ISOLATED PEPTIDE AND ISOLATED POLYNUCLEOTIDE THAT ENCODES IT

Abstract

This document relates to a peptide derived from glucagon and a composition for preventing or treating obesity comprising the peptide as an active ingredient. The glucagon derivative, according to this document, exhibits a more excellent activating effect with respect to glucagon peptide-1 receptors and glucagon receptors compared to native glucagon, and consequently can be widely used as an effective agent for treating obesity.

Description

The present invention relates to a novel glucagon derivative having an excellent effect on both glucagon-like peptide-1 (GLP-1) receptors and glucagon receptors, and to a composition for preventing or treating obesity containing the glucagon derivative as an active ingredient. [Background Art] Recent economic advances and lifestyle changes have been accompanied by significant shifts in dietary habits. In particular, today's busy population is becoming increasingly overweight and obese due to high-calorie diets and insufficient exercise. According to a World Health Organization (WHO) report, more than one billion adults worldwide are overweight, including more than three million who are clinically diagnosed with severe obesity, and 250,000 people in Europe and 2.5 million people worldwide die from being overweight or from obesity-related diseases each year (World Health Organization, Global Strategy on Diet, Physical Activity and Health, 2004). Being overweight or obese increases blood pressure and cholesterol levels, thus becoming a cause of various diseases, including heart disease, diabetes, and arthritis, or exacerbating existing conditions. Furthermore, being overweight or obese is among the leading factors that increase the risk of diseases such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease in children and adolescents, as well as in adults. As such, obesity is now recognized as a serious and prevalent disease worldwide and a cause of various illnesses. However, because obesity is often perceived as being overcome through self-help efforts, patients with obesity are often assessed as having poor self-control. Yet, obesity is not easily curable because it is a complex disease closely linked to appetite control and the mechanisms of energy metabolism. Therefore, treating obesity requires both individual efforts to control appetite and addressing the underlying mechanisms of energy metabolism related to appetite. In this regard, there is a need for the development of a drug capable of treating these abnormal mechanisms. As a result of previous efforts, anti-obesity drugs such as Rimonabant® (Sanofi-Aventis), Sibutramin® (Abbott), Contrave® (Takeda), and Orlistat® (Roche) have been developed. However, these drugs have had drawbacks such as fatal adverse reactions or limited efficacy in treating obesity. For example, Rimonabant® has been shown to cause central nervous system disorders, Sibutramin® and Contrave® have shown adverse cardiovascular effects, and Orlistat® has shown a weight loss of only about 4 kg after one year of administration. Consequently, there does not appear to be any truly safe anti-obesity drug that can be prescribed to patients with obesity. As such, active research has been conducted to develop a new drug to address the shortcomings of conventional anti-obesity medications, and recently, considerable attention has been paid to glucagon derivatives. Glucagon is secreted by the pancreas when blood glucose levels fall low due to, for example, drug treatment, disease, hormones, or enzyme deficiency. Glucagon signals the liver to break down glycogen into glucose and raise blood glucose levels back to normal. Furthermore, glucagon has been reported to have an anti-obesity effect, in addition to its blood glucose-raising effect, by suppressing appetite and activating hormone-sensitive lipase in fat cells, thereby promoting fat breakdown. Glucagon-like peptide-1 (hereafter referred to as 'GLP-1'), a glucagon derivative, is a substance under development as a drug to improve hyperglycemia in diabetic patients. GLP-1 has the functions of increasing insulin synthesis and promoting its secretion, inhibiting glucagon secretion, inhibiting gastric emptying, improving glucose utilization, and inhibiting food intake. In addition, exendin-4, which is secreted by lizard venom and shows approximately 50% amino acid sequence homology with GLP-1, is known to alleviate hyperglycemia in diabetic patients by activating the GLP-1 receptor. However, anti-obesity drugs containing GLP-1 or exendin-4 have been reported to cause nausea and vomiting as adverse effects. In this regard, oxyntomodulin has been highlighted as an alternative to GLP-1, as it can bind to GLP-1 and glucagon peptides. Oxyntomodulin is a peptide derived from pre-glucagon, the precursor of glucagon, and has the same effects as GLP-1, such as inhibiting food intake, promoting satiety, and breaking down fat, thus increasing its potential as an