabetes, hyperglycemia, and obesity, but is not limited thereto.

[0104] When insulin is administered to treat insulin-related diseases, while the desired therapeutic effect is obtained, there is a problem that unintended side effects such as hypoglycemia or weight gain occur, causing further illness or pain to the patient. The pharmaceutical composition according to the present invention can exert a therapeutic effect against insulin-related diseases while alleviating hypoglycemia, a side effect of insulin, and suppressing weight gain by using insulin and glucagon in combination.

[0105] The pharmaceutical composition of the present invention contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a content ratio that exhibits a therapeutic effect on insulin-related diseases and alleviates side effects, specifically, a weight ratio of 0.1:1 to 100:1, or a molar ratio of 1:1 to 30:1, 1:1 to 20:1, or 1:1 to 16:1, but is not limited thereto.

[0106] The pharmaceutical composition according to the present invention exhibits excellent blood glucose regulating effects with suppressed side effects by containing insulin or its long-acting conjugate and glucagon or its long-acting conjugate in a specific ratio. If the weight ratio is deviated from the above range, for example, if an excessive amount of insulin or its long-acting conjugate is contained, there is a high risk of significant insulin side effects, and if an excessive amount of glucagon or its long-acting conjugate is contained, there is a problem that the therapeutic effect of insulin-related diseases is reduced.

[0107] In the present invention, "insulin" refers to a type of hormone secreted from pancreatic β cells, and generally plays a role in promoting glucose uptake into cells, inhibiting fat decomposition, and regulating blood glucose in the body. Insulin is converted to insulin having blood glucose regulating function through processing from the form of a proinsulin precursor that does not have blood glucose regulating function. Insulin consists of two polypeptide chains, namely, A chain and B chain, which contain 21 and 30 amino acid residues, respectively, and are linked to each other by two disulfide bridges. The A chain and B chain of natural insulin contain the amino acid sequences represented by 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)

[0108] In the present invention, "proinsulin" refers to a precursor molecule of insulin. The proinsulin may include insulin A and B chains and a C-peptide therebetween. The proinsulin may be human proinsulin.

[0109] The insulin in the present invention may be a naturally occurring insulin, or an analog, derivative, or fragment in which at least one amino acid of naturally occurring insulin has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof, but is not limited thereto.

[0110] In the present invention, the term "insulin analog" refers to a non-natural insulin that is different from natural insulin.

[0111] The insulin analogues include those modified by addition, deletion or substitution of some amino acids of natural insulin. For example, but not limited to, insulin analogues in which at least one amino acid selected from the group consisting of the 1st, 2nd, 3rd, 5th, 8th, 10th, 12th, 16th, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, and 30th amino acids of the B chain and the 1st, 2nd, 5th, 8th, 10th, 12th, 14th, 16th, 17th, 18th, 19th, and 21st amino acids of the A chain of natural insulin is replaced with another amino acid or deleted. The substitution of the amino acid may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue. Such conservative substitutions with amino acids having similar properties are expected to exhibit the same or similar activity.

[0112] The insulin analogues of the present invention are disclosed in Patent Documents 2 and 3.

[0113] The insulin analogue used in the present invention may be in the form of a single chain or a double chain (two polypeptide chains), and the double chain form is more preferable, but is not particularly limited thereto. The double chain may be composed 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 one corresponding to the A chain or B chain of natural insulin means, when comparing the sequence identity of either one of the chains of the double chain polypeptide with the A chain or B chain of natural insulin, 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 of sequence identity, but is not particularly limited thereto, and can be easily understood by a person skilled in the art by comparing the sequence constituting the double chain with the sequence of the A chain or B chain of natural insulin.

[0114] In the present invention, "homology" refers to the degree of similarity to the amino acid sequence of a wild type protein or the nucleotide sequence encoding the same, and includes sequences having a sequence that is identical to the amino acid sequence or nucleotide sequence of the present invention by the above percentage or more. Such homology can be determined by visually comparing two sequences, or by using a bioinformatics algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences is expressed as a percentage. Useful automated algorithms can be found in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Sequence algorithms automated in the modules include the Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence algorithms. Other useful algorithms for sequence and homology determination are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

[0115] Information on the insulin and glucagon sequences used in the present invention and the nucleotide sequences encoding them can be obtained from known databases such as NCBI.

[0116] In one specific embodiment, the insulin analogue of the present invention is a composition comprising an A chain of SEQ ID NO: 48 represented by general formula 3 and a B chain of SEQ ID NO: 49 represented by general formula 4, but is not limited thereto.

[0117] [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)

[0118] 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.

[0119] [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)

[0120] In general formula 4, Xaa8 is alanine or glycine, Xaa16 is tyrosine, glutamic acid, serine, threonine, aspartic acid, or absent, Xaa23 is glycine or alanine, Xaa24 is alanine or phenylalanine, Xaa25 is alanine, phenylalanine, aspartic acid, glutamic acid, or absent, Xaa27 is threonine or absent, and Xaa28 is proline, glutamic acid, aspartic acid, or absent (excluding peptides comprising chain A of SEQ ID NO: 48 and chain B of SEQ ID NO: 49).

[0121] More specifically, the insulin analogue has one amino acid selected from the group consisting of the 8th, 23rd, 24th and 25th amino acids of the B chain and the 1st, 2nd and 19th amino acids of the A chain of natural insulin substituted with alanine, or the 14th amino acid of the A chain substituted with glutamic acid or asparagine, in particular comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67, or consisting of the above amino acid sequence, but not limited thereto.

[0122] Furthermore, the insulin analogue has the 16th amino acid of the B chain of natural insulin substituted with glutamic acid, the 25th amino acid deleted, or the 14th amino acid of the A chain of natural insulin substituted with glutamic acid or alanine, and in particular comprises the amino acid sequence of SEQ ID NO: 69 or 71, or consists of the above amino acid sequence, but is not limited to these.

[0123] Furthermore, said insulin analogues have the 16th amino acid of the B chain of natural insulin substituted with glutamic acid, serine, threonine or aspartic acid, the 25th amino acid substituted with aspartic acid or glutamic acid, the 14th amino acid of the A chain of natural insulin substituted with histidine, lysine, alanine or aspartic acid or the 19th amino acid substituted with glutamic acid, serine or threonine, in particular comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123, but are not limited thereto.

[0124] Even if the present invention describes a peptide as being "represented by a specific sequence number," it does not exclude meaningless sequence additions, naturally occurring mutations, or silent mutations before or after the amino acid sequence of the sequence number, as long as the peptide has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number. Needless to say, such sequence additions or mutations are also included in the present invention.

[0125] The above also applies to other embodiments or aspects of the present invention, but is not limited thereto.

[0126] On the other hand, the insulin analogue of the present invention may be in the form of a single-chain insulin from which the C-peptide has not been removed, and may be a substance having the activity and function of insulin, comprising an A chain of SEQ ID NO: 48 represented by general formula 3 and a B chain of SEQ ID NO: 49 represented by general formula 4.

[0127] The insulin analogue of the present invention may also be prepared by removing the C-peptide from proinsulin containing the C-peptide between the A-chain and the B-chain. The C-peptide can be removed by a method known in the art, specifically, by treatment with trypsin and carboxypeptidase B, but is not limited thereto.

[0128] Specifically, the insulin analogue of the present invention is composed of an A chain of SEQ ID NO: 48 represented by general formula 3 and a B chain of SEQ ID NO: 49 represented by general formula 4, and more specifically, is in the form of a double-chain insulin in which the A chain and the B chain are linked to each other by two disulfide bridges, but is not limited thereto.

[0129] Needless to say, the present invention also includes insulin analogs in a double-chain form produced by removing the C-peptide from an insulin analog in the form of proinsulin, which is represented by a specific sequence number.

[0130] The insulin analogues include peptides with at least one difference in amino acid sequence compared to natural insulin, peptides altered by modifying the natural insulin sequence, and mimetics of natural insulin that regulate blood glucose regulation in vivo like natural insulin. The insulin analogues in the present invention are used interchangeably with insulin derivatives. The derivatives of natural insulin may have blood glucose regulation in vivo.

[0131] Specifically, insulin derivatives may be insulin in which some of the amino acids of natural insulin have been altered by any of substitution, addition, deletion and modification, or a combination of these methods.

[0132] Specifically, it is a form that shows at least 80% amino acid sequence homology with the A-chain and B-chain of natural insulin, respectively, and / or in which some groups of one of the amino acid residues of insulin have been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination) or modified (e.g., N-methylation), but is not limited to these.

