EA040165B1 - GLUCAGON DERIVATIVE AND COMPOSITION CONTAINING A LONG-ACTING CONJUGATE OF THIS DERIVATIVE - Google Patents

Description

Technical field

The invention relates to a derivative of glucagon, a long-acting conjugate of this derivative of glucagon and their use.

Prior Art

Due to recent economic growth and changes in dietary habits, etc. the number of cases of diseases associated with metabolic syndrome, including various diseases such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, and liver disease, is rapidly increasing. These diseases can occur independently, but, as a rule, they occur in close relationship with each other and are accompanied by various symptoms.

In particular, according to the World Health Organization (WHO), more than one billion adults worldwide are overweight, of which over 3 million are clinically obese, and 250,000 people die every year in Europe and more than 2.5 million people around the world die every year due to weight-related diseases.

Overweight and obesity are responsible for the increase in blood pressure and cholesterol levels and the occurrence or aggravation of various diseases such as heart disease, diabetes, arthritis, etc. In addition, the problem of obesity is also becoming the main reason for the increase in the incidence of arteriosclerosis, hypertension, hyperlipidemia or heart disease, not only in adults, but also in children or adolescents.

Although obesity is a serious condition that is the cause of various diseases throughout the world, as mentioned above, it is believed that it can be overcome with individual efforts, and it is also believed that patients with obesity lack self-control. However, obesity is difficult to treat because it is a complex disease involving appetite control and energy metabolism mechanisms. Accordingly, the treatment of obesity requires not only the efforts of obese patients, but also a method capable of treating abnormal mechanisms related to appetite control and energy metabolism. Therefore, efforts have been made to develop drugs to treat the abnormal mechanisms.

As a result of these efforts, drugs such as Rimonabant® (Sanofi-Aventis), Sibutramine® (Abbott), Contrave® (Takeda), Orlistat® (Roche) etc. have been developed, but their disadvantages are serious adverse effects or very weak anti-obesity effects. For example, Rimonabant® is reported to have central nervous system side effects, Sibutramine® and Contrave® have cardiovascular side effects, and Orlistat® only shows a weight loss of about 4 kg when taken for one year. Accordingly, there are no anti-obesity therapeutic agents that can be safely administered to obese patients.

Many comprehensive studies have been carried out to develop new anti-obesity therapeutic agents that can solve the problems of conventional anti-obesity drugs. Recently, glucagon derivatives have attracted much attention. Glucagon is produced by the pancreas when blood glucose levels drop as a result of other medications or diseases, or hormone or enzyme deficiencies. Glucagon sends a signal for the breakdown of glycogen in the liver and subsequent release of glucose and is involved in increasing blood glucose levels to the normal range. In addition to the effect of increasing blood glucose levels, glucagon reduces appetite and activates the hormone-sensitive adipocyte lipase, promoting lipolysis and thus has an anti-obesity effect. However, the use of glucagon as a therapeutic agent is limited because it has low solubility and precipitates at neutral pH.

Accordingly, the improved glucagon itself can be effectively used to treat severe hypoglycemia, non-alcoholic steatohepatitis (NASH), dyslipidemia, and the like. due to its activity in fat breakdown and β-oxidation in the liver.

One glucagon derivative, glucagon-like peptide-1 (GLP-1), is in development as a therapeutic agent for the treatment of hyperglycemia in diabetic patients. GLP-1 has the functions of promoting insulin synthesis and secretion, inhibiting glucagon secretion, slowing gastric emptying, increasing glucose utilization, and inhibiting nutrient absorption.

Exendin-4, derived from lizard venom and having approximately 50% amino acid sequence homology with GLP-1, has also been reported to activate the GLP-1 receptor, thereby controlling hyperglycemia in diabetic patients (J. Biol. Chem. 1992 Apr 15; 267 (11): 7402-5). However, anti-obesity drugs containing GLP-1 are reported to have side effects such as nausea and vomiting.

Therefore, as an alternative to GLP-1, much attention has been paid to oxyntomodulin, which can bind to the receptors of two peptides, GLP-1 and glucagon. Oxyntomodulin is a peptide derived from the precursor of glucagon, pre-glucagon, and has the functions of inhibiting nutrient absorption and enhancing GLP-1 saturation, and has lipolytic activity similar to glucagon, which increases its potency in anti-obesity therapy.

However, oxyntomoduine or its derivatives have a serious disadvantage in that it is necessary to administer an excessive amount of the drug daily, since it has low efficacy and a short in vivo half-life.

In addition, when there is GLP-1 activity and glucagon activity in one peptide, the ratio of these activities becomes fixed, and therefore it is difficult to use a dual-acting agonist in different ratios. Accordingly, a combination therapy that allows different ratios of activities by regulating GLP-1 and glucagon levels may be more effective. However, combination therapy requires improvement in the physical characteristics of glucagon, which aggregates at neutral pH and precipitates over time, thus showing poor solubility.

In view of these circumstances, the present inventors have developed glucagon derivatives with partial modifications of the amino acid sequence of glucagon to enhance the therapeutic effects of glucagon in hypoglycemia and obesity by improving the physical properties of glucagon, and it has been found that these glucagon derivatives, due to altered pI values that differ from pI values of native glucagon have improved solubility and higher stability at neutral pH, and the developed glucagon derivatives have been confirmed to activate its receptors in an in vitro assay, thereby carrying out the invention.

Description of the invention

Technical task.

The object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of metabolic syndrome, containing a derivative of glucagon and at least one compound or substance having therapeutic activity against metabolic syndrome.

The aim of the present invention is also to create new derivatives of glucagon.

It is also an object of the present invention to provide an isolated polynucleotide encoding a glucagon derivative, a vector comprising the polynucleotide, and an isolated cell comprising the polynucleotide or vector.

It is also an object of the present invention to provide an isolated conjugate in which a glucagon derivative and a biocompatible substance which is capable of increasing the in vivo half-life are linked.

It is also an object of the present invention to provide a composition containing a glucagon derivative and an isolated conjugate.

The object of the present invention is also to provide a pharmaceutical composition for the treatment or prevention of hypoglycemia or metabolic syndrome, containing a derivative of glucagon or isolated conjugate.

It is also an object of the present invention to provide a method for preventing or treating hypoglycemia or metabolic syndrome, comprising administering to a subject in need thereof the above composition.

It is also an object of the invention to provide the use of a glucagon derivative or isolated conjugate or composition in the preparation of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome.

Solving a technical problem

In order to achieve the above objectives, in one aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing metabolic syndrome, comprising a glucagon derivative and at least one compound or substance that has therapeutic activity against metabolic syndrome.

More specifically, in one aspect of the present invention, there is provided a pharmaceutical composition for the treatment or prevention of metabolic syndrome, comprising: 1) a peptide containing the amino acid sequence of the following general formula 1, and 2) at least one compound or substance having therapeutic activity in relation to metabolic syndrome:

X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23X24-W-L-X27-X28-X29-X30 (general formula 1, SEQ ID NO: 45).

In the general formula 1

X1 is histidine, deaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, β-carboxyimidazopropionyl, tryptophan, or tyrosine, or is absent;

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

X7 is threonine, valine or cysteine;

X10 is tyrosine or cysteine;

- 2 040165

X12 is lysine or cysteine;

X13 is tyrosine or cysteine;

X14 is leucine or cysteine;

X15 is aspartic acid, glutamic acid or cysteine;

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

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

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

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

X20 is lysine, histidine, glutamine, aspartic acid, lysine, arginine, α-methyl-glutamic 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-methyl-glutamic acid, or leucine, or is absent;

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

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

X29 is lysine, alanine, glycine or threonine or is absent; And

X30 is cysteine or absent; provided that when the amino acid sequence of general formula 1 is identical to SEQ ID NO: 1, then it is excluded.

In another specific embodiment in the general formula 1

X1 is histidine, tryptophan or tyrosine or is absent;

X2 is serine or aminoisobutyric acid (Ab);

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;

X20 is glutamine, aspartic acid, lysine or cysteine;

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

X23 is isoleucine, valine or arginine;

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

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

X28 is glutamine, lysine, asparagine or arginine;

X29 is threonine; And

X30 is cysteine or absent.

In yet another specific embodiment in the general formula 1

X1 is histidine, tryptophan or tyrosine or is absent;

X2 is serine or aminoisobutyric acid (Aib);

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;

- 3 040165

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;

X29 is threonine; And

X30 is cysteine or absent.

In yet another specific embodiment in the general formula 1

X1 is tyrosine;

X2 is aminoisobutyric acid (Aib);

X7 is threonine;

X10 is tyrosine;

X12 is a 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;

X24 is valine or arginine;

X27 is methionine;

X28 is asparagine or arginine;

X29 is threonine; And

X30 is missing.

In another specific embodiment, the above peptide is characterized in that it is a peptide containing the amino acid sequence of the following general formula 2

Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X (general formula 2, SEQ III NO: 46).

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 absent, where among the peptides containing the amino acid sequence of General formula 2, the peptides corresponding to SEQ ID NO: 14, 19, 20, 25, 27, 31 and 33 may be excluded.

In yet another specific embodiment, the peptide comprising the amino acid sequence of general formula 1 is characterized in that it has a pI value different from that of native glucagon, for example pI of 6.5 or lower or pI of 7.0 or higher.

In yet another specific embodiment, the peptide comprising the amino acid sequence of general formula 1 is characterized in that at least one pair of amino acids from the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 is replaced by glutamic acid or lysine, which are capable of forming a ring, respectively.

In yet another specific embodiment, the peptide comprising the amino acid sequence of general formula 1 is characterized in that the amino acid pair X12 and X16 or the amino acid pair X16 and X20, respectively, is replaced by glutamic acid or lysine, which are capable of forming a ring.

In yet another specific embodiment, the peptide comprising the amino acid sequence of general formula 1 is characterized in that at least one pair of amino acids from the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 forms a ring (eg lactam ring).

In yet another specific embodiment, a peptide containing the amino acid sequence

- 4 040165 of general formula 1, differs in that the C-terminus of the peptide is amidated.

In yet another specific embodiment, the peptide comprising the amino acid sequence of the following general formula 1 is characterized in that it is a glucagon derivative capable of activating the glucagon receptor.

In another specific embodiment, the peptide is characterized in that it contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-44.

In another specific embodiment, the peptide is characterized in that it contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30 and 32-44.

In yet another specific embodiment, the peptide is characterized in that it contains the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 20.

In another specific embodiment, a compound or substance having therapeutic activity against metabolic syndrome is characterized in that it is selected from the group consisting of an insulinotropic peptide, a glucagon-like peptide-1 (GLP-1) receptor agonist, a leptin receptor agonist, a dipeptidyl peptidase inhibitor IV (DPP-IV), Y5 receptor antagonist, melanin-concentrating hormone (MCH) receptor antagonist, Y2/4 receptor agonist, melanocortin 3/4 receptor agonist (MC 3/4), gastric/pancreatic lipase inhibitor, 5-hydroxytryptamine 2C receptor agonist (5HT2C), β3Ά receptor agonist, amylin receptor agonist, ghrelin antagonist, ghrelin receptor antagonist, peroxisome proliferator-activated receptor alpha (PPARa) agonist, peroxisome proliferator-activated delta receptor (PΡΆΚδ) agonist, farnesoid X receptor (FXR) agonist, acetyl inhibitor -CoA carboxylase, peptide YY, cholecystokinin (CCK), xenin, glycentine, obest atin, secretin, nesfatin, insulin and glucose-dependent insulinotropic peptide (GIP).

In yet another specific embodiment, the insulinotropic peptide is characterized in that it is selected from the group consisting of GLP-1, exendin-3, exendin-4, an agonist thereof, a derivative thereof, a fragment thereof, a variant thereof, and a combination thereof.

In yet another specific embodiment, the insulinotropic peptide is characterized in that it is an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is replaced by a residue selected from the group consisting of deaminohystidil, Ndimethylhistidil, β-hydroxyimidazopropionyl, 4-imidazoacetyl, and β- carboxyimidazopropionyl.

In yet another specific embodiment, the insulinotropic peptide is characterized in that it is selected from the group consisting of native exendin-4; an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed; a derivative of exendin-4, in which the N-terminal amino group of exendin-4 is replaced by a hydroxyl group; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is modified with a dimethyl group; an exendin-4 derivative in which the α-carbon of the 1st amino acid of exendin-4, histidine, has been removed; an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, is replaced by serine; and an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, is replaced by arginine.