anti-obesity agent. However, oxyntomodulin or its derivatives have the disadvantage that they must be administered at a high daily dose due to their short in vivo half-life and low efficacy. [Description] [Technical Problem] The present inventors, in an effort to improve the effect of the treatment of obesity, while reducing the dose, have developed a glucagon derivative with a partial modification in its amino acid sequence, and it is confirmed that the glucagon derivative has an excellent effect acting on glucagon receptors and GLP-1 receptors, thus completing the present invention. [Technical solution] The present invention has been made taking into account the previous problems produced in the prior art, and one object of the present invention is to provide a new peptide that shows an excellent treatment effect for obesity. Another object of the present invention is to provide a composition containing the peptide for the prevention or treatment of obesity. [Advantageous effects] The novel peptide of the present invention can markedly activate both GLP receptors and glucagon receptors compared to native glucagon, exhibit an excellent anti-obesity effect even with a lower administration amount, and consequently can be widely used as a safe and effective agent for treating obesity. [Best way] In order to achieve the above objectives, in one aspect, the present invention provides a novel peptide having an amino acid sequence of the following Formula 1: (Formula 1) 4-imidazoacetyl, β-carboxy imidazopropionyl, or tyrosine; X2 is α-methyl-glutamic acid, aminoisobutyric acid (Aib), D-alanine, glycine, Sar(N-methylglycine), serine, or D-serine; X15 is cysteine, aspartic acid, or glutamic acid; X16 is glutamic acid, aspartic acid, serine, α-methylglutamic acid, or absent; X17 is cysteine, glutamine, glutamic acid, lysine, arginine, serine, or absent; X18 is cysteine, alanine, arginine, valine, or absent; Χ19 is alanine, arginine, serine, valine, or absent; X20 is lysine, histidine, glutamine, arginine, α-methylglutamic acid, or absent; X21 is aspartic acid, glutamic acid, euccine, or absent; X23 is isoleucine, valine, or absent; X24 is arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methylglutamic acid, or absent; X27 is valine, alanine, lysine, methionine, glutamine, arginine, or absent; X28 is glutamine, lysine, asparagine, or absent; and X29 is lysine, alanine, glycine, threonine, or absent; provided that the amino acid sequence is identical to the SEC ID NO: 1 is excluded. The peptide of the present invention may include peptides, peptide derivatives, and peptide mimetics thereof, which can activate GLP-1 receptors and glucagon receptors by modifying part of the amino acids through substitution. As used herein, the term “native glucagon” refers to native human glucagon having the amino acid sequence His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-AI a-GIn-Asp-Phe-Val-GIn-Trp-Leu-Met-Asn-Thr (SEC ID NO: 1) The present invention provides the peptides defined above as derivatives of natural glucagon, and in the definition of the peptides provided in the present invention, the peptides are intended to differ from native glucagon only at the X positions of the sequences by alteration. In the Formula 1 sequence according to the present invention, the amino acids may be considered to be numbered consecutively from the first amino acid to the 29a amino acid in the conventional direction from the N-terminal to the C-terminal end. Consequently, the position description in the Formula 1 sequence should be interpreted in the same way as in the position descriptions of native human glucagon and other molecules. As used herein, the term “peptide” refers to a compound in the form in which two or more amino acids are linked by peptide bonds. For the purposes of the present invention, the peptide may refer to one that exhibits an anti-obesity effect by activating both GLP-1 receptors and glucagon receptors. Throughout the present invention, the generally permitted three-letter codes for different amino acids, such as α-aminoisobutyric acid (Aib), Sar (N-methylglycine), and α-methylglutamic acid, are used in conjunction with the conventional one-letter or three-letter codes for natural amino acids. Furthermore, the amino acids described in the present invention are abbreviated as follows according to IUPAC-IUB nomenclature. alanine (A) asparagine (N) cysteine ​​(C) arginine (R) aspartic acid (D) glutamic acid (E) glutamine (Q) glycine (G) histidine (H) leucine (L) methionine (M) proline (P) threonine (T) tyrosine (Y) isoleucine (I) lysine (K) phenylalanine (F) serine (S) tryptophan (W) valine (V) The peptide, having the amino acid sequence of Formula 1 according to the present invention, may include any peptide that can activate both glucagon receptors and GLP-1 receptors, through introduction, substitution, addition, deletion, or post-translation modification (e.g., methylation, acylation, ubiquitination, and intramolecular covalent