[0133] Derivatives of natural insulin for use in the present invention can be produced by a combination of various methods for producing derivatives.

[0134] Furthermore, such modifications for the production of insulin derivatives include all modifications using L- or D-amino acids and / or unnatural amino acids, and / or modifications by modifying the native sequence or post-translational modifications (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonds, etc.).

[0135] Furthermore, insulin having one or more amino acids added to the N- and / or C-terminus is all included.

[0136] The substituted or added amino acids can be any of the 20 amino acids commonly found in human proteins, as well as unusual or unnatural amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides and typical peptide sequences containing these amino acids can be synthesized and purchased from private peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea, but are not limited thereto.

[0137] In the present invention, the term "fragment of natural insulin, insulin analogue or insulin derivative" refers to a form in which at least one amino acid is deleted at the N-terminus or C-terminus of natural insulin, insulin analogue or derivative of natural insulin. Such fragments have a blood glucose regulating function in the body.

[0138] The insulin analogues of the present invention may be produced using any of the methods used to produce the derivatives and fragments of natural insulin described above, either alone or in combination.

[0139] Specifically, the insulin analogues according to the present invention are those in which specific amino acid residues in the A-chain and B-chain of such natural insulin have been modified, more specifically, those in which specific amino acid residues in the A-chain and / or specific amino acid residues in the B-chain of natural insulin have been modified.

[0140] The pharmaceutical composition according to the present invention may contain insulin as one of the active ingredients, which may be (i) natural insulin, (ii) an insulin analogue, (iii) an insulin derivative, (iv) a fragment thereof, or (v) a combination thereof, so long as it has the blood glucose regulating ability of insulin.

[0141] In the present invention, "glucagon" refers to a type of hormone secreted from α cells of the pancreas, which plays a role in increasing the glucose concentration and raising blood sugar levels in the body by promoting the decomposition of glycogen. Native glucagon has 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)

[0142] The glucagon in the present invention may be, but is not limited to, a native glucagon, or an analog, derivative, or fragment in which at least one amino acid of native glucagon has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0143] The term "glucagon analog" as used herein refers to a non-natural glucagon that differs from natural glucagon.

[0144] Such glucagon analogs include analogs in which some amino acids of native glucagon have been modified in the form of addition, deletion or substitution.

[0145] The glucagon analog of the present invention has 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 sequence identity when compared with natural glucagon, but is not limited to a specific sequence as long as it has the effect of increasing blood glucose level. The glucagon analog of the present invention is disclosed in Patent Documents 4, 5, and 6.

[0146] In one specific embodiment, the glucagon analogue may be a peptide comprising the amino acid sequence of general formula 1.

[0147] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (General formula 1, arrangement number 46)

[0148] In the above formula, X1 is histidine, desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan, tyrosine, or absent, and X2 is α-methyl-glutamic acid, Aib (aminoisobutyricX7 is threonine, valine or cysteine, X10 is tyrosine or cysteine, X12 is lysine or cysteine, X13 is tyrosine or cysteine, X14 is leucine or cysteine, X15 is aspartic acid, glutamic acid or cysteine, and X16 is glutamic acid, asparagine X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine ​​or valine or is absent, X18 is alanine, aspartic acid, glutamine, glutamic acid, arginine, valine or cysteine ​​or is absent, X19 is alanine, arginine, serine, valine or cysteine; or absent, X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, α-methylglutamic acid or cysteine ​​or is absent, X21 is aspartic acid, glutamic acid, leucine, valine or cysteine ​​or is absent, X23 is isoleucine, valine or arginine or is absent, X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methylglutamic acid or leucine or is absent, X27 isoleucine, valine, alanine, lysine, methionine, glutamine or arginine or is absent, X28 is glutamine, lysine, asparagine or arginine or is absent, and X30 is cysteine ​​or is absent (excluding the case where the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1).

[0149] More specifically, in general formula 1, X1 is histidine, tryptophan, or tyrosine, or is absent, X2 is serine or Aib (aminoisobutyric acid), X7 is threonine, valine, or cysteine, X10 is tyrosine or cysteine, X12 is lysine or cysteine, X13 is tyrosine or cysteine, X14 is leucine or cysteine, X15 is aspartic acid or cysteine, X16 is 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, and X20 is glutamic acid. 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 may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1).

[0150] For example, the glucagon analogue includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, specifically, but not limited to, an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 2 to 45. The glucagon analogue has a pI of 6 to 7 and / or a relative in vitro activity of 200% or more compared to native glucagon, more specifically, includes an amino acid sequence of SEQ ID NO: 37, and even more specifically, but not limited to, an amino acid sequence of SEQ ID NO: 37 (essential).

[0151] Specifically, in the general formula 1, X1 is histidine, tryptophan or tyrosine, and X2 is serine or Aib (aminoisobutyric X15 is aspartic acid or cysteine, X16 is 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 is 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.

[0152] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11 to 17, 19 to 27, 29, 31, 33, and 35 to 44, specifically, the peptide consists of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 3, 11 to 17, 19 to 27, 29, 31, 33, and 35 to 44, but is not limited thereto.

[0153] Specifically, in the general formula 1, X1 is tyrosine and X2 is Aib (aminoisobutyric acid 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 absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1).

[0154] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, 40 to 42, and 44, specifically, the peptide consists of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, 40 to 42, and 44, but is not limited thereto.

[0155] Specifically, in general formula 1, X1 is histidine, tryptophan, or tyrosine, or is absent, X2 is serine or Aib (aminoisobutyric acid), X7 is threonine, valine, or cysteine, X10 is tyrosine or cysteine, X12 is lysine or cysteine, X13 is tyrosine or cysteine, X14 is leucine or cysteine, X15 is aspartic acid or cysteine, X16 is glutamic acid, serine, or cysteine, X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine, and X18 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine. X19 is alanine or cysteine, X20 is 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 may be absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1).

[0156] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 45, specifically, the peptide consists of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 45, but is not limited thereto.

[0157] Specifically, in general formula 1, X1 is tyrosine, or X2 is Aib (aminoisobutyric acid), X7 is threonine, X10 is tyrosine, X12 is lysine, X13 is tyrosine, X14 is leucine, X15 is aspartic acid or cysteine, X16 is 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 may be absent.

[0158] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25 and 31, specifically, the peptide consists of an amino acid sequence (essentially) selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25 and 31, but is not limited thereto.

[0159] More specifically, the peptide may be a peptide comprising an amino acid sequence of general formula 2.

[0160] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula 2, sequence number 47)

[0161] In general formula 2, X7 is threonine, valine or cysteine, X10 is tyrosine or cysteine, X12 is lysine or cysteine, X15 is aspartic acid or cysteine, X16 is glutamic acid or serine, X17 is lysine or arginine, X20 is glutamine or lysine, X21 is aspartic acid or glutamic acid, X24 is valine or glutamine, and X30 is cysteine ​​or may be absent.

[0162] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, specifically, the peptide consists of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36 to 44, but is not limited thereto. More specifically, the peptide includes an amino acid sequence of SEQ ID NO: 12, 20, or 37, or consists of the amino acid sequence (essential), but is not limited thereto.

[0163] Specifically, in general formula 2, X7 is threonine, valine or cysteine, X10 is tyrosine or cysteine, X12 is lysine, X15 is aspartic acid, X16 is glutamic acid or serine, X17 is lysine or arginine, X20 is glutamine or lysine, X21 is aspartic acid or glutamic acid, X24 is glutamine, and X30 is cysteine ​​or absent, but is not particularly limited thereto.

[0164] For example, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 36 to 38, 40 to 42, and 44, specifically, the peptide consists of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 12, 36 to 38, 40 to 42, and 44, but is not limited thereto.

[0165] The glucagon analog may include an intramolecular bridge (e.g., a covalent or non-covalent bridge), and may specifically include a ring. For example, the glucagon analog may include a ring between the 16th and 20th amino acids, but is not limited thereto.

[0166] Examples of such rings include, but are not limited to, lactam bridges (or lactam rings).

[0167] The glucagon analogues also include all those modified to contain a ring by including ring-forming amino acids at the desired position.

[0168] Such a ring may be formed between the side chains of amino acids in the glucagon analogue, for example, a lactam ring may be formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0169] For example, a peptide comprising the amino acid sequence of general formula 1 or 2 may be one in which the amino acids in each amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 or 2 are substituted with glutamic acid or lysine, respectively, but is not limited thereto. In the above, n in Xn (n is a natural number) indicates the amino acid position from the N-terminus of the presented amino acid sequence.