In another specific embodiment, the peptide containing the amino acid sequence of general formula 1 is characterized in that the peptide containing the amino acid sequence of general formula 1 is in the form of a long-acting conjugate, which is associated with a biocompatible substance capable of increasing the half-life of the peptide in vivo; and the insulinotropic peptide is in the form of a long-acting conjugate to which is associated a biocompatible substance capable of increasing the half-life of the insulinotropic peptide in vivo.

In yet another specific embodiment, the biocompatible substance is characterized in that it is selected from the group consisting of polyethylene glycol, fatty acid, cholesterol, albumin and fragment thereof, albumin binder, polymer of repeating units of a specific amino acid sequence, antibody, antibody fragment, FcRn (neonatal Fc -receptor)-binding substance, connective tissue in vivo or their derivative, nucleotide, fibronectin, transferrin, saccharide and polymer.

In yet another specific embodiment, the peptide containing the amino acid sequence of general formula 1 and the insulinotropic peptide differ in that they are respectively linked to the biocompatible substance via a linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol , polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer such as polylactic acid (ΡΙΆ) and polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, fatty acid, polymer, small molecule compound, nucleotide, and combinations thereof.

In yet another specific embodiment, the biocompatible substance is characterized in that it is an FcRn-binding substance, and the peptide containing the amino acid sequence of general formula 1 and the insulinotropic peptide, respectively, are linked to the biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of 5 040165 ethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, dextran, biodegradable polymer such as polylactic acid (PIA) and polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid and combinations thereof.

In yet another specific embodiment, the FcRn binding agent is characterized in that it is a polypeptide containing the Fc region of an immunoglobulin.

In yet another specific embodiment, the Fc region of an immunoglobulin is characterized in that it is aglycosylated.

In another specific embodiment, the Fc region of an immunoglobulin 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) combinations between one or two or more domains from a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, and the hinge region or part of the hinge region of an immunoglobulin; and (e) a dimer between each domain of the heavy chain constant region and the light chain constant region.

In another specific embodiment, the polypeptide containing the Fc region of the immunoglobulin is in the form of a dimer.

In yet another specific embodiment, the Fc region of an immunoglobulin is characterized in that it is a derivative of native Fc in which the region capable of forming a disulfide bond has been removed, a derivative of native Fc in which part of the amino acid(s) at the N-terminus has been removed, a derivative of native An Fc in which methionine is added to the N-terminus, a native Fc derivative in which the complement-binding site has been removed, or a native Fc derivative in which the antibody-dependent cell-mediated cytotoxicity (ADCC) site has been removed.

In yet another specific embodiment, the Fc region of an immunoglobulin is characterized in that it is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

In yet another specific embodiment, the Fc region of an immunoglobulin is characterized in that it is an Fc region of IgG4.

In yet another specific embodiment, the Fc region of an immunoglobulin is characterized in that it is an aglycosylated Fc region of human IgG4.

In yet another specific embodiment, the non-peptide linker is characterized in that it is linked to a cysteine residue of a peptide containing the amino acid sequence of general formula 1.

In yet another specific embodiment, the non-peptide linker is characterized in that both ends of the non-peptide linker are respectively linked to an amino group or a thiol group of a peptide that contains the amino acid sequence of general formula 1, or an insulinotropic peptide and a biocompatible substance.

In yet another specific embodiment, the metabolic syndrome is characterized in that it is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, non-alcoholic steatohepatitis (NASH), dyslipidemia-induced atherosclerosis, atherosclerosis, arteriosclerosis, coronary disease heart and stroke.

In another aspect of the present invention, a novel glucagon derivative is provided.

In a specific embodiment, the glucagon derivative is characterized in that it is an isolated peptide containing the amino acid sequence of the following general formula 2

Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X (general formula 2, SEQ W NO: 46)

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 absent, where among the peptides containing the amino acid sequence of General formula 2, the peptides corresponding to SEQ ID NO: 14, 19, 20, 25, 27, 31 and 33 may be excluded.

- 6 040165

In yet another specific embodiment, the peptide containing the amino acid sequence of general formula 2 is characterized in that the amino acid pair X16 and X20 is replaced by glutamic acid or lysine, which are capable of forming a ring.

In yet another specific embodiment, the peptide comprising the amino acid sequence of general formula 2 is characterized in that the C-terminus of the peptide is amidated.

In yet another specific embodiment, the peptide is characterized in that it is a glucagon derivative capable of activating the glucagon receptor.

In yet another specific embodiment, the peptide is characterized in that it contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15 and 36-44.

In yet another specific embodiment, the peptide is characterized in that it contains the amino acid sequence of SEQ ID NO: 12.

In yet another aspect, the present invention provides an isolated polynucleotide encoding a glucagon derivative, a vector comprising the polynucleotide, and an isolated cell comprising the polynucleotide or vector.

In yet another aspect of the present invention, an isolated conjugate is provided wherein a glucagon derivative and a biocompatible substance capable of increasing in vivo half-life are linked.

In a specific embodiment, the biocompatible substance is characterized in that the biocompatible substance is selected from the group consisting of polyethylene glycol, fatty acid, cholesterol, albumin and its fragment, albumin binding substance, a polymer of repeating units of a specific amino acid sequence, an antibody, an antibody fragment, an FcRn-binding substance, a connective in vivo tissue or derivative thereof, nucleotide, fibronectin, transferrin, saccharide and polymer.

In a particular embodiment, the isolated peptide is characterized in that it is linked to a biocompatible substance via a linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, such as polylactic acid (PIA) and polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, fatty acid, polymer, small molecule, nucleotide, and combinations thereof.

In a specific embodiment, the biocompatible substance is characterized in that it is an FcRn-binding substance, and the isolated peptide and insulinotropic peptide, respectively, are linked to the biocompatible substance via a peptide linker or a non-peptide linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, dextran, biodegradable polymer such as polylactic acid (PIA) or polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, and combinations thereof.

In a specific embodiment, the FcRn binding agent is characterized in that it is a polypeptide containing the Fc region of an immunoglobulin.

In yet another aspect of the present invention, a composition is provided comprising a glucagon derivative or isolated conjugate.

In a specific embodiment, the composition is characterized in that it is a pharmaceutical composition for the treatment or prevention of hypoglycemia or metabolic syndrome.

In yet another aspect of the present invention, there is provided a method for preventing or treating hypoglycemia or metabolic syndrome, comprising administering a composition to a subject in need thereof.

In yet another aspect of the present invention, the use of a glucagon derivative or isolated conjugate or composition in the preparation of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome is provided.

Advantageous Effects of the Invention.

The glucagon derivatives of the present invention have improved physical properties compared to those of native glucagon and therefore can be effectively used as therapeutic agents for the treatment of hypoglycemia by improving patient compliance with the regimen and treatment regimen. Additionally, the glucagon derivatives of the present invention can be effectively used in the prevention and treatment of hypoglycemia and metabolic syndrome such as obesity, diabetes, and non-alcoholic steatohepatitis (NASH).

Brief description of graphic materials

In FIG. 1 is a graph illustrating body weight changes in animal models of obesity (rats) that were obtained from feeding a high fat diet during single or co-administration of a long-acting insulinotropic peptide conjugate (referred to as long-acting exendin-4 derivative) and long-term administration to rats. 7 040165 active glucagon derivative conjugate (referred to as long acting derivative SEQ ID

NO: 12) using the established dose in rats at intervals of 3 days for 15 days;

in fig. 2 is a result illustrating the amount of mesenteric fat in obese animal models (rats) that were obtained from feeding a high fat diet during separate or co-administration of a long acting insulinotropic peptide conjugate (referred to as a long acting exendin-4 derivative) and a long acting glucagon derivative conjugate (referred to as long-acting derivative of SEQ ID NO: 12) using a fixed dose in rats for 15 days (*p<0.05, **p<0.01 relative to vehicle as analyzed by ANOVA (analysis of variance));

in fig. 3 is a result illustrating the difference in liver weight in obese animal models (rats) that were obtained from feeding a high fat diet during single or co-administration of a long acting insulinotropic peptide conjugate (referred to as long acting exendin-4 derivative) and long-term active glucagon derivative conjugate (referred to as long-acting derivative of SEQ ID NO: 12) using a fixed dose in rats for 15 days (xp<0.01, xp<0.001 relative to vehicle as analyzed by ANOVA);

in fig. 4 is a graph of change in body weight (BW) of animal models of obesity (mice) that were obtained as a result of feeding a diet with a high fat content, after separate or joint administration to rats of a long-acting insulinotropic peptide conjugate (referred to as a long-acting exendin-4 derivative) and long-term an active glucagon derivative conjugate (referred to as a long acting derivative of SEQ ID NO: 20) using a set dose in rats for 22 days;

in fig. 5 is a result illustrating changes in blood cholesterol levels in obese animal models (mice) that were obtained by feeding a high-fat diet after single or co-administration of a long-acting insulinotropic peptide conjugate (referred to as long-acting exendin-4 derivative) and long-term active glucagon derivative conjugate (referred to as long acting derivative of SEQ ID NO: 20) using a set dose in rats for 22 days.

The best embodiment of the invention.

Specific details of the present invention may be explained as follows. In particular, explanations and embodiments disclosed in the present invention are applicable to other explanations and embodiments, respectively. That is, all combinations of the various elements disclosed in the present invention are within the scope of the present invention. In addition, the scope of the present invention is not limited to the specific features described below.

Throughout the text of the description of the present invention, not only the conventional 1-letter codes and 3-letter codes for natural amino acids are used, but also 3-letter codes such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), commonly used for other amino acids . In addition, the amino acids abbreviated in the present specification are described according to the IUPAC-IUB nomenclature.

alanine A arginine R asparagine N aspartic acid D cysteine C glutamic acid E glutamine Q glycine G histidine H isoleucine I leucine L lysine K methionine M phenylalanine F proline R serine S threonine T tryptophan W tyrosine Y valine V

In one aspect, the present invention provides a composition comprising a glucagon derivative and at least one compound or substance having therapeutic activity for metabolic syndrome, and more specifically, a pharmaceutical composition for treating or preventing metabolic syndrome comprising a glucagon derivative and at least at least one compound or substance having therapeutic activity in relation to the metabolic syndrome.

The glucagon derivative of the present invention comprises a peptide having at least one amino acid sequence difference compared to native glucagon; peptide in which the sequence of native glucagon is modified by modification

- 8 040165 native glucagon; and a native glucagon mimetic that can activate glucagon receptors similar to native glucagon.

Such a glucagon derivative may be a glucagon derivative having improved physical properties in that it has an altered pI compared to native glucagon. Additionally, the glucagon derivative may be a solubility-improved glucagon derivative while retaining glucagon receptor activating activity, but is not limited to this.

Additionally, the glucagon derivative may be a non-naturally occurring glucagon.

In particular, native glucagon may have the following amino acid sequence: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-AlaGln-Asp -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1)

As used herein, the term pI or isoelectric point refers to the pH value at which a macromolecule, such as a polypeptide, has no net charge (0). In the case of a polypeptide with different charged functional groups, the net charge of the polypeptide as a whole is 0 at the point where the pH value is the same as the pI value. The net charge of the polypeptide at pH higher than pI will be negative, and the net charge of the polypeptide at pH lower than pI will be positive.

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

As used herein, the term altered pI refers to a pI that differs from the pI of native glucagon due to the replacement of a portion of the amino acid sequence of native glucagon with an amino acid residue having a negative charge or a positive charge, ie. to a lower or higher pI value. A peptide with such an altered pI can exhibit improved solubility and high stability at neutral pH as a glucagon derivative.

More specifically, the glucagon derivative may have an altered pI value but not the pI (6.8) value of native glucagon, and even more specifically may have a pI value of less than 6.8, more specifically 6.7 or less, more specifically 6.5 or less, and furthermore, a pI value greater than 6.8, 7 or higher, more specifically 7.5 or higher, but not limited to these values, and any pI value different from the pI value of native glucagon will be within the scope of the present invention. In particular, when the pI value differs from that of native glucagon and therefore exhibits improved solubility at neutral pH compared to that of native glucagon, thereby exhibiting a low level of aggregation, then the derivative will be within the scope of the present invention.