[0170] Furthermore, a peptide comprising the amino acid sequence of general formula 1 or 2 may have each amino acid of the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 substituted with glutamic acid or lysine forming a ring, but is not limited thereto.

[0171] Furthermore, at least one of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 or 2 may form a ring (e.g., a lactam ring) between each amino acid in each amino acid pair, but is not limited thereto.

[0172] Furthermore, in 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 thereto.

[0173] The "glucagon derivative" in the present invention includes peptides with at least one difference in amino acid sequence compared to native glucagon, peptides modified by modifying the native glucagon sequence, and mimetics of native glucagon that can activate glucagon receptors like native glucagon. The derivatives of native glucagon may have the function of regulating blood glucose in vivo. The glucagon derivatives in the present invention are used in combination with glucagon analogs.

[0174] The glucagon analogs or glucagon derivatives of the present invention have an altered pI relative to native glucagon, exhibit improved physical properties, have activity to activate the glucagon receptor, and have improved solubility, but are not limited to these.

[0175] The glucagon analogue or glucagon derivative may also be non-naturally occurring.

[0176] As used herein, "isoelectric point" or "pI" refers to the pH value at which a molecule, such as a polypeptide or peptide, has a total net charge of zero (0). For a polypeptide having various charged functional groups, the sum of the charges is zero at the pI. At pH values ​​higher than the pI, the total net charge of the polypeptide is negative, and at pH values ​​lower than the pI, the total net charge of the polypeptide is positive.

[0177] The pI is determined by isoelectric focusing on immobilized pH gradient gels composed of polyacrylamide, starch or agarose, or by calculation from the amino acid sequence using, for example, the pI / MW tool (Non-Patent Document 1) on the ExPASy server.

[0178] In the present invention, "altered pI" means that a part of the amino acid sequence of native glucagon is replaced with negatively and positively charged amino acid residues, resulting in a pI that is different from the pI of native glucagon, i.e., a pI that is decreased or increased compared to the pI of native glucagon. Peptides having such an altered pI exhibit improved solubility and / or high stability at neutral pH. However, the altered pI is not particularly limited thereto.

[0179] Specifically, the glucagon analog or derivative has a pI value that is different from that of native glucagon (6.8), more specifically, a pI value less than 6.8, even more specifically, 6.7 or less, even more specifically, 6.5 or less, or more specifically, a pI value greater than 6.8, 7 or more, even more specifically, 7.5 or more, but is not limited thereto, and is included in the present invention as long as it has a pI value different from that of native glucagon. In particular, it is included in the present invention as long as it has a pI value different from that of native glucagon and exhibits improved solubility at neutral pH compared to native glucagon, thereby reducing the degree of aggregation.

[0180] Specifically, the pI value is 4 to 6.5 and / or 7 to 9.5, more specifically 7.5 to 9.5, and even more specifically 8.0 to 9.3, but is not limited thereto. In this case, since the pI is higher or lower than that of native glucagon, it shows improved solubility and higher stability at neutral pH than native glucagon. However, the pI value is not limited thereto.

[0181] Specifically, glucagon derivatives may be those in which some amino acids of natural glucagon have been modified by any of substitution, addition, deletion and modification, or a combination of these methods. The amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Conservative substitutions with amino acids having such similar properties are expected to exhibit the same or similar activity.

[0182] Examples of glucagon derivatives prepared by combining the above-mentioned methods include peptides that have at least one amino acid sequence different from that of native glucagon, have deamidated N-terminal amino acid residues, and have activation function for the glucagon receptor, but are not limited to these. Derivatives of native glucagon used in the present invention can be prepared by combining various methods for preparing derivatives.

[0183] Such modifications to generate derivatives of native glucagon also include all modifications with L- or D-amino acids and / or non-natural amino acids, and / or modifications of the native sequence, such as modifications of side chain functional groups, covalent bonds within the molecule, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. Furthermore, said modifications also include all substitutions with non-natural compounds.

[0184] Further included are all those in which at least one amino acid has been added to the N- and / or C-terminus of native glucagon.

[0185] The substituted or added amino acids can be any of the 20 amino acids commonly found in human proteins, as well as unusual or unnatural amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides and typical peptide sequences containing these amino acids can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0186] Amino acid derivatives can also be obtained in the same manner, one example being 4-imidazoacetic acid.

[0187] In the present invention, "a fragment of native glucagon, a glucagon analogue or a glucagon derivative" means a form of native glucagon, a glucagon analogue or a derivative of native glucagon in which at least one amino acid at the N-terminus or C-terminus has been deleted, which has a blood glucose increasing effect in the body.

[0188] The glucagon analogues of the present invention may be produced by using any of the methods used for producing the above-mentioned derivatives and fragments of native glucagon, either alone or in combination.

[0189] The pharmaceutical composition according to the present invention may contain glucagon as one of the active ingredients, which may be (i) native glucagon, (ii) a glucagon analogue, (iii) a glucagon derivative, (iv) a fragment thereof, or (v) a combination thereof, so long as the composition has the blood glucose regulating ability of glucagon.

[0190] Furthermore, the peptides according to the present invention (e.g., insulin, insulin analogs, glucagon, glucagon analogs, etc.) may have unmodified N- and / or C-termini, but also include those in which the N- and / or C-termini are chemically modified, protected with an organic group, or modified by the addition of an amino acid to the peptide terminus, etc., in order to protect them from in vivo protease and increase their stability. When the C-terminus is unmodified, the terminus of the peptide according to the present invention has a carboxy group, but is not particularly limited thereto.

[0191] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, and therefore, in order to remove such charges, acetylation of the N-terminus and / or amidation of the C-terminus may be performed, but is not limited to these.

[0192] Specifically, the N-terminus or C-terminus of the insulin or analog thereof or glucagon or analog thereof of the present invention has an amino group (-NH2) or a carboxy group (-COOH), and the C-terminus of glucagon or analog thereof has an amino group, but is not limited to these.

[0193] Unless otherwise specified in the present specification, the detailed description of the specification and the claims regarding the "peptide" according to the present invention or the "conjugate" in which the peptide is covalently linked to a biocompatible substance are applicable to the category including not only the peptide or conjugate, but also the salt of the peptide or conjugate (e.g., a pharma- ceutically acceptable salt of the peptide), or a solvate thereof. Thus, even if the specification only describes a "peptide" or a "conjugate", the description also applies to a specific salt thereof, a specific solvate thereof, or a specific solvate of a specific salt thereof. These salt forms may be, for example, any pharma-ceutically acceptable salt. The type of the salt is not particularly limited. However, it is preferable that the form is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0194] The term "pharmacologically acceptable" refers to a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reactions, and the like, within the scope of medical judgment.

[0195] The "pharmaceutically acceptable salt" in the present invention includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Suitable salts derived from bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0196] In addition, the term "solvate" in the present invention means a complex formed between the peptide, conjugate or salt thereof according to the present invention and a solvent molecule.

[0197] Furthermore, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0198] Specifically, the peptides of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other method known in the art. Thus, the glucagon derivatives of the present invention can be synthesized in a number of ways, including, for example, (a) synthesizing the peptide stepwise in solid or liquid phase methods or by fragment assembly, and isolating and purifying the final peptide product, (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture, (c) expressing a nucleic acid construct encoding the peptide in a cell-free test tube and recovering the expression product, or any combination of (a), (b) and (c) to obtain fragments of the peptide, and then recovering the peptide by ligating the fragments to obtain the peptide.

[0199] As a more specific example, a fusion gene encoding a fusion protein containing a fusion partner and insulin or glucagon or an analog thereof can be prepared by genetic engineering, transformed into a host cell, and then expressed in the form of a fusion protein, and the insulin or glucagon or an analog thereof can be cleaved and separated from the fusion protein using a protease or a compound to produce a desired peptide. For this purpose, a DNA sequence encoding an amino acid residue that can be cleaved by a protease such as Factor Xa or enterokinase, or a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and a polynucleotide encoding insulin or glucagon or an analog thereof.

[0200] As a specific example, the insulin or glucagon of the present invention may be in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

[0201] In the present invention, the term "long-acting conjugate" or "conjugate" of insulin or glucagon means a structure in which insulin or glucagon is conjugated with a biocompatible substance, and has a longer duration of efficacy than insulin or glucagon to which the biocompatible substance is not conjugated. The biocompatible substance in the long-acting conjugate may be covalently linked to insulin or glucagon, but is not particularly limited thereto. In the present invention, the insulin or glucagon, which is one component of the conjugate, may be insulin or glucagon having a native sequence, or may be an analog, derivative, or fragment of insulin or glucagon in which at least one amino acid in the native sequence has been mutated by substitution, addition, deletion, modification, or a combination thereof, but any one having the blood glucose lowering / elevating effect of native insulin or native glucagon can be used as one component of the conjugate of the present invention.

[0202] In the present invention, the term "biocompatible substance" refers to a substance that is bound to a physiologically active substance (e.g., glucagon derivatives, insulinotropic peptides, etc.) and can improve the duration of the effect of the physiologically active substance compared to a physiologically active substance that is not bound to a biocompatible substance moiety or carrier. The biocompatible substance may be linked to the physiologically active substance by a covalent bond, but is not particularly limited thereto.

[0203] Specifically, the conjugate is represented by Chemical Formula 1.

[0204] [Chemical formula 1] X-La-F

[0205] Here, X is insulin or glucagon, L is a linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others, and F is a substance capable of extending the half-life of X.

[0206] The composition of the present invention may comprise (i) insulin and glucagon, (ii) a long-acting insulin conjugate and glucagon, (iii) insulin and a long-acting glucagon conjugate, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate. The insulin or glucagon in the form of a long-acting conjugate exhibits an excellent blood glucose regulating effect due to its improved persistence in the body and can ameliorate the side effects of insulin.

[0207] F in the conjugate is a substance capable of increasing the half-life of X, ie, insulin or glucagon, and is one component that forms part of the conjugate of the present invention.

[0208] The F's may be bonded to X by a covalent or non-covalent chemical bond, or may be bonded to X via L by a covalent or non-covalent chemical bond, or a combination thereof.

[0209] The substance capable of extending the half-life of X is a biocompatible substance, for example, selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0210] In the case of elastin, it may be human tropoelastin, which is a water-soluble precursor, or it may be a polymer of a partial sequence or a partial repeating unit thereof, for example, an elastin-like polypeptide, but is not limited to these.

[0211] Examples of the polymer include a polymer 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, and the polysaccharide is dextran, but is not particularly limited thereto.

[0212] The polyethylene glycol is a term that encompasses the forms of ethylene glycol homopolymer, PEG copolymer, and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

[0213] The biocompatible material also includes polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid, but is not limited to these.

[0214] Furthermore, the fatty acid may be one that has a binding ability to albumin in the body, but is not particularly limited thereto.

[0215] As a more specific example, the FcRn-binding substance may be an immunoglobulin Fc region, and more specifically, an IgG Fc region, but is not particularly limited thereto.

[0216] At least one amino acid side chain in the peptides of the invention is conjugated to such biocompatible substances to improve solubility and / or half-life in vivo and / or increase bioavailability. Such modifications also reduce the clearance of therapeutic proteins and peptides.

[0217] Such biocompatible materials may be water soluble (amphiphilic or hydrophilic), non-toxic and / or pharma- ceutical acceptable.

[0218] The F may be directly linked to X (ie, in Chemical Formula 1, a is 0) or may be linked to X via a linker L.

[0219] In the present invention, the "immunoglobulin Fc region" refers to a portion excluding the variable regions of the heavy and light chains of an immunoglobulin and including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). The immunoglobulin Fc region is a component that forms a part of the conjugate of the present invention.

[0220] Such an immunoglobulin Fc region includes, but is not limited to, a hinge region in a heavy chain constant region. Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region including a part or the whole of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding the variable regions of the heavy and light chains of an immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0221] For example, the immunoglobulin Fc region of the present invention is 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region, but is not limited thereto.

[0222] In addition, as a specific example, the immunoglobulin Fc region may be in a dimeric form, and may have one X1 molecule covalently linked to one Fc region in the dimeric form, where the immunoglobulin Fc and X may be linked to each other via a non-peptidic polymer. On the other hand, the immunoglobulin Fc region in the dimeric form may have one X2 molecule symmetrically linked to it. Here, the immunoglobulin Fc and X may be linked to each other via a non-peptidic linker. However, the present invention is not limited thereto.

[0223] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means a sequence in which at least one amino acid residue of the naturally occurring amino acid sequence is different due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0224] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as sites suitable for modification.

[0225] Also, various kinds of derivatives are used, such as derivatives in which the site forming disulfide bonds has been removed, derivatives in which several amino acids have been deleted from the N-terminus of native Fc, and derivatives in which a methionine residue has been added to the N-terminus of native Fc. Furthermore, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in Patent Documents 7 and 8, etc.

[0226] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 2). The most common exchanges are between the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, the amino acid residues may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0227] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc region.

[0228] In addition, such an Fc region may be obtained from a natural source isolated from the living body of an animal such as a human or a cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant product obtained from a transformed animal cell or a microorganism, or a derivative thereof. Here, the method of obtaining from a natural source may be a method of obtaining by isolating the whole immunoglobulin from the living body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. These can be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0229] Furthermore, the immunoglobulin Fc region may be in a form having a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. Such increase, decrease, or removal of the immunoglobulin Fc sugar chain may be performed using conventional methods such as chemical methods, enzymatic methods, and genetic engineering techniques using microorganisms. Here, an immunoglobulin Fc region from which the sugar chain has been removed from Fc has a significantly reduced binding ability to complement (c1q) and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, an immunoglobulin Fc region from which the sugar chain has been removed or which has been deglycosylated is suitable for its original purpose as a drug carrier.

[0230] In the present invention, "deglycosylation" refers to an Fc region from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0231] On the other hand, the immunoglobulin Fc region may be of human origin or animal origin, such as bovine, goat, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment is of human origin.

[0232] The immunoglobulin Fc region 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 in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend 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 a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0233] On the other hand, the term "combination" in the present invention means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is bound to a single-chain polypeptide of a different origin, i.e., a dimer or multimer can be prepared from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0234] The conjugates described above may also have improved durability of efficacy compared to native insulin or glucagon, or to X where F is not modified. Such conjugates include not only the above-mentioned forms, but also forms encapsulated in biodegradable nanoparticles, etc.

[0235] Furthermore, the L may be a peptidic linker or a non-peptidic linker.

[0236] When the L is a peptide linker, it may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not limited thereto. In the present invention, various known peptide linkers are used to link F and X, for example, [GS]x linker, [GGGS]x linker, [GGGGS]x linker, etc., where x is a natural number of 1 or more. However, it is not limited to the above examples.

[0237] The "non-peptidic linker" of the present invention includes a biocompatible polymer having at least two repeating units bonded together. The repeating units are linked to each other by any covalent bond except a peptide bond. The non-peptidic linker may be a part of the conjugate of the present invention and corresponds to L in Chemical Formula 1. The non-peptidic linker of the present invention is used in combination with a non-peptidic polymer.

[0238] The non-peptidic linker in the present invention contains a reactive group at its terminal and can form a conjugate by reacting with other components constituting the conjugate. When a non-peptidic linker having reactive functional groups at both terminals forms a conjugate by binding to X and F in Chemical Formula 1 via each reactive group, the non-peptidic linker or non-peptidic polymer is also called a non-peptidic linker linking moiety or a non-peptidic polymer linking moiety.

[0239] In the above La, a may be 1 or more, and when a is 2 or more, each L may be independent.

[0240] In a specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker comprising reactive groups at both ends that are coupled to F, specifically an immunoglobulin Fc region, and X, specifically a peptide drug.

[0241] Specifically, the non-peptide linker may be selected from the group consisting of fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0242] In particular, although not limited thereto, the molecular weight of the high molecular weight polymer in the present invention is in the range of more than 0 and not more than about 100 kDa, specifically in the range of about 1 to about 100 kDa, and more specifically in the range of about 1 to about 20 kDa.

[0243] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0244] In particular, but not limited to, the 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, and the polysaccharide is dextran. In a more specific embodiment, the L is polyethylene glycol, but is not limited thereto. In addition, derivatives thereof known in the art and derivatives that can be easily prepared at the technical level of the art are also included in the present invention.

[0245] The non-peptidyl linker used in the present invention may be any polymer that is resistant to endogenous proteases. The molecular weight of the non-peptidyl polymer is in the range of about 1 to about 100 kDa, specifically about 1 to about 20 kDa, but is not limited thereto. Furthermore, the non-peptidyl linker of the present invention that is bonded to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0246] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0247] In a specific embodiment, both ends of the non-peptidic linker can be bound to an amino or thiol group of F, for example an immunoglobulin Fc region, and an amino or thiol group of X, respectively.