More specifically, the glucagon derivative may have a pI value of 4 to 6.5 and/or 7 to 9.5, specifically 7.5 to 9.5 and more specifically 8.0 to 9.3, but the pI value is not limited. these values. In this case, due to the lower or higher pI value compared to native glucagon, improved solubility and high stability at neutral pH compared to the solubility and stability of native glucagon can be demonstrated.

Specifically, the native glucagon derivative may be modified by any means of substitution, addition, deletion and modification, or combinations thereof, of the native glucagon amino acid.

Examples of glucagon derivatives obtained by the combination of the above methods include a peptide that differs in at least one amino acid residue of an amino acid sequence compared to the amino acid sequence of native glucagon, and in which the N-terminal amino acid residue is deaminated, having the function of activating the glucagon receptor, without limitation, and native glucagon derivatives may be obtained by a combination of various derivatization methods.

Additionally, such modification to produce native glucagon derivatives may include all modifications using L-type or D-type amino acids and/or non-native type amino acids; and/or native sequence modification, eg, functional group modification, intermolecular covalent bonding (eg, ring formation by side chains), methylation, acylation, ubiquination, phosphorylation, aminohexanation, biotinylation, etc.

Additionally, the modification may also include all modifications where one or more amino acids are added to the amino and/or carboxy terminus of native glucagon.

During the replacement or addition of amino acids, not only the 20 amino acids commonly found in human proteins, but also atypical or non-naturally occurring amino acids can be used. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptides containing these amino acids and atypical peptide sequences can be synthesized and can be purchased from commercial suppliers, for example

- 9 040165

American Peptide Company, Bachem (USA) or Anygen (Korea).

Since glucagon has a pH of about 7, it does not dissolve in a solution having physiological pH (pH 4-8) and tends to precipitate at neutral pH. In an aqueous solution of pH 3 or less, glucagon dissolves initially but precipitates after one hour to form a gel. Since gelatinous glucagon is composed primarily of β-structural fibrils, administration of thus precipitated glucagon through an injection needle or by intravenous injection will block the vessels and hence be unsuitable for use as an injectable agent. In order to slow down the precipitation process, acidic (pH 2-4) preparations are usually used, in which case glucagon can remain in a relatively unaggregated state for a short period of time. However, glucagon can form fibrils very rapidly at low pH values, and therefore such acidic formulations should not be injected once prepared.

In this regard, the authors of the present invention have developed derivatives of glucagon with long-acting profiles by modifying the pI of native glucagon by replacing amino acid residues having negative charges and positive charges. Glucagon derivatives of the present invention having an altered pI compared to that of native glucagon are characterized by improved solubility and/or high stability at neutral pH compared to native glucagon.

In a specific embodiment of the invention, the glucagon derivative may be a peptide that contains the amino acid sequence of the following general formula 1

X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W

-L-X27-X28-X29-X30 (general formula 1, SEQ III NO: 45).

In the above formula

X1 is histidine, deaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, β-carboxyimidazopropionyl, tryptophan, or tyrosine, or is absent;

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

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, glutamic acid or cysteine;

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

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

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

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

X20 is lysine, histidine, glutamine, aspartic acid, lysine, arginine, α-methyl-glutamic 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-methyl-glutamic acid, or leucine, or is absent;

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

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

X29 is lysine, alanine, glycine or threonine or is absent; And

X30 is cysteine or absent;

provided that when the amino acid sequence of general formula 1 is identical to SEQ ID NO: 1, then it is excluded.

In the above, when the amino acid sequence of general formula 1 is identical to any amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15 and 36-44, and in particular the amino acid sequence of any of the amino acid sequences of SEQ ID NO: 13 , 15, 36 and 38-43, then it is possible that the peptide can be excluded from the scope of peptides that contain the amino acid sequence of General formula 1, but is not limited to this.

More specifically, in the general formula 1

X1 is histidine, tryptophan or tyrosine or is absent;

- 10 040165

X2 is serine or aminoisobutyric acid (Ab);

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;

X20 is glutamine, aspartic acid, lysine or cysteine;

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

X23 is isoleucine, valine or arginine;

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

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

X28 is glutamine, lysine, asparagine or arginine;

X29 is threonine; And

X30 is cysteine or absent, provided that when the amino acid sequence of general formula 1 is identical to SEQ ID NO: 1, then it is excluded.

For example, a peptide may be a peptide that contains an amino acid sequence selected from the group consisting of SEQ ID NO: 2-44, and specifically a peptide that (essentially) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: : 2-44, but not limited to this.

In addition, although in the present invention a peptide is described as a peptide consisting of a particular SEQ ID NO, such expression does not exclude a mutation in the peptide that may result from an irrelevant addition of a sequence to the left or right of the amino acid sequence of the corresponding SEQ ID NO, or a naturally occurring mutation or a silent mutation, provided that the peptide having such a mutation has the same activity as that of a peptide that consists of the amino acid sequence of the corresponding SEQ ID NO or the corresponding activity of that peptide. Even when there is a sequence addition or mutation, the peptide is clearly within the scope of the present invention.

Conversely, in another aspect, when the amino acid sequence of general formula 1 is identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15 and 36-44, and in particular any of the amino acid sequences of SEQ ID NO: 13, 15, 36 and 38-43, the peptide may optionally be excluded from the scope of peptides that contain the amino acid sequence of General formula 1, but is not limited to this. What is described above may also be applicable to other specific embodiments or aspects, but without limitation.

Specifically, in the general formula 1

X1 is histidine, tryptophan or tyrosine or is absent;

X2 is serine or aminoisobutyric acid (Aib);

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;

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;

X29 is threonine; And

X30 is cysteine or absent;

- 11 040165 provided that when the amino acid sequence of general formula 1 is identical to SEQ ID

NO: 1, then it is excluded.

For example, a peptide may be a peptide that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30, and 32-44, and specifically a peptide that (according to essentially) consists of an amino acid sequence selected from, but not limited to, SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30 and 32-44.

Specifically, in the general formula 1

X1 is tyrosine;

X2 is aminoisobutyric acid;

X7 is threonine;

X10 is tyrosine;

X12 is a 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;

X24 is valine or arginine;

X27 is methionine;

X28 is asparagine or arginine;

X29 is threonine; And

X30 is missing.

For example, a peptide may be a peptide that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, and specifically a peptide that (essentially) consists of an amino acid sequence selected from the group consisting of but not limited to SEQ ID NOs: 14, 16, 18, 19, 25 and 31.

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

Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X (general formula 2, SEQ III NO: 46).

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 absent, provided that when the amino acid sequence of general formula 1 is identical to any of SEQ ID NOs: 14, 19, 20, 25, 27, 31 and 33, then it can be excluded, but not limited to this.

For example, the peptide may be a peptide that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 15, and 36-44, and specifically a peptide that (essentially) consists of an amino acid sequence selected from the group , consisting of SEQ ID NO: 12, 13, 15 and 36-44, but is not limited to this. More specifically, the peptide may be a peptide that contains the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 20 or (essentially) consists of the corresponding amino acid sequence, but is not limited to this.

Additionally, a peptide containing an amino acid sequence of general formula 1 or general formula 2 may be a peptide in which at least one amino acid pair from amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24 , and X24 and X28 in the general formula 1 or the general formula 2 can be replaced by glutamic acid or lysine, which are capable of forming a ring, respectively, but are not limited to this.

More specifically, a peptide containing the amino acid sequence of general formula 1 or

- 12 040165 of general formula 2, may be a peptide in which the amino acid pair X12 and X16 or the amino acid pair X16 and X20, respectively, is replaced by glutamic acid or lysine, which are capable of forming a ring.

More specifically, at least one amino acid pair of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 may be a peptide that forms a ring (e.g., a lactam ring) , but is not limited to this.

In particular, the peptide may be modified at its amino-terminus or carboxy-ring or protected with various organic groups to protect the peptide from protein-cleaving enzymes in vivo while increasing its stability, for example, it may be a peptide in which its C-terminus is amidated.

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

Specifically, the peptide of the present invention can be obtained by a standard synthetic method using a recombinant expression system, or by any other method known in the art. Accordingly, the glucagon derivative of the present invention can be synthesized by various methods, including, for example, the methods described below:

(a) a method for synthesizing a peptide by a solid-phase or liquid-phase method in a step-by-step or fragment assembly method, followed by isolation and purification of the final peptide product; or (b) a method for expressing a nucleic acid construct encoding a peptide in a host cell and isolating the expression product from a culture of the host cell; or (c) a method for performing cell-free expression in vitro of a nucleic acid construct encoding a peptide and isolating the expression product; or a method for obtaining peptide fragments by carrying out any combination of methods (a), (b) and (c) to obtain a peptide by linking peptide fragments and then isolating the peptide.

In a more specific example, the described glucagon derivative can be obtained by genetic manipulation, which includes obtaining a fusion gene encoding a fusion protein, including a fusion partner and a glucagon derivative, transforming the resulting product into a host cell, expressing it in the form of a fusion protein, and purifying the glucagon derivative from the fusion protein. using a protease or a compound that is capable of cleaving a protein, followed by separation. To this end, for example, a DNA sequence encoding an amino acid residue, which can be purified by a protease such as Factor Xa or enterokinase, CNBr, or a compound such as hydroxylamine, can be inserted between a fusion partner and a polynucleotide encoding a glucagon derivative.

In a specific embodiment of the present invention, it is confirmed that the peptide of the present invention has a different pI compared to the pI of native glucagon (see table). As a result, the peptide of the present invention has improved solubility and higher stability at neutral pH. Accordingly, the peptide of the present invention can promote patient compliance when used as a hypoglycemic agent, and is also suitable for co-administration with other anti-obesity agents, and therefore can be effectively used to prevent and treat hypoglycemia and obesity.

In this regard, the peptide of the present invention may provide an attractive therapeutic choice regarding hypoglycemia, obesity or related diseases.

For example, the peptide of the present invention is a major insulin response controlling hormone and can be effectively used to treat severe hypoglycemia in diabetic patients.

In addition, the peptide of the present invention can be used as a pharmaceutical drug not only to prevent weight gain, promote weight loss, reduce overweight and obesity, including unhealthy obesity (for example, by controlling appetite, food intake, absorption of nutrients). substances, calorie intake and/or energy intake), but also for the treatment of obesity-associated inflammation, obesity-associated gallbladder disease and obesity-induced sleep apnea, but without limitation, and can be used to treat associated diseases or health conditions.

The peptide of the present invention can also be used to treat metabolic syndrome other than obesity, i. obesity-related diseases such as impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, non-alcoholic steatohepatitis (non-alcoholic steatohepatitis, NASH), dyslipidemia-induced atherosclerosis, atherosclerosis, arteriosclerosis, coronary heart disease, stroke, hypoglycemia, etc. d. However, the effects of the peptide of the present invention may be mediated in whole or in part by the body weight-related effects described above, or may be independent of them.

Examples of a compound or substance having therapeutic activity against metabolic syndrome and intended to be included in a co-administration or composition according to

- 13 040165 of the present invention may include an insulinotropic peptide, a glucagon-like peptide-1 (GLP-1) receptor agonist, a leptin receptor agonist, a dipeptidyl peptidase-IV (DPP-IV) inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, an agonist Y2/4 receptor agonist, melanocortin receptor 3/4 MC 3/4), gastric/pancreatic lipase inhibitor, 5-hydroxytryptamine 2C (5HT2C) receptor agonist, β3Ά receptor agonist, amylin receptor agonist, ghrelin antagonist, ghrelin receptor antagonist, activated peroxisome proliferator receptor alpha (PPARa), peroxisome proliferator-activated delta receptor (PΡΡΆΚδ) agonist, farnesoid X receptor (FXR) agonist, acetyl-CoA carboxylase inhibitor, peptide YY, cholecystokinin (CCK), xenin, glycentin, obestatin, secretin, nesfatin, insulin and glucose-dependent insulinotropic peptide (GIP), but are not limited to them. Additionally, all drugs that are effective in the treatment of obesity and drugs capable of inhibiting hepatic inflammation and fibrosis may be included.