[0248] Specifically, the non-peptidyl polymer contains reactive groups at both ends that are bonded to F (e.g., an immunoglobulin Fc region) and X, respectively, more specifically, a reactive group that is bonded to X, or an amino group located at the N-terminus or lysine of F (e.g., an immunoglobulin Fc region), or a thiol group of cysteine, but is not limited thereto.

[0249] Furthermore, the reactive group of the non-peptidyl polymer bound to F, for example, an immunoglobulin Fc region, and X is selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0250] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0251] In the above, examples of succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0252] The non-peptide linker is linked to X and F via such a reactive group, but is not particularly limited thereto.

[0253] Furthermore, the final products generated by reductive amination with aldehyde bonds are much more stable than those linked with amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0254] The reactive groups at both ends of the non-peptidic linker may be the same or different. For example, one end may have a maleimide group and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. However, the non-peptidic linker is not particularly limited to these, as long as F, specifically, the immunoglobulin Fc region and X are bonded to each end of the non-peptidic linker.

[0255] For example, the non-peptide linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end.

[0256] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups can be activated into the various reactive groups described above by known chemical reactions, or a commercially available polyethylene glycol having modified reactive groups can be used to prepare the long-acting protein conjugate of the present invention.

[0257] In a specific embodiment, the non-peptidyl polymer is linked to a cysteine ​​residue of X, more specifically, to the -SH group of cysteine, but is not limited thereto.

[0258] Examples of the non-peptide polymer include, but are not limited to, those in which the non-peptide polymer is linked to the 10th cysteine ​​residue, the 13th cysteine ​​residue, the 15th cysteine ​​residue, the 17th cysteine ​​residue, the 19th cysteine ​​residue, the 21st cysteine ​​residue, the 24th cysteine ​​residue, the 28th cysteine ​​residue, the 29th cysteine ​​residue, the 30th cysteine ​​residue, the 31st cysteine ​​residue, the 40th cysteine ​​residue, or the 41st cysteine ​​residue in the peptide corresponding to X.

[0259] Specifically, the reactive group of the non-peptidic polymer may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X by a thioether bond, and the aldehyde group can be linked to F, specifically, the -NH2 group of immunoglobulin Fc by a reductive amination reaction, but this is not limited thereto and is merely an example.

[0260] Furthermore, in the above-mentioned conjugate, the reactive group of the non-peptidyl polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is merely one example.

[0261] In the present invention, "prevention" means any action of suppressing or delaying the onset of an insulin-related disease by administering the composition, and "treatment" means any action of improving or favorably changing the symptoms of an insulin-related disease by administering the composition.

[0262] In the present invention, "administration" means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition is not particularly limited, but may be any common route that can deliver the composition to a target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0263] The pharmaceutical composition according to the present invention may include a pharma- ceutically acceptable carrier.

[0264] In the present invention, "pharmacologically acceptable" means a sufficient amount to exert a therapeutic effect and to cause no side effects, and can be easily determined by a person skilled in the art based on factors known in the medical field, such as the type of disease, the patient's age, weight, health condition, sex, sensitivity to the drug, administration route, administration method, number of doses, treatment period, combination, and drugs used concomitantly.

[0265] The pharma- ceutically acceptable carrier may be a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye, flavoring, etc., for oral administration, a buffer, preservative, analgesic, solubilizer, isotonicity agent, stabilizer, etc., for injection, a base, excipient, lubricant, preservative, etc., for topical administration. The pharmaceutical composition of the present invention may be prepared in various forms by mixing with the pharma- ceutically acceptable carrier as described above. For example, for oral administration, it may be prepared in the form of a tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc., and for injection, it may be prepared in the form of a disposable ampoule or a multiple dose form. It may also be prepared in the form of a solution, suspension, tablet, pill, capsule, sustained release preparation, etc.

[0266] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, etc.

[0267] Furthermore, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized agents, and suppositories.

[0268] Furthermore, the conjugate can be used by mixing it with various pharmaceutical carriers such as physiological saline or organic solvents, and in order to improve stability and absorbability, carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, other stabilizers, etc. can be used as pharmaceutical agents.

[0269] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease.

[0270] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredients depending on the severity of the disease. Specifically, the total dose of the conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the dose of the conjugate is determined by taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these into consideration, a person having ordinary knowledge in the art would be able to determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0271] The pharmaceutical composition of the present invention is one in which insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are co-administered simultaneously, sequentially or in the reverse order, but is not limited thereto.

[0272] As described above, the combination administration may be, but is not limited to, a) administration of (i) insulin or a long-acting conjugate thereof and (ii) glucagon or a long-acting conjugate thereof in a single mixture, or b) administration of (i) insulin or a long-acting conjugate thereof and (ii) glucagon or a long-acting conjugate thereof in separate forms.

[0273] When insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are in a separated form, insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof may be formulated into separate preparations and administered simultaneously, separately, sequentially, or in the reverse order.

[0274] Another aspect of the present invention is a pharmaceutical composition for preventing or treating hypoglycemia, comprising insulin and glucagon.

[0275] Specifically, the compositions include (i) a composition comprising insulin and glucagon, (ii) a composition comprising a long-acting insulin conjugate in which insulin is conjugated with a biocompatible substance capable of extending its half-life in vivo, and glucagon, (iii) a composition comprising insulin and a long-acting glucagon conjugate in which glucagon is conjugated with a biocompatible substance capable of extending its half-life in vivo, or (iv) a composition comprising a long-acting insulin conjugate and a long-acting glucagon conjugate.

[0276] As described above, the insulin or glucagon of the present invention may be insulin or glucagon having a native sequence, or it may be an analog, derivative, or fragment thereof in which at least one amino acid has been mutated by substitution, addition, deletion, modification, or a combination thereof.

[0277] The pharmaceutical composition for preventing or treating hypoglycemia according to the present invention contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and has the effect of alleviating hypoglycemia that occurs as a side effect of insulin administration, as well as the effect of suppressing weight gain.

[0278] In the examples of the present invention, not only was a blood glucose lowering effect confirmed in db / db mice administered a composition containing insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, but also weight gain inhibition and hypoglycemia alleviation effects were confirmed, demonstrating that the pharmaceutical composition according to the present invention can improve the side effects of insulin while maintaining its efficacy.

[0279] The pharmaceutical composition of the present invention is a composition in which insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are co-administered simultaneously, sequentially, or in the reverse order, but is not limited thereto. Co-administration is as described above.

[0280] Yet another aspect of the present invention is a composition for improving insulin side effects, comprising insulin and glucagon.

[0281] Specifically, the compositions include (i) a composition comprising insulin and glucagon, (ii) a composition comprising a long-acting insulin conjugate in which insulin is conjugated with a biocompatible substance capable of extending its half-life in vivo, and glucagon, (iii) a composition comprising insulin and a long-acting glucagon conjugate in which glucagon is conjugated with a biocompatible substance capable of extending its half-life in vivo, or (iv) a composition comprising a long-acting insulin conjugate and a long-acting glucagon conjugate.

[0282] As described above, the insulin or glucagon of the present invention may be insulin or glucagon having a native sequence, or it may be an analog, derivative, or fragment thereof in which at least one amino acid has been mutated by substitution, addition, deletion, modification, or a combination thereof.

[0283] The composition for improving insulin side effects according to the present invention contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, and has the effect of alleviating the side effects of insulin administration (e.g., hypoglycemia or weight gain).

[0284] The pharmaceutical composition of the present invention is a composition in which insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof are co-administered simultaneously, sequentially, or in the reverse order, but is not limited thereto. Co-administration is as described above.

[0285] Yet another aspect of the present invention is a combination preparation for improving hypoglycemia in a patient with an insulin-related disease, comprising (i) insulin and glucagon, (ii) a long-acting insulin conjugate and glucagon, (iii) insulin and a long-acting glucagon conjugate, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate.

[0286] In one embodiment, the combination formulation according to the present invention may include a pharma- ceutically acceptable carrier.

[0287] The terms "pharmacologically acceptable" and "pharmacologically acceptable carrier" in the present invention are as defined above.

[0288] The combined preparation according to the present invention is a combined preparation for treating insulin-related diseases, and has the effect of alleviating the side effects of insulin (e.g., weight gain, hypoglycemia). Specifically, the combined preparation can improve hypoglycemia, which is a side effect of insulin, and can suppress weight gain.

[0289] The composite preparation of the present invention contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a weight ratio of 0.1:1 to 100:1, or in a molar ratio of 1:1 to 30:1, 1:1 to 20:1, or 1:1 to 16:1, but is not limited thereto.

[0290] Yet another aspect of the present invention is a method for preventing or treating an insulin-related disorder, comprising the step of administering (i) insulin and glucagon, (ii) a long-acting insulin conjugate and glucagon, (iii) insulin and a long-acting glucagon conjugate, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate to an individual suffering from or at risk of developing an insulin-related disorder.

[0291] The step of administering can be performed by co-administering (i) insulin and glucagon, (ii) a long-acting insulin conjugate and glucagon, (iii) insulin and a long-acting glucagon conjugate, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate. The substances may be administered simultaneously, sequentially, or in the reverse order, and may be administered simultaneously in an appropriate effective amount, but are not limited to a specific administration method or order. In addition, the preventive or therapeutic method of the present invention is a method of administering a composition or a combined preparation containing any of (i) to (iv) to an individual, but is not limited thereto. As described above, the insulin or its long-acting conjugate and the glucagon or its long-acting conjugate administered may be individual preparations or a combined preparation.

[0292] The composition or composite preparation of the present invention, which contains insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof, replenishes the activity or function of insulin, significantly lowers blood glucose, and suppresses insulin side effects such as weight gain and hypoglycemia, and is therefore highly effective in treating insulin-related diseases.

[0293] Furthermore, the composition or composite preparation can be formulated into a single-dose preparation suitable for administration to the patient's body by a conventional method in the pharmaceutical field, specifically into a preparation form useful for administration of protein pharmaceuticals, and administered orally or via a parenteral administration route, including dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, but is not limited to these.

[0294] The method of the invention may include administering a pharmaceutical composition comprising said peptide in a pharma- ceutical effective amount. A suitable total daily dose will be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and extent of the response to be achieved, whether or not other formulations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0295] Yet another aspect of the present invention is a method for ameliorating insulin side effects comprising the step of administering (i) insulin and glucagon, (ii) a long-acting insulin conjugate and glucagon, (iii) insulin and a long-acting glucagon conjugate, or (iv) a long-acting insulin conjugate and a long-acting glucagon conjugate to an individual experiencing or at risk of experiencing insulin side effects.

[0296] A further object of the present invention is to provide a use of the composition or combined preparation in the manufacture of a medicament for the treatment of insulin-related diseases.

[0297] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. EXAMPLES

[0298] Preparation of glucagon analogues The present inventors attempted to confirm the therapeutic effect of diabetes by combined administration of insulin and glucagon, and further attempted to confirm whether said combined administration could reduce the risk of hypoglycemia, a side effect of insulin.

[0299] Therefore, first, natural glucagon and glucagon analogs having activity equivalent thereto were prepared as shown in Table 1 below. Glucagon analogs were synthesized with reference to Patent Documents 4, 5 and 6, and their pI and in vitro activity were measured.

[0300] [Table 1] JPEG2024178193000003.jpg199161 JPEG2024178193000004.jpg215161 JPEG2024178193000005.jpg149161

[0301] In the sequences of Table 1, the amino acid marked with X represents the unnatural amino acid aminoisobutyric acid (Aib), the underline in the amino acid symbol represents the formation of a lactam ring between the side chains of the underlined amino acid pair, and "-" represents the absence of an amino acid residue at that position. In the column indicating the presence or absence of ring formation, "-" represents the absence of a ring in the sequence. EXAMPLES

[0302] Preparation of insulin analogues The present inventors have prepared insulin analogs to be administered in combination with glucagon or a long-acting conjugate thereof. Specifically, the analogs shown in Table 2 were prepared with reference to Patent Document 2.

[0303] Table 2 shows the modified amino acid sequence and analog name of each A chain or B chain. That is, Analog 1 is a form in which the first glycine in A chain is replaced with alanine, and Analog 4 is a form in which the eighth glycine in B chain is replaced with alanine.

[0304] [Table 2]

[0305] Table 3 shows the DNA and protein sequences of each of the insulin analogues 1 to 9.

[0306] [Table 3] JPEG2024178193000008.jpg209161 JPEG2024178193000009.jpg208161 JPEG2024178193000010.jpg211161 JPEG2024178193000011.jpg201161

[0307] The present inventors also prepared the insulin analogues shown in Table 4 with reference to Patent Document 3.

[0308] [Table 4]

[0309] Table 5 shows the DNA and protein sequences of each of the insulin analogues 10 and 11.

[0310] [Table 5] JPEG2024178193000014.jpg55150

[0311] Furthermore, the present inventors produced the insulin analogs shown in Table 6. Specifically, to produce insulin analogs in which one amino acid in the A or B chain was modified, forward and reverse oligonucleotides were synthesized using the native insulin expression vector as a template, and then PCR was performed to amplify each analog gene.

[0312] [Table 6]

[0313] Primers for amplifying insulin analogues are shown in Table 7.

[0314] [Table 7]

[0315] The PCR conditions for amplifying insulin analogs were 95°C for 30 seconds, 55°C for 30 seconds, and 68°C for 6 minutes, followed by 18 cycles. The insulin analog fragments obtained under these conditions were inserted into a pET22b vector to be expressed in the form of inclusion bodies within cells, and the expression vectors thus obtained were named pET22b-insulin analogs 1-13. The expression vectors contain nucleic acids encoding the amino acid sequences of insulin analogs 1-13 under the control of a T7 promoter. In a host containing the expression vector, insulin analog proteins were expressed in the form of inclusion bodies.

[0316] Table 4 shows the DNA and protein sequences of each insulin analogue 1 to 13.

[0317] Each sequence modification was confirmed by DNA sequence analysis, and the results confirmed that each insulin analog had the intended sequence modification.

[0318] [Table 8] JPEG2024178193000018.jpg209161 JPEG2024178193000019.jpg211161 JPEG2024178193000020.jpg211161 JPEG2024178193000021.jpg209161 JPEG2024178193000022.jpg211161 JPEG2024178193000023.jpg210161 JPEG2024178193000024.jpg211161 JPEG2024178193000025.jpg226161 EXAMPLES

[0319] Expression of recombinant insulin analog fusion peptides The recombinant insulin analogs were expressed under the control of the T7 promoter. E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λ(DE3); Novagen) was transformed with each recombinant insulin analog expression vector. The transformation method was according to the method recommended by Novagen. A single colony transformed with each recombinant expression vector was obtained, inoculated into 2X Luria Broth (LB) medium containing ampicillin (50 μg / ml), and cultured at 37°C for 15 hours. The recombinant strain culture liquid and 2XLB medium containing 30% glycerin were mixed at a ratio of 1:1 (v / v), and 1 ml of each was dispensed into cryotubes and stored at -140°C. This was used as a cell stock for the production of recombinant fusion proteins.

[0320] For the expression of recombinant insulin analogs, one vial of each cell stock was dissolved and inoculated into 500 ml of 2X Luria Broth medium and cultured at 37°C for 14-16 hours with shaking. When the OD600 value reached 5.0 or more, the culture was terminated and used as the seed culture. The seed culture was inoculated into 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, BEMARUBISHI, Japan) to start initial bath fermentation. The culture conditions were a temperature of 37°C, an air volume of 20 L / min (1 vvm), an agitation speed of 500 rpm, and pH was maintained at 6.70 using 30% aqueous ammonia. The fermentation was performed as a fed-batch culture by adding a feeding solution when the nutrients in the culture solution were exhausted. The growth of the strain was monitored by the OD value, and when the OD value reached 100 or more, IPTG was introduced at a final concentration of 500 μM. Cultivation was continued for approximately 23 to 25 hours after introduction, and after completion of the cultivation, the recombinant strain was isolated using a centrifuge and stored at -80°C until use. EXAMPLES

[0321] Recovery and refolding of recombinant insulin analogues To convert the recombinant insulin analogue expressed in Example 3 into a soluble form, the cells were disrupted and refolded. 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). The cells were disrupted at 15,000 psi pressure using a Microfluidizer processor (Microfluidic Corp. Model M-110EH-30). The disrupted cell lysate was centrifuged at 7,000 rpm for 20 min at 4-8°C, the supernatant was discarded, and the cells were resuspended in 3 L of wash buffer (0.5% Triton X-100, 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, and 1 mM EDTA). The mixture was centrifuged at 4-8°C and 7,000 rpm for 20 minutes, and the pellet was resuspended in distilled water, and then centrifuged in the same manner. The pellet was obtained and resuspended in a buffer solution (1M L-glycine, 3.78 g L-cysteine-HCl, pH 10.6) and stirred at room temperature for 1 hour. To recover the resuspended recombinant insulin analog, 8M urea was added and then stirred for 3-5 hours. To refold the solubilized recombinant proinsulin analog, the mixture was centrifuged at 4-8°C and 7,000 rpm for 30 minutes to obtain a supernatant, which was then treated with a reducing agent (15 mM L-cysteine-HCL) for 1 hour, and a specified amount of distilled water was added using a peristaltic pump and stirred at 4-8°C for 12 hours or more. EXAMPLES