Specifically, the insulinotropic peptide may be selected from the group consisting of GLP-1, exendin-3, exendin-4, an agonist thereof, a derivative thereof, a fragment thereof, a variant thereof, and a combination thereof.

More specifically, the insulinotropic peptide may be an insulinotropic peptide derivative in which the N-terminal histidine of the insulinotropic peptide is replaced by a residue selected from, but not limited to, desaminohystidil, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4imidazoacetyl, and β-carboxyimidazopropionyl.

More specifically, the insulinotropic peptide may be selected from the group consisting of native exendin-4; an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed; a derivative of exendin-4, in which the N-terminal amino group of exendin-4 is replaced by a hydroxyl group; a derivative of exendin-4, in which the N-terminal amino group of exendin-4 is modified with a dimethyl group; an exendin-4 derivative in which the α-carbon of the 1st amino acid of exendin-4, histidine, is excluded; an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, is replaced by serine; and an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, is replaced by arginine, but is not limited thereto.

It should be noted that, as an example of an insulinotropic peptide or long acting conjugate thereof, the full disclosure of US Patent Application Publication No. 2010-0105877 is disclosed herein by reference.

In a more specific embodiment, a glucagon derivative, for example, a peptide containing the amino acid sequence of general formula 1 or general formula 2, may be in the form of a long-acting conjugate, which is associated with a biocompatible substance that can increase the half-life in vivo, but is not limited to this. The biocompatible substance can be used interchangeably with the carrier.

Additionally, the insulinotropic peptide may also be in the form of a long-acting conjugate to which is associated a biocompatible substance capable of increasing the in vivo half-life, but is not limited to this.

In a specific embodiment of the present invention, the duration of action of the above conjugate is increased compared to native glucagon or a glucagon derivative to which the carrier is not bound. In the present invention, the protein conjugate is referred to as a long-acting conjugate.

Examples of a biocompatible substance may include polyethylene glycol, fatty acid, cholesterol, albumin and fragment thereof, albumin binder, polymer of repeating units of a specific amino acid sequence, antibody, antibody fragment, FcRn binder, in vivo connective tissue or derivative thereof, nucleotide, fibronectin, transferrin, saccharide and polymer, but are not limited to. For example, at least one amino acid side chain in a peptide of the present invention may be attached to a biocompatible substance in order to increase in vivo solubility and/or half-life and/or increase its bioavailability. These modifications are known to reduce the clearance of therapeutic proteins and peptides.

For a biocompatible polymer, soluble (amphipathic or hydrophilic), non-toxic and pharmaceutically inert polymers are suitable and may include, for example, PEG (polyethylene glycol), PEG homopolymers or copolymers, monomethyl-substituted polymers (mPEGs), and poly-amino acids such as poly -lysine, but not limited to poly-aspartic acid and poly-glutamic acid.

It is a known fact for a person skilled in the art that a modified glucagon derivative in this way will have a better therapeutic effect compared to native glucagon. Accordingly, variants of the glucagon derivative as described above are also within the scope of the present invention.

In a more specific embodiment, a glucagon derivative, for example a peptide that contains the amino acid sequence of general formula 1 or general formula 2, and an insulinotropic peptide can respectively be linked to a biocompatible substance via a linker selected

- 14 040165 from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer such as polylactic acid (PIA) and polylactic-glycolic acid (PIGA) , lipid polymer, chitin, hyaluronic acid, fatty acid, polymer, low molecular weight compound, nucleotide, and combinations thereof, but without limitation.

In an even more specific embodiment, the biocompatible substance may be an FcRn-binding substance, and a glucagon derivative, for example, a peptide that contains the amino acid sequence of General Formula 1 or General Formula 2, and the insulinotropic peptide may be respectively linked to the biocompatible substance via a peptide linker or a non-peptide linker, but without limitation to this.

As a specific example, the FcRn binding agent may be a polypeptide containing the Fc region of an immunoglobulin.

As used herein, a non-peptide linker includes a biocompatible polymer to which at least two repeat units are linked. The repeating units are linked to each other by an arbitrary covalent bond instead of a peptide bond. The non-peptidic linker may be a single structure that forms the moiety of the long acting conjugate of the present invention.

As used herein, the term non-peptide linker may be used interchangeably with a non-peptide polymer.

Additionally, in a specific embodiment, the conjugate may be a conjugate in which the protein drug is covalently linked to the immunoglobulin Fc region via a non-peptide linker containing a reactive group that can bind to the immunoglobulin Fc region and the protein drug at both ends of the conjugate.

Without particular limitation, the non-peptidic linker may be a non-peptidic linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer such as polylactic acid (PIA ) and polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, polysaccharide, and combinations thereof. In a more specific embodiment, the non-peptide polymer may be, but is not limited to, polyethylene glycol. Additionally, derivatives that are already known in the art and derivatives that can be easily obtained by known techniques in the art are within the scope of the present invention.

The non-peptide linker that is used in the present invention can be any polymer that is protease resistant in vivo, without limitation. The molecular weight of the non-peptide polymer may range from 1 to 100 kDa, and specifically from 1 to 20 kDa, but is not limited to these values. Additionally, the non-peptide linker of the present invention, which is linked to a polypeptide containing an immunoglobulin Fc region, may include not only a single type of polymer, but also a combination of different types of polymers.

In a specific embodiment, both ends of the non-peptide linker can be respectively linked to the amino group or thiol group of a peptide that contains the amino acid sequence of general formula 1, or an insulinotropic peptide and a biocompatible substance.

Specifically, the non-peptidic polymer may contain a reactive group at both ends, respectively, which may bind to an immunoglobulin Fc fragment and a glucagon derivative or an insulinotropic peptide; and specifically, the reactive group may be linked to the amino group of the N-terminus of lysine or the thiol group of cysteine of a glucagon derivative, or an insulinotropic peptide, or an immunoglobulin Fc fragment.

Additionally, the reactive group of the non-peptide polymer that can bind to the Fc region of an immunoglobulin, a glucagon derivative, and an insulinotropic peptide can be selected from, but is not limited to, an aldehyde group, a maleimide group, and a succinimide derivative.

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

In the above, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionoate, succinimidyl butanoate, succinimidyl propionoate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidylcarboxymethyl, or succinimidyl carbonate can be used as the succinimidyl derivative as the succinimidyl derivative.

Additionally, the end product resulting from reductive alkylation via an aldehyde bond is more stable than the product linked via an amide bond. The aldehyde reactive group reacts selectively at the N-terminus at low pH, although it may form a covalent bond with the lysine residue at high pH, eg at pH 9.0.

The reactive groups at both ends of the non-peptide linker may be the same

- 15 040165 or may be different from each other, for example, a maleimide reactive group may be at one end, and an aldehyde group, a propionaldehyde group or a butyraldehyde group may be at the other end. However, if the Fc region of an immunoglobulin and a glucagon derivative or an insulinotropic peptide can be conjugated at each end of the non-peptide linker, this is not particularly limited.

For example, a non-peptide polymer may have a maleimide group at one end and an aldehyde group, propionaldehyde group, or butyraldehyde group at the other end.

When used as a non-peptide polymer, polyethylene glycol having a reactive hydroxy group at both of its ends, the hydroxy group can be activated by converting it into various reactive groups by known chemical reactions, or commercially available polyethylene glycol can be used to obtain a long-acting protein conjugate of the present invention, having a modified reactive group.

In a specific embodiment, the non-peptide polymer may be a non-peptide polymer that can bind to, but is not limited to, a cysteine residue of a glucagon derivative, and more specifically to a -SH group of cysteine.

In a specific embodiment, the conjugate may be a conjugate in which a peptide comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 20 is linked to the Fco region of an immunoglobulin via a non-peptide polymer, and in particular, the non-peptide polymer may be a non-peptide polymer that linked to, but not limited to, a cysteine residue located at the 30th position of the amino acid sequence of SEQ ID NO: 12, or a cysteine residue located at the 17th position of the amino acid sequence of SEQ ID NO: 20.

When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the SH group of the glucagon derivative via a thioether bond, and the aldehyde group can be linked to the -NH ₂ Fc of an immunoglobulin via reductive alkylation, but is not limited to this, and the above is only an embodiment.

In the present invention, an immunoglobulin Fc region refers to a region containing a heavy chain constant region 2 (CH2) and/or a heavy chain constant region 3 (CH3), excluding the immunoglobulin heavy chain and light chain variable regions. The Fc region of an immunoglobulin may be the structure that assembles the protein conjugate of the present invention.

The Fc region of an immunoglobulin may include, but is not limited to, a hinge region in a heavy chain constant region. Additionally, the Fc region of an immunoglobulin of the present invention may be an extended Fc region containing part or all of the heavy chain 1 (CH1) constant region and/or the light chain 1 (CL1) constant region, excluding the heavy chain and light chain variable regions. immunoglobulin, provided that the Fc region of the immunoglobulin has an effect substantially the same or improved compared to the native type. Additionally, the Fc region of an immunoglobulin of the present invention may be one in which a sufficiently long portion of the amino acid sequence corresponding to CH2 and/or CH3 has been deleted.

For example, the Fc region of an immunoglobulin of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) CH1 domain and CH2 domain; 3) CH1 domain and CH3 domain; 4) CH2 domain and CH3 domain; 5) a combination between one or two or more domains from a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, and an immunoglobulin hinge region (or part of a hinge region); and 6) a dimer between, but not limited to, each heavy chain constant region domain and the light chain constant region.

Additionally, in a specific embodiment, the immunoglobulin Fc region may be in dimeric form, and one molecule of the glucagon derivative or insulinotropic peptide may be covalently linked to the Fc region in dimeric form, and in particular, the immunoglobulin Fc and the glucagon derivative or insulinotropic peptide may be linked through a non-peptide polymer. In addition, two molecules of a glucagon derivative or an insulinotropic peptide may optionally be conjugated in a symmetrical manner to a single Fc region in dimeric form. In particular, an immunoglobulin Fc and a glucagon derivative or an insulinotropic peptide may be linked via a non-peptide linker, but the embodiment described above is not a limitation.

Additionally, the Fc region of an immunoglobulin of the present invention contains not only a native amino acid sequence, but also a derivative of this sequence. An amino acid sequence derivative refers to an amino acid sequence that differs at least one amino acid residue due to a deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

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

Additionally, various other derivatives are possible, including a derivative that has a deletion of a region capable of forming a disulfide bond, or a deletion of some amino acid residues at the N-terminus of a native Fc, or an addition of a methionine residue at the N-terminus of a native Fc. In addition, to remove effector functions, a deletion can take place at a complement-binding site, such as the C1q binding site and the antibody-dependent cell-mediated cytotoxicity (ADCC) site. Methods for obtaining such derivatives of the immunoglobulin Fc region sequence are disclosed in international patent applications WO 97/34631, WO 96/32478 and others.

Amino acid substitutions in proteins and peptides that do not normally alter the activity of the proteins or peptides are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). In most cases, common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly in both directions. In addition, the Fc region can be modified by phosphorylation, sulfanation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., as needed.

The Fc derivatives described above exhibit identical biological activity to the Fc region of the present invention and have improved structural stability with respect to heat, pH, etc.

Further, the Fc region of an immunoglobulin may be derived from native forms isolated in vivo from humans or animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or may be recombinants or derivatives derived from transformed animal or microorganism cells. Here, the Fc region can be obtained from native immunoglobulin by isolating the complete immunoglobulin from a living human or animal body and treating the isolated immunoglobulin with a protease. When the complete immunoglobulin is treated with papain, it is cleaved into the Fab and Fc regions, and when the complete immunoglobulin is treated with pepsin, it is cleaved into pF'c and F(ab) ₂ fragments. Fc or pF'c can be isolated by size exclusion chromatography, etc. In a more specific embodiment, the human Fc region is a recombinant immunoglobulin Fc region derived from a microorganism.

In addition, the immunoglobulin Fc region may have natural glycans with increased or decreased glycan content compared to the natural type, or may be in a deglycosylated form. The increase, decrease or removal of immunoglobulin Fc glycans can be carried out by standard methods such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism. The Fc region of an immunoglobulin obtained by removing glycans from the Fc region exhibits a significant reduction in binding affinity for the C1q portion and a reduction or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and therefore does not induce unwanted immune responses in vivo. In this regard, an immunoglobulin Fc region in a deglycosylated or aglycosylated immunoglobulin Fc region may be a more stable form, fulfilling the original purpose of the present invention, as a drug carrier.