[0322] Cation exchange chromatography purification The refolded sample was bound to an SP FF (GE Healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and the insulin analog protein was then eluted with a linear concentration gradient of 10 column volumes from 0% to 100% using 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol. EXAMPLES

[0323] Trypsin and Carboxypeptidase B Treatment Salts were removed from the eluted sample using an ultrafiltration membrane, and the solution was exchanged for a buffer solution (10 mM Tris-HCl, pH 8.0). Trypsin equivalent to a molar ratio of about 30,000 and carboxypeptidase B equivalent to a molar ratio of about 3,000 based on the protein amount of the obtained sample were added, and the mixture was stirred at 4 to 8°C for 16 hours or more. EXAMPLES

[0324] Cation exchange chromatography purification After the reaction was completed, the sample was bound again to an SP HP (GE Healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and then the insulin analog protein was eluted with a linear concentration gradient of 10 column volumes from 0% to 100% using 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol. EXAMPLES

[0325] Reverse phase chromatography purification To separate the pure insulin analog from the sample obtained in Example 7, the sample was bound to a reversed-phase chromatography Source30RPC (GE Healthcare, USA) equilibrated with a buffer containing sodium phosphate and isopropanol, and the insulin analog protein was then eluted with a linear concentration gradient using a buffer containing sodium phosphate and isopropanol. EXAMPLES

[0326] Preparation of long-acting conjugates of insulin and glucagon The insulin and glucagon analogs prepared in the above examples were bound to the immunoglobulin Fc region using polyethylene glycol as a linker to prepare a long-acting conjugate of insulin and glucagon. The glucagon analog used to prepare the long-acting glucagon conjugate has a pI of 6 to 7 and a relative in vitro activity of 200% or more compared to that of native glucagon. The insulin used to prepare the long-acting insulin conjugate is native insulin or an analog in which one amino acid is substituted in the A chain of native insulin.

[0327] Specifically, to PEGylate the N-terminus of the B chain of insulin (Biocon, India) with 3.4K propion-ALD(2) PEG (3.4 kDa PEG with one propionaldehyde group at each end, NOF, USA), the insulin:PEG molar ratio was set to 1:4, the insulin concentration was set to 5 mg / ml, and the reaction was carried out at 4°C for about 2 hours. The reaction was carried out in a mixed solvent of 50 mM sodium citrate buffer (pH 5.0) and 45% isopropanol, with the addition of a 3 mM sodium cyanoborohydride (NaCNBH3) reducing agent. The reaction solution was purified on an SP-HP (GE Healthcare) column using a buffer containing sodium citrate (pH 3.0), 45% EtOH, and a concentration gradient of KCl.

[0328] Next, in order to bind the insulin-bound PEG to the N-terminus of the immunoglobulin Fc fragment, the purified mono-PEGylated insulin and the immunoglobulin Fc fragment were reacted at a molar ratio of 1:1.2 at a total protein concentration of 20 mg / ml for 15 hours at 25° C. The reaction solution was 100 mM HEPES buffer (pH 8.2) and sodium chloride, to which 20 mM sodium cyanoborohydride was added as a reducing agent.

[0329] After the reaction was completed, the reaction solution was applied to a Q-HP (GE, USA) column using a concentration gradient of Tris-HCl (pH 7.5) buffer and NaCl, and then applied to a Source 15ISO (GE, USA) using a concentration gradient of ammonium sulfate and Tris-HCl (pH 7.5) to purify the insulin-3.4K PEG-immunoglobulin Fc conjugate.

[0330] In addition, to prepare a long-acting glucagon conjugate, 10 kDa PEG having a maleimide group and an aldehyde group at each end, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), was PEGylated to the cysteine ​​residue of the glucagon analogue at a molar ratio of 1:1-5, a protein concentration of 3-10 mg / ml, and reacted at low temperature for 1-3 hours. Here, the reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative mono-PEGylated to cysteine.

[0331] Next, the purified mono-PEGylated glucagon analog and immunoglobulin Fc were reacted at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg / ml at 4-8° C. for 12-18 hours. The reaction was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was supplemented with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-20% isopropanol. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify a conjugate containing a glucagon analog and immunoglobulin Fc. EXAMPLES

[0332] Confirmation of the hypoglycemic effect of combined administration of glucagon and insulin in DIO / STZ rats The long-acting conjugate of glucagon and natural insulin prepared in Example 9 was administered to DIO / STZ rats, known as a model of obesity and type 2 diabetes. In order to confirm the hypoglycemic therapeutic effect of the combined administration of the long-acting conjugate of glucagon and natural insulin, the test was divided into a vehicle control group, a group administered only the long-acting insulin conjugate (53.9 nmol / kg (2997 ug / kg), Q3D), and a group administered the long-acting insulin conjugate (53.9 nmol / kg, Q3D) and the long-acting glucagon conjugate (3.4 nmol / kg (182 ug / kg), Q3D) in combination, and the substance was repeatedly administered subcutaneously for 2 weeks. During the two-week repeated administration, blood was collected from the tail vein of each rat, and the degree of change in blood glucose level was measured using a blood glucose meter (OneTouch (registered trademark) Ultra (registered trademark).

[0333] After repeated administration of the long-acting insulin conjugate for two weeks, it was confirmed that the hyperglycemia values ​​measured in the vehicle control group dropped to normal levels and to hypoglycemia levels (70 mg / dL or less) (Figure 1). Thus, it was confirmed that high doses of insulin have a blood glucose lowering effect but can induce hypoglycemia. However, it was confirmed that coadministration of the long-acting insulin conjugate and the long-acting glucagon conjugate continuously normalized blood glucose without causing hypoglycemia.

[0334] In addition, blood glucose changes during the administration period were quantified using the AUC (area under curve) and analyzed as shown in Figure 2. The results confirmed that when a long-acting glucagon conjugate was co-administered with a long-acting insulin conjugate, the co-administration of the long-acting insulin conjugate and the long-acting glucagon conjugate did not significantly affect the overall blood glucose lowering effect of the long-acting insulin conjugate, and hypoglycemia, a side effect of administration of the long-acting insulin conjugate, could be prevented (Figure 2). Thus, it was confirmed that the risk of hypoglycemia caused by the long-acting insulin conjugate could be reduced and the blood glucose lowering effect could be maintained by co-administering a long-acting glucagon conjugate with a long-acting insulin conjugate. EXAMPLES

[0335] Combined administration of glucagon and insulin improves insulin-induced side effects in DIO / STZ rats The long-acting conjugate of natural insulin used in Example 10, like other long-acting insulins, has side effects of hypoglycemia and weight gain.

[0336] In fact, as shown in Figure 3, repeated administration of long-acting insulin conjugates to DIO / STZ rats for 2 weeks resulted in significant weight gain compared to the vehicle control group. However, coadministration of a long-acting glucagon conjugate with a long-acting insulin conjugate not only neutralized the weight gain, but also reduced body weight compared to the vehicle control group. Thus, it was confirmed that coadministration of a long-acting insulin conjugate and a long-acting glucagon conjugate alleviates hypoglycemia and weight gain, which are side effects of long-acting insulin conjugate administration.

[0337] From the above examples, it was confirmed that the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate not only provides a blood glucose lowering effect but also suppresses the side effects (hypoglycemia and weight gain) caused by insulin administration. Therefore, it was confirmed that the combined administration of a long-acting insulin conjugate and a long-acting glucagon conjugate according to the present invention not only provides a therapeutic effect for insulin-related diseases but also improves hypoglycemia and insulin side effects.

[0338] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A pharmaceutical composition for preventing or treating an insulin-related disease, comprising insulin and glucagon.

2. The composition of claim 1 , wherein the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

3. The composition of claim 1, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

4. the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound; The composition of claim 1, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

5. The composition according to any one of claims 1 to 4, wherein the insulin-related disease is selected from the group consisting of insulin resistance disease, diabetes, hyperglycemia and obesity.

6. The composition according to any one of claims 1 to 4, which alleviates hypoglycemia, a side effect of insulin, and suppresses weight gain.

7. The composition according to any one of claims 1 to 4, comprising insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a weight ratio of 0.1:1 to 100:

1.