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

At the same time, the Fc region of an immunoglobulin can be isolated from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs. In a more specific embodiment, she is isolated from humans.

In addition, the Fc region of an immunoglobulin (Ig) may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is of IgG or IgM origin, which are among the most abundant proteins in human blood, and in an even more specific embodiment, it is of IgG origin, which is known to increase the half-life of ligand-binding proteins. In an even more specific embodiment, an immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, an IgG4 Fc region is, but is not limited to, an aglycosylated Fc region from human IgG4.

In particular, the term combination as used herein means that polypeptides encoding single chain immunoglobulin Fc regions of the same origin are linked to a single chain polypeptide of a different origin to form a dimer or multimer. That is, a dimer or multimer may be formed from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

The composition of the present invention can be used to prevent or treat hypoglycemia or metabolic syndromes.

As used herein, the term prevention refers to all activities associated with inhibiting or slowing the onset of hypoglycemia or the metabolic syndrome when a peptide or composition is administered, and the term treatment refers to all activities associated with improvement or beneficial changes in symptoms. hypoglycemia or metabolic syndrome, with the introduction of the peptide or composition.

As used herein, the term administration refers to the administration of a particular substance to a patient in an appropriate manner. The composition may be administered by a conventional route that provides in vivo delivery of the composition to the target tissue, such as, but not specifically limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and intrarectal administration.

As used herein, the term metabolic syndrome refers to the symptoms of a single or complex occurrence of various diseases due to a chronic metabolic disorder, and in particular, examples of the metabolic syndrome may include impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, non-alcoholic steatohepatitis (NASH ), atherosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, stroke, etc., but not limited to.

As used herein, the term obesity refers to a medical condition with excess body fat, and a person having a body mass index (BMI; body weight (kg) divided by the square of height (m)) of 25 or higher is diagnosed as obese. Obesity usually results from a long-term energy imbalance in which energy intake exceeds energy expenditure. Obesity is a metabolic disease that affects the entire body, increases the risk of diabetes, hyperlipidemia, sexual dysfunction, arthritis and cardiovascular disease, and in some cases it is associated with cancer.

Hypoglycemia can be an acute symptom of diabetes.

As used herein, the term hypoglycemia refers to an acute symptom of diabetes in which blood glucose levels are lower than in normal individuals, and generally refers to a condition where blood glucose levels are 50 mg/dl or less. Hypoglycemia often occurs when a person who is taking an oral hypoglycemic agent or insulin eats less than usual or engages in more activity or exercise than usual. In addition, hypoglycemia can occur due to glucose-lowering drugs, severe physical illness, hormone deficiency such as adrenal cortex hormones and glucagon, due to an insulin-producing pancreatic tumor, autoimmune insulin syndrome in patients undergoing gastrectomy , hereditary disorders of carbohydrate metabolism, etc.

Symptoms of hypoglycemia include weakness, trembling, pale skin, cold sweat, dizziness, agitation, anxiety, palpitations, empty stomach, headache, fatigue, etc. In the case of persistent hypoglycemia, it can lead to convulsions or seizures and can cause shock and loss of consciousness.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, excipient or diluent. As used herein, the term pharmaceutically acceptable refers to the properties of having a sufficient amount to exhibit a therapeutic effect without causing adverse effects, and one of skill in the art would readily be able to determine this based on factors commonly known in the medical field, such as type of disease, age , body weight, health status, gender, drug sensitivity of the patient, route of administration, route of administration, frequency of administration, duration of treatment, drug that is mixed or administered simultaneously in combination, etc.

The pharmaceutical composition of the present invention containing the peptide of the present invention may further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may include: for oral administration, a binder, glidant, disintegrant, excipient, solubilizing agent, dispersing agent, stabilizing agent, suspending agent, coloring agent, flavoring agent, etc.; for injection buffer agent, preservative, analgesic, solubilizing agent, isotonic agent, stabilizing agent, etc., which can be used together; and for topical administration, a base, an excipient, a lubricant, a preservative, etc., although not limited to them.

Various dosage forms of the composition of the present invention can be obtained by combination with a pharmaceutically acceptable carrier, as described above. For example, for oral administration, the composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups, cachets, and the like. For injection, the composition may be prepared in single dose ampoules or multi-dose containers. The composition may also be formulated as solutions, suspensions, tablets, capsules and sustained release preparations.

While examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone , water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the composition may additionally contain a filler,

- 18 040165 anticoagulant, lubricant, humectant, flavor, preservative, etc.

Additionally, the pharmaceutical composition of the present invention may be formulated into any type of dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile injectables, non-aqueous solvents, lyophilized preparations and suppositories.

Additionally, the composition may be formulated in a single dosage form suitable for the body of the patient, and preferably in the form of a preparation useful for peptide drugs, by a typical method used in the pharmaceutical field, for administration by the oral or parenteral route, such as through the skin, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, intraventricularly, into the lungs, transdermally, subcutaneously, intraperitoneally, intranasally, into the stomach, topically, sublingually, vaginally or rectally, but without limitation.

Additionally, the peptide can be used by mixing with various pharmaceutically acceptable carriers such as saline or organic solvents. Carbohydrates such as glucose, sucrose or dextrans can be used to increase stability and absorption; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers.

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

The total effective dose of the composition of the present invention may be administered to a patient in a single dose, or may be administered over an extended period of time in multiple doses according to a divided treatment protocol. In the pharmaceutical composition of the present invention, the content of the active(s) ingredient(s) may vary depending on the severity of the disease. Specifically, the preferred total daily dose of the peptide of the present invention may be from about 0.0001 μg to 500 mg per 1 kg of patient body weight. However, the effective dose of the peptide is determined taking into account various factors, including the age, body weight, health status, sex of the patient, severity of the disease, diet and elimination rate, as well as the route and frequency of administration of the pharmaceutical composition. In this regard, those skilled in the art will readily be able to determine the effective dose appropriate for a particular application of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention is not limited to a specific dosage form, a specific route and method of administration, provided that it has the effects of the present invention.

The pharmaceutical composition of the present invention shows a very good duration of efficacy and titer in vivo, and therefore the amount and frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced.

In particular, since the pharmaceutical composition of the present invention contains, as an active ingredient, a glucagon derivative having an altered pI different from that of native glucagon, it exhibits improved solubility and high stability depending on the pH of the solution, and therefore, the pharmaceutical composition of the present invention can be effectively used in obtaining a stable preparation of glucagon for the treatment of hypoglycemia or obesity.

In another aspect of the present invention, a novel glucagon derivative is provided.

The glucagon derivative is as explained above.

More specifically, this derivative is characterized in that it is an isolated peptide containing the amino acid sequence of the following general formula 2:

Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-TX30 (general formula 2, SEQ ID NO: 46).

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 absent, provided that when the amino acid sequence of general formula 2 is identical to any of

- 19 040165

SEQ ID NOs: 14, 19, 20, 25, 27, 31 and 33, then it can be excluded.

More specifically, the amino acid pair X16 and X20 in the general formula 2 can be replaced by glutamic acid or lysine, respectively, which are capable of forming a ring, thereby forming a ring (for example, a lactam ring) with the amino acid pair X16 and X20, but not limited to this.

Additionally, the C-terminus of the peptide containing the amino acid sequence of General formula 2 may be amidated, but not limited to this.

Additionally, the peptide may be a glucagon derivative capable of activating the glucagon receptor, but is not limited to this.

More specifically, the peptide may contain an amino acid sequence selected from, but not limited to, SEQ ID NOs: 12, 13, 15, and 36-44.

In yet another aspect, the present invention provides an isolated polynucleotide encoding a glucagon derivative, a vector comprising the polynucleotide, and an isolated cell comprising the polynucleotide or vector.

The glucagon derivative is as explained above.

Additionally, an isolated polynucleotide encoding a glucagon derivative includes, within the scope of the present invention, a polynucleotide sequence having a homology of 75% or more, specifically 85% or more, more specifically 90% or more, and even more specifically 95% or more with the corresponding sequence.

As used herein, the term homology refers to the sequence similarity of a wild-type amino acid sequence or a wild-type nucleotide sequence, and homology comparisons can be made with the naked eye or using a commercially available comparison program. Using a commercially available comparison program, homology between two or more sequences can be expressed as a percentage (%), and % homology between opposing sequences can be calculated.

As used herein, the term recombinant vector refers to a DNA construct containing a polynucleotide sequence encoding a target peptide, such as a glucagon derivative, that is operably linked to an appropriate regulatory sequence to allow expression of the target peptide, such as a glucagon derivative, in a host cell.

The regulatory sequence contains a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable ribosomal binding domain of the mRNA, and a sequence regulating the termination of transcription and translation. The recombinant vector, once transformed into a suitable host cell, may be replicated, or may be functionally independent of the host genome, or may be integrated into the host genome itself.

The recombinant vector used in the present invention is not particularly limited as long as the vector can be replicated in the host cell, and it can be constructed using any vector known in the art. Examples of a commonly used vector may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. The vectors used in the present invention may be any expression vector known in the art.

The recombinant vector is used to transform a host cell to produce the glucagon derivatives of the present invention. Additionally, these transformed cells, as part of the present invention, may be used to amplify nucleic acid fragments and vectors, or may be cultured cells or cell lines used in the recombinant production of glucagon derivatives of the present invention.

As used herein, the term transformation refers to the process of introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell, thereby allowing expression of the protein encoded by the polynucleotide in the host cell. With respect to the transformed polynucleotide, it does not matter whether it is inserted into the chromosome of the host cell and localized there or localized outside the chromosome, as long as it can be expressed in the host cell, and both cases are covered.

Additionally, the polynucleotide includes DNA and RNA that encode the target protein. The polynucleotide may be inserted in any form as long as it can be introduced into and expressed in the host cell. For example, a polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all the essential elements required for self-expression. The expression cassette can typically contain a promoter operably linked to the polynucleotide, a transcription terminator, a ribosomal binding domain translation terminator. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide can be introduced into the host cell when it is operably linked to a sequence necessary for its expression in the host cell, but is not limited to this.

- 20 040165

Additionally, as used herein, the term operably linked refers to a functional link between a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target peptide of the present invention and the aforementioned gene sequence.

A suitable host for use in the present invention may not be particularly limited as long as it can express the polynucleotide of the present invention. Examples of a suitable host may include bacteria of the genus Escherichia such as E. coli; bacteria of the genus Bacillus such as Bacillus subtilis; bacteria of the genus Pseudomonas such as Pseudomonas putida; yeasts such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe; insect cells such as Spodoptera frugiperda (Sf9); and animal cells such as CHO, COS and BSC.

In yet another aspect of the present invention, an isolated conjugate is provided wherein a glucagon derivative and a biocompatible substance that is capable of increasing in vivo half-life are linked. The conjugate may be a long acting conjugate.

With regard to the glucagon derivative, biocompatible substance and conjugate structure, all that is described above applies to them.

Specifically, the biocompatible substance may be selected from the group consisting of polyethylene glycol, fatty acid, cholesterol, albumin and a fragment thereof, an albumin binder, a polymer of repeating units of a particular amino acid sequence, an antibody, an antibody fragment, an FcRn binder, in vivo connective tissue, or their derivative, nucleotide, fibronectin, transferrin, saccharide and polymer, but is not limited to them.

Additionally, the isolated peptide may be linked to a biocompatible substance via a linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol/propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer such as polylactic acid. (PIA) and polylactic-glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, fatty acid, polymer, small molecule compound, nucleotide, and combinations thereof.

Additionally, the biocompatible agent may be an FcRn binding agent, and the isolated peptide may be linked to the biocompatible agent via a peptide linker or a non-peptide linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide , dextran, polyvinyl ethyl ether, biodegradable polymer such as polylactic acid (PIA) and polylactic glycolic acid (PIGA), lipid polymer, chitin, hyaluronic acid, fatty acid, polymer, small molecular weight compound, nucleotide, and combinations thereof, but not limited to them.

Additionally, the FcRn binding agent may be a polypeptide containing the Fc region of an immunoglobulin, but is not limited to.

In yet another aspect of the present invention, a composition is provided comprising a glucagon derivative or isolated conjugate.

The glucagon derivative and the isolated conjugate are as explained above.

Specifically, the composition may be a pharmaceutical composition for the treatment or prevention of hypoglycemia or metabolic syndrome, but is not limited to. The pharmaceutical composition is as described above.

Additionally, the composition may be a composition containing a peptide from the amino acid sequence of the following general formula 2:

Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-TX30 (general formula 2, SEQ ID NO: 46).

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 absent, provided that when the amino acid sequence of general formula 1 is identical to any of SEQ ID NOs: 14, 19, 20, 25, 27, 31 and 33, then it can be excluded.

In yet another aspect of the invention, a method for preventing or treating hypoglycemia is provided.

- 21 040165 or metabolic syndrome, including the introduction to the subject of the above composition.

Composition, hypoglycemia, metabolic syndrome, prevention and treatment are as explained above.

In the present invention, the term "subject" refers to subjects suspected of having hypoglycemia or metabolic syndrome, and the term refers to mammals, including humans, mice, and livestock, having hypoglycemia or metabolic syndrome or at risk of hypoglycemia or metabolic syndrome. However, any subject to be treated with a glucagon derivative of the present invention or a composition containing a glucagon derivative is within the scope of the invention without limitation. In addition, a subject suspected of having hypoglycemia or obesity can be effectively treated by administering a pharmaceutical composition containing the glucagon derivative of the present invention. Hypoglycemia and obesity are as explained above.

The method of the present invention may include administering a pharmaceutical composition containing the peptide in a pharmaceutically effective amount. The total daily dose will be determined by the attending physician based on appropriate medical judgment and administered in single or multiple divided doses. With regard to the objects of the present invention, a specific therapeutically effective dose for each particular patient can preferably be used differently depending on various factors commonly known in medicine, including the type and degree of response to be achieved, specific compositions containing other agents or not, age, body weight, general health, sex and diet of the patient, time and route of administration, rate of secretion of the composition, duration of treatment, other drugs used in combination or in parallel with the composition of the present invention, and similar factors known in medicine.

In yet another aspect of the present invention, the use of a glucagon derivative or isolated conjugate or composition in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of hypoglycemia or metabolic syndrome is provided.

The glucagon derivative, isolated conjugate, composition, hypoglycemia and metabolic syndrome are as explained above.

Implementation of the invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are presented for illustrative purposes only, and the scope of the present invention is not limited thereto in any way.

Example 1 Preparation of a cell line demonstrating cAMP (cyclic adenosine monophosphate) response to glucagon.

PCR (polymerase chain reaction) was performed using the open reading frame (ORF) region of the cDNA (OriGene Technologies, Inc., USA) of the human glucagon receptor gene as a template along with the following forward and reverse primers (SEQ ID NO: 47 and 48, respectively), each containing EcoRI and XhoI restriction sites.

In particular, PCR was carried out for a total of 30 cycles under the following conditions: denaturation at 95°C for 60 s, annealing at 55°C for 60 s, and polymerization at 68°C for 30 s. The amplified PCR products were subjected to 1.0% agarose gel electrophoresis and the 450 bp band was eluted. (base pairs).

Forward primer (SEQ ID NO: 47)

5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3'

Reverse primer (SEQ ID NO: 48):

5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3'

The PCR product was cloned into a known animal cell expression vector, x0GC/dhfr (dihydrofolate reductase), to obtain a recombinant x0GC/GCGR vector.

CHO DG44 cell line (Chinese hamster ovary cell line defective in dhfr) cultured in DMEM/F12 (Dulbecco's Modified Eagle's Medium with Ham's F12) (10% FBS (fetal bovine serum)) was transfected with recombinant vector x0GC/ GLPIR using Lipofectamine® (lipofectamine) (Invitrogen, USA) and cultured in a selective medium containing G418 (1 mg/ml) and methotrexate (10 nM) From this, cell lines were selected from one clone using the limiting dilution method, and from they were finally selected for a cell line demonstrating an excellent cAMP response to glucagon in a concentration-dependent manner.

Example 2 Synthesis of a glucagon derivative.

In order to obtain glucagon derivatives with improved physical properties, the amino acid sequence of the native glucagon of SEQ ID NO: 1 was replaced by amino acid residues having positive and negative charges, and thus glucagon derivatives were synthesized, which are shown in the table below. Relative activity in

- 22 040165 vitro, indicated below, was measured by the method described in example 4.

Amino acid sequences of native glucagon and glucagon derivatives

SEQ GO NO Peptide sequence Education- ring pl Activity in vitro (relative activity SEQ ID NO: 1, %) SEQID NO: 1 HSQGTFTSD YSKYLD SRRAQDF VQWLMNT - 6.8 100 SEQID NO: 2 HSQGTFTSDYSKYLDCDRAQDF VQWLMNT - 4.56 0.6 SEQID NO: 3 HSQGTFTSDYSKYLDCERAQDF VQWLMNT - 4.66 6.1 SEQID NO: 4 HSQGTFTSD YSKYLD SCD AQDF VQWLMNT - 4.13 <0.1 SEQID NO: 5 HSQGTFTSD YSKYLD SCEAQDF VQWLMNT - 4.22 0.3 SEQID NO: 6 HSQGTFTSD YSKYLD SCEADDF VQWLMNT - 4.03 <0.1 SEQID NO: 7 YSQGTFTSD YSKYLD SCEADDF VQWLMNT - 3.71 <0.1 SEQID NO: 8 YXQGTFTSDYSKYLDSCDAQDF VQWLINT - 3.77 <0.1 SEQID NO: 9 YXQGTFTSDYSKYLDSCDAQDF VVWLINT - 3.77 <0.1

- 23 040165

SEQ GO NO: 10 YXQGTFTSDYSKYLDSCDADDDF VVWLINT - 3.66 <0.1 SEQ GO NO:11 YXQGTFTSDYSKYLDEKCAKEEF VQWLMNT - 4.78 4.6 SEQ ID NO:12 YXQGTFTSDYSKYLDEKRAKEF VQWLMNTC formed ring 6.20 56.3 SEQ ID NO:13 YXQGTFTSDYSCYLDSRRAQDF VQWLMNT - 4.43 5.2 SEQ ID NO:14 YXQGTFTSDYSKYLDCKRAKEF VQWLMNT - 8.12 18.1 SEQ ID NO:15 YXQGTFTSDYSKYLCEKRAQDF VVWLMNT - 6,I 1.1 SEQ ID NO:16 YXQGTFTSDYSKYLDCRRAQVF VQWLMRT - 9,I 4.2 SEQ ID NO:17 YXQGTFTSDYSKYLDCVRAQDF VQWLMRT - 6.03 23.2 SEQ ID NO:18 YXQGTFTSDYSKYLDSRRACDF RLWLMNT - 8.15 <0.1 SEQ ID NO:19 YXQGTFTSDYSKYLCEKRAKEF VQWLMNT formed ring 8.12 12.1 SEQ ID NO: 20 YXQGTFTSDYSKYLDECRAKEF VQWLMNT formed ring 4.78 299.7 SEQ ID NO: 21 YXQGTFTSDYSKYLDEKCAKEEF VQWLMNT formed ring 4.78 57.8 SEQ ID NO: 22 YXQGTFTSDYSKYLDEKRCKEEF VQWLMNT formed ring 6.20 147.8 SEQ ID NO: 23 YXQGTFTSDYSKYCDEKRAKEF VQWLMNT formed ring 6.20 76.8 SEQ ID NO: 24 YXQGTFTSDYSKCLDEKRAKEF VQWLMNT formed ring 6.21 58.0 SEQ ID NO: 25 YXQGTFTSDYSKYLDEKRAKCF VQWLMNT formed ring 8.12 46.9 SEQ ID WXQGTFTSDYSKYLDECRAKDF formed 4.68 1.0

- 24 040165

NO: 26 VQWLMNT ring SEQ ID NO: 27 YXQGTFVSDYSKYLDECRAKDF VQWLMNT formed ring 4.68 93.6 SEQ ID NO: 28 WXQGTFVSDYSKYLDECRAKD FVQWLMNT formed ring 4.68 <0.1 SEQ GO NO: 29 YXQGTFTSDYSKCLDERRAKDF VQWLMNT formed ring 6.15 61.3 SEQ ID NO: 30 WXQGTFTSDYSKCLDERRAKDF VQWLMNT formed ring 4.44 oh, s SEQ ID NO:31 YXQGTFTSDYSKYLDCKRAKEF VQWLMNT formed ring 8.12 6.3 SEQ ID NO:32 -SQGTFTSDYSKYLDECRAKEFV QWLMNT formed ring 4.78 0.7 SEQ ID NO:33 YXQGTFTSDYSKYLDSRRAQDF VQWLMNT - 6.04 108.2 SEQ ID NO:34 WXQGTFTSDYSKYCDERRAKEF VQWLMNT formed ring 6.21 0.2 SEQ ID NO:35 YXQGTFTSDYSKYCDERRAKEF VQWLMNT formed ring 6.2 17.7 SEQ ID NO:36 YXQGTFTSDCSKYLDERRAKEF VQWLMNT formed ring 6.21 9.9 SEQ ID NO:37 YXQGTFTSDYSKYLDERRAKEF VQWLMNTC formed ring 6.21 225.5 SEQ ID NO:38 YXQGTFCSDYSKYLDERRAKEF VQWLMNT formed ring 6.15 167.3 SEQ ID NO:39 YXQGTFVSDCSKYLDERRAKDF VQWLMNT formed ring 6.15 3.7 SEQ ID NO:40 YXQGTFVSDYSKYLDERRAKDF VQWLMNTC formed ring 6.15 40.8 SEQ ID NO:41 YXQGTFCSDYSKYLDERRAKDF VQWLMNT formed ring 6.03 45.2 SEQ ID NO:42 YXQGTFCSDYSKYLDSRRAQDF VQWLMNT - 6.03 37.9 SEQ ID NO:43 YXQGTFT SDC SKYLD SRRAQDF VQWLMNT - 6.03 1.6 SEQ ID NO:44 YXQGTFTSDYSKYLDSRRAQDF VQWLMNTC - 6.21 75.4

In the amino acid sequences shown in the table, the amino acid designated X is the non-native amino acid, aminoisobutyric acid (Aib); underlined amino acid residues indicate ring formation; and - in an amino acid sequence indicates that there is no amino acid residue at the corresponding position.

Example 3 Measurement of pI of glucagon derivatives.

In order to measure the improved physical properties of the glucagon derivatives synthesized in Example 2, pI values were calculated from amino acid sequences using the pI/Mw tool (http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003 ) on the ExPASy server.

As shown in the table above, while native glucagon SEQ ID NO: 1 has a pI of 6.8, some glucagon derivatives of the present invention showed pI values ranging from

- 25 040165 about 4 to about 6. Since the glucagon derivatives of the present invention have pI values lower or higher than the pI value of native glucagon, they may exhibit improved solubility and higher stability under neutral pH conditions compared to native glucagon.

Accordingly, when using the glucagon derivatives of the present invention as a therapeutic agent for the treatment of hypoglycemia, they can improve patient compliance and treatment regimen, and are also suitable for administration in combination with other anti-obesity or anti-diabetic agents, and therefore the glucagon derivatives of the present invention can be effectively used as therapeutic agents for the treatment of hypoglycemia and metabolic syndromes, including obesity, diabetes, non-alcoholic steatohepatitis (NASH), dyslipidemia and coronary heart disease.

Example 4 Measurement of cAMP activity of glucagon derivatives.

The activity of the glucagon derivatives synthesized in example 2 was measured in cell lines having human glucagon receptors obtained in example 1.

Specifically, the transfected cell line was subcultured 3-4 times per week, aliquoted into a 384-well plate at 6x10 cells per well, and cultured for 24 hours. Native glucagon and glucagon derivatives were suspended in Hanks' balanced salt buffer solution (HBSS) containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1% bovine serum albumin (BSA), and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), with cell cultures at 200 concentrations and 1600 nM, respectively, were continuously subjected to 4-fold dilution 10 times, a cAMP assay kit (LANCE cAMP 384 kit, PerkinElmer) was used and added to cultured cells, and their fluorescence value was measured. After measurement, the highest fluorescence value was taken as 100% and from this the EC ₅₀ values (50% effective concentration) of the glucagon derivatives were calculated and compared with the EC ₅₀ of native glucagon. The results are presented in the table above.