8. The composition of any one of claims 1 to 4, wherein the glucagon is a native glucagon or a glucagon analogue in which at least one amino acid of the native glucagon has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification and combinations thereof.

9. The composition of claim 8 , wherein the glucagon analog comprises an amino acid sequence of general formula 1: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number 46) In the above formula, X1 is histidine (H), desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan (W), tyrosine (Y), or absent; X2 is α-methyl-glutamic 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), α-methylglutamic acid, cysteine ​​(C), or is absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), cysteine ​​(C), or absent; X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine ​​(C), or absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, cysteine ​​(C), or is absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), cysteine ​​(C), or absent; X23 is isoleucine (I), valine (V), arginine (R), or is absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid, leucine (L), or absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent; X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 1 is the same as SEQ ID NO:1).

10. In the general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y), or is absent; X2 is 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); X30 is cysteine ​​(C) or absent; The composition of claim 9.

11. In the general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y); X2 is 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 is methionine (M), X28 is asparagine (N) or arginine (R); X30 is cysteine ​​(C) or absent; The composition of claim 9.

12. In the general formula 1, X1 is tyrosine (Y); X2 is Aib (aminoisobutyric acid), X7 is cysteine ​​(C), threonine (T) or valine (V); X10 is tyrosine (Y) or cysteine ​​(C); X12 is 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 is methionine (M), X28 is asparagine (N) or arginine (R); X30 is cysteine ​​(C) or absent; The composition of claim 9.

13. In the general formula 1, X1 is histidine (H), tryptophan (W) or tyrosine (Y), or is absent; X2 is 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); X30 is cysteine ​​(C) or absent; The composition of claim 9.

14. In the general formula 1, X1 is tyrosine (Y); X2 is Aib (aminoisobutyric acid), X7 is threonine (T); X10 is tyrosine (Y); X12 is 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 is arginine (R); X19 is 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 is methionine (M), X28 is asparagine (N) or arginine (R); X30 does not exist, The composition of claim 9.

15. 10. The composition of claim 9, wherein the glucagon analog comprises an amino acid sequence of general formula 2: Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-RA-X20-X21-FV-X24-WL-M-N-T-X30 (General formula 2, SEQ ID NO: 47) In the 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 is 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); X30 is cysteine ​​(C) or absent.

16. The composition according to claim 9, wherein in the general formula 1, a ring is formed between each amino acid in at least one amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28.

17. The composition according to claim 16, wherein each amino acid of at least one of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 is substituted with glutamic acid or lysine to form a ring.

18. The composition of claim 9, wherein the glucagon analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-45.

19. The composition of claim 18 , wherein the glucagon analog comprises the amino acid sequence of SEQ ID NO:

37.

20. The composition according to any one of claims 1 to 4, wherein the insulin is a natural insulin or an insulin analogue in which at least one amino acid of natural insulin has been mutated in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

21. 21. The composition of claim 20, wherein the insulin analogue is an insulin analogue in which at least one amino acid selected from the group consisting of the 1st, 2nd, 3rd, 5th, 8th, 10th, 12th, 16th, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, and 30th amino acids of the B chain and the 1st, 2nd, 5th, 8th, 10th, 12th, 14th, 16th, 17th, 18th, 19th, and 21st amino acids of the A chain of native insulin is substituted with another amino acid or deleted.

22. The composition of claim 20, wherein the insulin analog comprises 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 the 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; 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 the general formula 4, Xaa8 is alanine or glycine; Xa16 is tyrosine, glutamic acid, serine, threonine, aspartic acid, or is absent; Xaa23 is glycine or alanine; Xaa24 is alanine or phenylalanine; Xaa25 is alanine, phenylalanine, aspartic acid, glutamic acid, or is absent; Xaa27 is threonine or absent, Xaa28 is proline, glutamic acid, aspartic acid, or absent (Herein excluding peptides comprising an A chain of SEQ ID NO:48 and a B chain of SEQ ID NO:49).

23. 23. The composition of claim 22, wherein the insulin analogue has one amino acid selected from the group consisting of the 8th, 23rd, 24th, and 25th amino acids of the B chain and the 1st, 2nd, and 19th amino acids of the A chain of native insulin substituted with alanine, or the 14th amino acid of the A chain substituted with glutamic acid or asparagine.

24. 24. The composition of claim 23, wherein the insulin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67.

25. 23. The composition of claim 22, wherein the insulin analogue has the 16th amino acid of the B chain of native insulin substituted with glutamic acid, the 25th amino acid deleted, or the 14th amino acid of the A chain of native insulin substituted with glutamic acid or alanine.

26. The composition of claim 25 , wherein the insulin analog comprises the amino acid sequence of SEQ ID NO: 69 or 71.

27. 23. The composition of claim 22, wherein the insulin analogue has the 16th amino acid of the B chain of natural insulin substituted with glutamic acid, serine, threonine or aspartic acid, the 25th amino acid substituted with aspartic acid or glutamic acid, the 14th amino acid of the A chain of natural insulin substituted with histidine, lysine, alanine or aspartic acid, or the 19th amino acid substituted with glutamic acid, serine or threonine.

28. 28. The composition of claim 27, wherein the insulin analog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123.

29. The composition of claim 22, wherein the insulin analogue is in the form of a double chain consisting of an A chain of sequence number 48 represented by the general formula 3 and a B chain of sequence number 49 represented by the general formula 4.

30. 30. The composition of claim 29, wherein the A and B chains are linked by a disulfide bond.

31. The composition according to any one of claims 2 to 4, wherein the conjugate is represented by the following chemical formula 1: [Chemical formula 1] X-La-F Where: X is insulin or glucagon; L is a linker, a is 0 or a natural number, but if a is 2 or more, each L is independent of the others; F is a substance capable of extending the in vivo half-life of X, The "-" represents a covalent or non-covalent bond.

32. The composition of claim 31, wherein F is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

33. The composition of claim 32, wherein the 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.

34. 32. The composition of claim 31 , wherein F is an immunoglobulin Fc region.

35. The composition of claim 34, wherein F is an IgG Fc region.

36. 35. The composition of claim 34, wherein the immunoglobulin Fc region is non-glycosylated.

37. The composition of claim 34, wherein the immunoglobulin Fc region is selected from the group consisting of: (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region; and (f) a dimer of each domain of a heavy chain constant region and a light chain constant region.

38. The composition of claim 34, wherein the immunoglobulin Fc region has a disulfide bond-forming site removed, some amino acids at the N-terminus of a native Fc region have been deleted, a methionine residue has been added to the N-terminus of a native Fc region, a complement binding site has been removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site has been removed.

39. 35. The composition of claim 34, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.

40. 35. The composition of claim 34, wherein the immunoglobulin Fc region is a hybrid of domains of different origin derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

41. 32. The composition of claim 31, wherein 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.

42. The polymer is 42. The composition of claim 41, wherein the polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, polyvinyl ethyl ethers, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

43. 32. The composition of claim 31 , wherein L is polyethylene glycol.

44. The composition according to claim 1, characterized in that insulin and glucagon are co-administered simultaneously, sequentially or in reverse order.

45. A pharmaceutical composition for preventing or treating hypoglycemia, comprising insulin and glucagon.

46. 46. ​​The composition of claim 45, wherein the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

47. 46. ​​The composition of claim 45, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

48. the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound; 46. ​​The composition of claim 45, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

49. 46. ​​The composition of claim 45, wherein insulin and glucagon are administered in combination simultaneously, sequentially or in reverse order.

50. A composition for improving insulin side effects, comprising insulin and glucagon.

51. 51. The composition of claim 50, wherein the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

52. 51. The composition of claim 50, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

53. the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound; 51. The composition of claim 50, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

54. 51. The composition of claim 50, wherein insulin and glucagon are administered in combination simultaneously, sequentially or in reverse order.

55. A combination preparation comprising insulin and glucagon for improving hypoglycemia in patients with insulin-related diseases.

56. The combination preparation according to claim 55, wherein the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

57. The combination preparation according to claim 55, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

58. the insulin is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound; The combination preparation according to claim 55, wherein the glucagon is in the form of a long-acting conjugate to which a biocompatible substance capable of extending its in vivo half-life is attached.

59. The combined preparation according to any one of claims 55 to 58, wherein the hypoglycemia is a side effect of insulin.

60. The composite preparation according to any one of claims 55 to 58, wherein the composite preparation inhibits weight gain.

61. The composite preparation according to any one of claims 55 to 58, comprising insulin or a long-acting conjugate thereof and glucagon or a long-acting conjugate thereof in a weight ratio of 0.1:1 to 100:1.

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