Example 5 Preparation of a conjugate containing a derivative of glucagon and an immunoglobulin Fc (conjugate of SEQ ID NO: 12 or 20 and an immunoglobulin Fc region).

For the pegylation of a cysteine residue of a glucagon derivative (SEQ ID NOs: 12 and 20) using a 10 kDa PEG having a maleimide group and an aldehyde group, respectively, at both ends (named maleimide-PEG-aldehyde, 10 kDa, NOF, Japan), glucagon derivatives and maleimide-PEG-aldehyde was subjected to interaction in a molar ratio of 1:1 to 1:5 at a protein concentration of 3 to 10 mg/ml at a low temperature for 1-3 hours. In particular, the reaction was carried out in a medium to which 20 -60% isopropanol. After completion of the reaction, the reaction mixture was applied to SP Sepharose HP (GE healthcare, USA) to purify cysteine mono-pegylated glucagon derivatives.

Then, the purified mono-pegylated glucagon derivatives and the immunoglobulin Fc region were interacted in a molar ratio of 1:2 to 1:10 at a protein concentration of 10 mg/ml to 50 mg/ml at a temperature of 4 to 8°C for 12-18 hours. This reaction was carried out in a medium in which sodium cyanoborohydride (NaCNBH ₃ ) and 10 to 20% isopropanol were added to 100 mM calcium phosphate buffer (pH 6.0). After completion of the reaction, the reaction mixture was applied to a Butyl sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA) to purify the conjugate containing the glucagon derivative and the immunoglobulin Fc region.

After preparation, the purity of the conjugates as analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography was shown to be 95% or higher.

Specifically, a conjugate in which a glucagon derivative of SEQ ID NO: 12 and an immunoglobulin Fc have been linked by PEG has been referred to as a conjugate containing a glucagon derivative of SEQ ID NO: 12 and an immunoglobulin Fc or a long-acting derivative of SEQ ID NO: 12, and herein the invention, these terms can be used interchangeably.

Specifically, a conjugate in which a glucagon derivative of SEQ ID NO: 20 and an immunoglobulin Fc have been linked by PEG has been referred to as a conjugate containing a glucagon derivative of SEQ ID NO: 20 and an immunoglobulin Fc or a long-acting derivative of SEQ ID NO: 20, and herein the invention, these terms can be used interchangeably.

Example 6 Preparation of a conjugate containing an exendin-4 derivative and an immunoglobulin Fc region.

PEG 3.4 kDa having a propionaldehyde group at both ends, i.e. 3.4k PropIonALD (2) PEG was reacted with Exendin-4 Lys CA, i.e. imidazo-acetyl-exendin-4 where the alpha carbon of the N-terminal histidine is removed (CA exendin-4, AP, USA) and then coupled on the isomeric peak at the very last part (Lys27) between the two Lys peaks, which is quite reactive and clearly differs from the N-terminal isomer.

The peptide and Fc of the immunoglobulin were reacted in a molar ratio of 1:8 at a total protein concentration of 60 mg/ml at 4°C for 20 h. The reactant was 100 mM K-P (potassium phosphate

- 26 040165 buffer) (pH 6.0) and the reducing agent 20 mM SCB (sodium cyanoborohydride) was added. The coupling reactants were purified by passing through two purification columns. First, a large amount of uncoupled immunoglobulin Fc was removed using SOURCE Q (XK 16 ml, Amersham Biosciences). After applying a salt gradient using 1 M NaCl at 20 mM Tris (pH 7.5), the immunoglobulin Fc, which has a relatively weak binding affinity, is immediately eluted, followed immediately by exendin-4-Fc immunoglobulin. As a result of this primary purification, the immunoglobulin Fc is removed to a certain extent, however, complete separation is not achieved on the ion exchange column due to the small difference in binding affinity between the immunoglobulin Fc and the immunoglobulin exendin-4-Fc. Accordingly, secondary purification was carried out using the hydrophobicity of two different substances. The primary purified sample was bound to SOURCE ISO (HR 16 ml, Amersham Biosciences) using 20 mM Tris (pH 7.5) and 1.5 M ammonium sulfate and then eluted with a gradual decrease in ammonium sulfate concentration. As a result, the immunoglobulin Fc region, which has a weak binding affinity for the HIC (Hydrophobic Interaction Chromatography) column, was eluted first, followed by the exendin-4-Fc immunoglobulin sample, which has a strong binding affinity for the tail, was eluted. The separation was much easier compared to the ion exchange column due to the greater difference in hydrophobicity.

Column: SOURCE Q (XK 16 ml, Amersham Biosciences).

Flow rate: 2.0 ml/min.

Gradient: A 0 ->25% 70 min B (A: 20 mM Tris, pH 7.5; B: A + 1M NaCl).

Column: SOURCE ISO (HR 16 ml, Amersham Biosciences).

Flow rate: 7.0 ml/min.

Gradient: B 100 ->0% 60 min B [A: 20 mM Tris (pH 7.5); B: A+1.5M ammonium sulfate ((NH ₄ ) ₂ SO ₄ )].

The conjugate thus obtained, in which the exendin-4 derivative and the immunoglobulin Fc region are linked by PEG, was named the long-acting exendin-4 derivative. In addition, this term in the present invention can be used interchangeably with a long-acting exendin derivative.

Experimental Example 1 Effect of weight loss in high-fat diet-induced obese rats.

In this experiment, we used rats with high-fat diet-induced obesity, which are widely used as animal models of obesity. The body weight of the rats before administration was approximately 600 g. During the experiment, the rats were kept separately and had unlimited access to water. Lighting was not provided from 6 am to 6 pm.

The test groups fed a high fat diet included: group 1, excipient (injection once every 3 days) - control group; group 2, long-acting exendin derivative from example 6 at a dose of 3.3 nmol/kg (injection once every 3 days); group 3, long-acting derivative of SEQ ID NO: 12 at a dose of 1.6 nmol/kg (injection once every 3 days); group 4, long-acting derivative of SEQ ID NO: 12 at a dose of 3.3 nmol/kg (injection once every 3 days); group 5, long-acting derivative of SEQ ID NO: 12 at a dose of 6.6 nmol/kg (injection once every 3 days); group 6, long-acting derivative of exendin from example 6 at a dose of 3.3 nmol/kg + long-acting derivative of SEQ ID NO: 12 at a dose of 1.6 nmol/kg (injection once every 3 days, respectively); group 7, long-acting derivative of the exendin from example 6 at a dose of 3.3 nmol/kg + long-acting derivative of SEQ ID NO: 12 at a dose of 3.3 nmol/kg (injection once every 3 days, respectively); group 8, long-acting derivative of exendin from example 6 at a dose of 3.3 nmol/kg + long-acting derivative of SEQ ID NO: 12 at a dose of 6.6 nmol/kg (injection once every 3 days, respectively); group 9, pair feeding with group 4; and group 10, paired feeding with group 7. The experiment was stopped on the 15th day, and during the experiment, changes in body weight of rats in each group were measured at intervals of 3 days. After stopping the experiment and autopsy, the amount of mesenteric fat and liver mass were measured. To compare the group treated with the excipient (control group) and the test groups, a statistical analysis was performed by 1-way ANOVA (ANOVA).

As a result of measuring changes in body weight, which are confirmed in FIG. 1, groups administered with either the long-acting exendin derivative alone or the long-acting derivative of SEQ ID NO: 12 alone showed a decrease in body weight from -8 and -7 to -22% compared to pre-administration body weight, and in groups to which the long-acting derivative of exendin and the long-acting derivative of SEQ ID NO: 12 were co-administered, the effect of reducing body weight further increased from -22 to -35%.

In addition, when comparing the effect of reducing body weight in the group administered only the long-acting derivative of SEQ ID NO: 12, and in the group administered the combination of the long-acting derivative of exendin and the long-acting derivative of SEQ ID NO: 12, with a decrease in body weight in the paired feeding group, respectively, showed a difference of about -11% and about -17%, respectively, confirming that the effect of weight loss

- 27 040165 of the body occurred when a glucagon derivative was administered alone or when administered together as a result of actions other than food intake.

That is, it has been confirmed that the long-acting glucagon derivative of the present invention can play an additional role in weight loss besides the effect of anorexia.

Additionally, as a result of measuring the amount of mesenteric fat and liver mass, as confirmed in FIG. 2 and 3, the co-administration of the long-acting exendin derivative and the long-acting derivative of SEQ ID NO: 12 showed a significant decrease in body fat as well as a decrease in liver weight compared to those in the excipient group. In particular, the reason for the increase/decrease in liver weight is usually an increase/decrease in fat present in the liver, and the above effect of reducing liver weight is indicative of the effect of reducing fat in the liver. Accordingly, the measurement of liver fat reduction can serve as a way to measure the therapeutic effect on metabolic syndrome such as obesity, diabetes, non-alcoholic steatohepatitis, etc.

Experimental Example 2 Effect of weight loss in high fat diet-induced obese mice.

In this experiment, mice were used with high fat diet-induced obesity, which are widely used as animal models of obesity. The body weight of the mice before administration was approximately 55 g. During the experiment, the mice were housed in 7 mice per group and had unlimited access to water. Lighting was not provided from 6 am to 6 pm.

The test groups fed a high fat diet included: group 1, excipient (injection once every 2 days) - control group; group 2, long-acting exendin derivative from example 6 at a dose of 4.3 nmol/kg (injection once every 2 days); group 3, long-acting derivative of SEQ ID NO: 20 at a dose of 4.4 nmol/kg (injection once every 2 days); group 4, long-acting derivative of SEQ ID NO: 20 at a dose of 8.8 nmol/kg (injection once every 2 days); group 5, long acting exendin derivative of Example 6 at 4.3 nmol/kg + long acting derivative of SEQ ID NO: 20 at 4.4 nmol/kg (injection once every 2 days); group 6, long acting exendin derivative of Example 6 at 2.1 nmol/kg + long acting derivative of SEQ ID NO: 20 at 6.6 nmol/kg (injection once every 2 days); and group 7, long acting exendin derivative of Example 6 at 0.8 nmol/kg + long acting derivative of SEQ ID NO: 20 at 8.0 nmol/kg (injection once every 2 days). The experiment was stopped on the 22nd day, and during the experiment, changes in body weight of mice in each group were measured at intervals of 2 days. After stopping the experiment and autopsy, the mass of the mouse liver was measured.

As a result of measuring changes in body weight, which are confirmed in FIG. 4, each of the groups administered only the long-acting derivative of SEQ ID NO: 20 (8.8 nmol/kg, injection once every 2 days) showed a decrease in body weight of -25 and -29%, respectively, compared to body weight. before the introduction. In addition, it was shown that the effect of reducing body weight increased further when administered in combination with a long-acting exendin derivative. It was also confirmed that the co-administration of the long-acting exendin derivative and the long-acting derivative of SEQ ID NO: 20 at a ratio of 1:1, 1:3 and 1:10 further increased the effect of reducing body weight by -50% or more. Additionally, the effect of reducing body weight depending on the ratio of long-acting exendin derivative and long-acting derivative of SEQ ID NO: 20 was insignificant, however, the effect of anorexia became higher with increasing percentage of long-acting exendin derivative, indicating that the long-acting derivative of glucagon by of the present invention may play an additional role in weight loss besides the effect of anorexia.

Additionally, as a result of measuring total blood cholesterol, as confirmed in FIG. 5, each of the groups receiving the long-acting exendin derivative (4.4 nmol/kg, injection once every 2 days) and the long-acting derivative of SEQ ID NO: 20 (8.8 nmol/kg, injection once every 2 days ), showed a cholesterol reduction of -35% and -71%, respectively. From the foregoing, it has been confirmed that the long-acting glucagon derivative of the present invention can play an additional role in lowering blood cholesterol levels besides the effect of anorexia. To compare the group treated with the excipient (control group) and the test groups, statistical analysis was performed by 1-way ANOVA.

Those skilled in the art will appreciate that the present invention may be embodied in other specific forms without departing from its spirit or essential features. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the present invention is defined by the appended claims and not by the foregoing description of the invention. All changes that occur within the meaning and equivalence range of the claims are within the scope of the present invention.

Claims (33)

CLAIM 1. Pharmaceutical composition for the treatment or prevention of metabolic syndromes, containing: 1) a peptide containing the amino acid sequence of native glucagon SEQ ID NO: 1, having one or more substitutions in positions 1, 2, 7, 10, 12, 15-17, 20 -21, 24 and optionally containing a cysteine at position 30, which is selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44; and 2) at least one compound or substance having therapeutic activity against metabolic syndrome, where the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, hypertension, non-alcoholic steatohepatitis (NASH), atherosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease and stroke. 2. The pharmaceutical composition of claim 1, wherein the compound or substance having therapeutic activity against metabolic syndrome is selected from the group consisting of an insulinotropic peptide, a glucagon-like peptide-1 (GLP-1) receptor agonist, a leptin receptor agonist, a dipeptidyl peptidase- IV (DPP-IV), Y5 receptor antagonist, melanin-concentrating hormone (MCH) receptor antagonist, Y2/4 receptor agonist, melanocortin 3/4 receptor agonist (MC 3/4), gastric/pancreatic lipase inhibitor, 5-hydroxytryptamine 2C receptor agonist (5HT2C), e3A receptor agonist, amylin receptor agonist, ghrelin antagonist, ghrelin receptor antagonist, peroxisome proliferator-activated receptor alpha (PPARa) agonist, peroxisome proliferator-activated receptor delta (PPARδ) agonist, farnesoid X receptor (FXR) agonist, acetyl inhibitor -CoA-carboxylase, peptide YY, cholecystokinin (CCK), xenin, glycentin, obestatin, secretin a, nesfatin, insulin, and glucose-dependent insulinotropic peptide (GIP). 3. The pharmaceutical composition of claim 2, wherein the insulinotropic peptide is selected from the group consisting of GLP-1, exendin-3, exendin-4, an agonist thereof, and a combination thereof. 4. The pharmaceutical composition according to claim 3, wherein the insulinotropic peptide is an insulinotropic peptide derivative wherein the N-terminal histidine residue is replaced by a residue selected from the group consisting of deaminohistidil, N-dimethylhistidil, β-hydroxyimidazopropionyl, 4-imidazoacetyl and β- carboxyimidazopropionyl. 5. The pharmaceutical composition according to claim 3, where the insulinotropic peptide is selected from the group consisting of native exendin-4; an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed; a derivative of exendin-4, in which the N-terminal amino group of exendin-4 is replaced by a hydroxyl group; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is modified with a dimethyl group; an exendin-4 derivative in which the α-carbon of the 1st amino acid of exendin-4, histidine, has been removed; an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, has been replaced by serine; and an exendin-4 derivative in which the 12th amino acid of exendin-4, lysine, is replaced by arginine. 6. The pharmaceutical composition according to claim 2, wherein the peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44 is associated with a biocompatible substance capable of increasing the half-life of the peptide in vivo, with the formation of the longest-acting conjugate, the duration of action of which is increased compared to native glucagon; and the insulinotropic peptide is coupled to a biocompatible substance capable of increasing the half-life of the insulinotropic peptide in vivo, thereby forming a long-acting conjugate, the duration of which is increased compared to the native insulinotropic peptide. 7. The pharmaceutical composition according to claim 6, wherein the biocompatible substance is an FcRn (neonatal Fc receptor) binder. 8. The pharmaceutical composition according to claim 6, wherein the peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44 and the insulinotropic peptide, respectively, are linked to a biocompatible substance via a polyethylene glycol linker. 9. Pharmaceutical composition according to claim 6, wherein the biocompatible substance is an FcRn-binding substance, and a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36-44, and an insulinotropic peptide, respectively, are linked to the biocompatible substance by means of a peptide linker or a non-peptide linker which is polyethylene glycol. 10. The pharmaceutical composition of claim 9, wherein the FcRn binding agent is a polypeptide containing the Fc region of an immunoglobulin. 11. The pharmaceutical composition of claim 10, wherein the Fc region of the immunoglobulin is aglycosylated. 12. The pharmaceutical composition of claim 10, wherein the immunoglobulin Fc region is selected from the group consisting of: - 29 040165 (a) CH1 domain, CH2 domain, CH3 domain and 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) combinations between one or at least two domains from the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and the hinge region or part of the hinge region of an immunoglobulin; and (e) a dimer between each domain of the heavy chain constant region and the light chain constant region. 13. The pharmaceutical composition of claim 10, wherein the polypeptide comprising the immunoglobulin Fc region is in the form of a dimer. 14. The pharmaceutical composition according to claim 10, where the Fc region of the immunoglobulin is a derivative of native Fc, in which the region capable of forming a disulfide bond is removed, a derivative of native Fc, in which part of the amino acid(s) at the N-terminus is removed, a derivative of native An Fc in which methionine is added to the N-terminus, a native Fc derivative in which the complement-binding site has been removed, or a native Fc derivative in which the antibody-dependent cell-mediated cytotoxicity (ADCC) site has been removed. 15. The pharmaceutical composition of claim 10, wherein the immunoglobulin Fc region is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM. 16. The pharmaceutical composition of claim 15, wherein the immunoglobulin Fc region is an IgG4 Fc region. 17. The pharmaceutical composition of claim 10, wherein the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4. 18. The pharmaceutical composition of claim 9, wherein the non-peptide linker is linked to a cysteine residue of a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44. 19. The pharmaceutical composition according to claim 9, wherein both ends of the non-peptide linker are respectively linked to the amino group or thiol group of a peptide that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44, or an insulinotropic peptide and biocompatible substance. 20. An isolated peptide for the treatment or prevention of metabolic syndrome, containing the amino acid sequence of native glucagon SEQ ID NO: 1, having one or more substitutions at positions 1, 2, 7, 10, 12, 15-17, 20-21, 24 and possibly containing a cysteine at position 30 selected from the group consisting of SEQ ID NOs: 13, 15 and 36-44, where the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, hypertension, non-alcoholic steatohepatitis (NASH ), atherosclerosis caused by dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease and stroke. 21. The isolated peptide of claim 20, wherein the C-terminus of the peptide is amidated. 22. The isolated peptide of claim 20, wherein the peptide is a glucagon derivative capable of activating the glucagon receptor. 23. An isolated polynucleotide encoding an isolated peptide according to any one of claims 20-22. 24. An expression vector containing the isolated polynucleotide according to claim 23. 25. An isolated conjugate for the treatment or prevention of metabolic syndrome, containing the isolated peptide according to claim 20, associated with a biocompatible substance capable of increasing the in vivo half-life, where the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity , hypertension, non-alcoholic steatohepatitis (NASH), dyslipidemia-induced atherosclerosis, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke. 26. The isolated conjugate of claim 25, wherein the biocompatible agent is an FcRn binder. 27. The isolated conjugate of claim 25, wherein the peptide is linked to the biocompatible substance via a polyethylene glycol linker. 28. The isolated conjugate of claim 25, wherein the biocompatible agent is an FcRn binder and the isolated peptide is linked to the biocompatible agent via a peptide linker or a non-peptide linker that is polyethylene glycol. 29. The isolated conjugate of claim 28, wherein the FcRn binding agent is a polypeptide containing an immunoglobulin Fc region. 30. Pharmaceutical composition for the treatment or prevention of metabolic syndrome, containing the isolated peptide according to claim 20 or the isolated conjugate according to claim 25, where the metabolic syndrome is selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, hypertension, non-alcoholic steatohepatitis (NASH), dyslipidemia-induced atherosclerosis, atherosclerosis, arteriosclerosis, coronary heart disease and stroke. 31. An isolated peptide for the treatment or prevention of hypoglycemia, containing amino acids - 30 040165 lot sequence of native glucagon SEQ ID NO: 1, having one or more substitutions at positions 1, 2, 7, 10, 12, 15-17, 20-21, 24 and possibly containing cysteine at position 30, selected from the group , consisting of SEQ ID NO: 13, 15 and 36-44. 32. An isolated conjugate for the treatment or prevention of hypoglycemia, comprising the isolated peptide of claim 32 linked to a biocompatible substance capable of increasing in vivo half-life. 33. Pharmaceutical composition for the treatment or prevention of hypoglycemia, containing the isolated peptide according to claim 31 or the isolated conjugate according to claim 32. excipient long-acting exendin-4 derivative (3.3 nmol/kg) long-acting derivative SEQ ID NO: 12 (1.6 nmol/kg) long-acting derivative SEQ ID NO: 12 (3.3 nmol/kg), long-acting derivative SEQ ID NO: 12 (6.6 nmol/kg) - ^-long acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (1.6 nmol/kg) ^-long-acting exendin-4 derivative (3.3 nmol/kg) + long acting derivative SEQ ID NO: 12 (3.3 nmol/kg) ♦ long acting derivative of exendin-4 (3.3 nmol/kg) + long acting derivative SEQ ID NO: 12 (6.6 nmol/kg) coupled feeding (to long-acting derivative of SEQ ID NO: 12 (3.3 nmol/kg) couple feeding (to long-acting derivative of exendin-4 (3.3 nmol/kg) + long-acting derivative of SEQ ID NO: 12 (6.6 nmol/kg) Fig. 1 - 31 040165 IM excipient SP long-acting exendin-4 derivative (3.3 nmol/kg) O long acting derivative SEQ ID NO: 12 (1.6 nmol/kg) long acting derivative SEQ ID NO: 12 (3.3 nmol/kg) III long acting derivative SEQ ID NO: 12 (6.6 nmol/kg) O long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (1.6 nmol/kg) O long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (3.3 nmol/kg) E3 long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (6.6 nmol/kg) pair feeding (to long-acting derivative SEQ ID NO: 12 (3.3 nmol/kg) kg) (77| pair feeding (to long acting derivative of exendin-4 (3.3 nmol/kg) + long acting derivative of SEQ ID NO: 12 (6.6 nmol/kg) Fig. 2 - 32 040165 Ml excipient α long acting exendin-4 derivative (3.3 nmol/kg) O long acting derivative SEQ ID NO: 12 (1.6 nmol/kg) long acting derivative SEQ ID NO: 12 (3.3 nmol/kg) long acting derivative SEQ ID NO: 12 (6.6 nmol/kg) o long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (1.6 nmol/kg) O long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (3.3 nmol/kg) O long acting derivative of exendin-4 (3.3 nmol/kg) + long acting derivative of SEQ ID NO: 12 (6.6 nmol/kg) pair feeding (to long acting derivative of SEQ ID NO: 12 (3.3 nmol/kg) kg) pair feeding (to long-acting exendin-4 derivative (3.3 nmol/kg) + long-acting derivative SEQ ID NO: 12 (6.6 nmol/kg) Fig. 3 O 2 4 6 8 10 12 14 16 18 20 22 Time (day) About the excipient In long-acting derivative of exendin-4 (4.3 nmol/kg) -fr- long-acting derivative SEQ ID NO: 20 (4.4 nmol/kg) ψ long-acting derivative SEQ ID NO: 20 (8.8 nmol/kg ) long acting exendin derivative (4.3 nmol/kg) + long acting derivative SEQ ID NO: 20 (4.4 nmol/kg) long acting exendin derivative (2.1 nmol/kg) + long acting derivative SEQ ID NO: 20 (6.6 nmol/kg) long acting exendin derivative (0.8 nmol/kg) + long acting derivative SEQ ID NO: 20 (8.0 nmol/kg) Fig. 4 - 33 040165 400 v NIN excipient ^□Long acting exendin-4 derivative (4.3 nmol/kg) ^Long acting derivative SEQ ID NO: 20 (4.4 nmol/kg) DC> Long acting derivative SEQ ID NO: 20 (8.8 nmol /kg) long acting derivative of Exendin (4.3 nmol/kg) + long acting derivative of SEQ ID NO: 20 (4.4 nmol/kg) SHL long acting Exendin derivative (2.1 nmol/kg) + long acting derivative SEQ ID NO: 20 (6.6 nmol/kg) '/v long acting Exendin derivative (0.8 nmol/kg) + long acting derivative SEQ ID NO: 20 (8.0 nmol/kg) Fig. 5 Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2