bonding) in the amino acid sequence of glucagon described by SEC ID NO: 1. • For amino acid substitution or addition, atypical or non-natural amino acids can be used in addition to the 20 amino acids conventionally found in human proteins. Commercial suppliers of atypical amino acids include Sigma-Aldrich, ChemPep, Genzyme Pharmaceuticals, etc. Peptide sequences that include these atypical amino acids and those of typical peptides can be synthesized or purchased from commercial peptide manufacturing companies, such as American Peptide Company or Bachem (USA) or Anygen (Korea), etc. To increase the effect of the peptide of the present invention on glucagon receptors and GLP-1 receptors, in the amino acid sequence represented by SEC ID NO: 1, the first amino acid, histidine, may be substituted with 4-imidazoacetyl by deletion of the a-carbon of histidine, substituted with desamino-histidiium by deletion of the N-terminal amino group, substituted with N-dimethyl-histidyl by modification of the N-terminal amino group with two methyl groups, substituted with (3-hydroxyimidazopropionyl) by substitution of the N-terminal amino group with a hydroxyl group, substituted with β-carboxyimidazopropionyl by substitution of the N-terminal amino group with a carboxyl group, or substituted with tyrosine. Furthermore, the domain that binds to a GLP-1 receptor can be substituted with an amino acid that strengthens the hydrophobic and ionic bonds. Additionally, a partial sequence of the glucagon sequence can be replaced with the GLP-1 amino acid sequence or the exendin-4 amino acid sequence to enhance GLP-1 receptor activity. Furthermore, a partial sequence of the glucagon sequence can be substituted with a sequence that can strengthen the α-helix. Preferably, the amino acids of Formula 1 at positions 10, 14, 16, 20, 24, and 28 can be substituted with the amino acids Tyr(4-Me), Phe, Phe(4-Me), Phe(4-Cl), Phe(4-CN), Phe(4-NO2), Phe(4-NH2), Phg, Pal, Nal, Ala(2-thienyl), or Ala(benzothienyl), which are known to assist in α-helix formation, or their derivatives. The type and number of amino acids or derivatives thereof to be added for this purpose are not limited. Furthermore, preferably, at least one amino acid in at least one amino acid pair at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 of the amino acid sequence of Formula 1 can be substituted with either glutamic acid or lysine, which produces a glutamic acid and lysine pair, which can form a ring, and the number of rings for insertion is also not limited. In an exemplary embodiment, the amino acid sequence of glucagon can be substituted with a sequence that has the ability to bind to GLP-1 receptors so that the peptide can exhibit an excellent effect on GLP-1 receptors and glucagon receptors. Preferably, the peptide of the present invention may be, in the amino acid sequence of Formula 1, a peptide, wherein X1 is histidine; X2 is alpha-methylglutamic acid; X15 is cysteine ​​or aspartic acid; X16 is serine, glutamic acid, or aspartic acid; X17 is arginine, lysine, glutamic acid, or cysteine; X18 is cysteine, valine, or arginine; X19 is alanine or valine; X20 is glutamine, lysine, or histidine; X21 is aspartic acid, glutamic acid, or leucine; X23 is isoleucine or valine; X24 is arginine, glutamic acid, or glutamine; X27 is valine, lysine, or methionine; X28 is glutamine, lysine, or asparagine; and X29 is lysine, glycine, or threonine; provided that the amino acid sequence is identical to the SEC ID NO: 1 is excluded. More preferably, the peptide of the present invention may be a peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences SEC ID NOS: 2 to 14. The peptide of the present invention can be prepared by a standard synthesis procedure, a recombinant expression system, or any other procedure known in the art. Accordingly, the glucagon analogue according to the present invention can be synthesized by numerous procedures, including the following: (a) synthesizing a peptide by a step-by-step process, by means of a solid-phase or liquid-phase process, or by assembly of fragments, separating the final peptide followed by purification; (b) express a nucleic acid construct encoding the peptide in a host cell, and retrieve the expression product from the host cell culture; (c) performing an expression of a nucleic acid construct encoding the peptide within a cell-free tube, and recovering the expression product; or a procedure for obtaining fragments of a peptide by a random combination of (a), (b) and (c), connecting the fragments, thereby recovering the corresponding peptide. The present inventors confirmed through in vitro experiments that the peptide of the present invention has an excellent effect on GLP-1 receptors and glucagon receptors, compared to native glucagon (see Table 2). Furthermore, in vitro experiments confirmed that the peptide of the present invention has an excellent inhibitory effect against food intake in an obese animal model, demonstrating that the peptide of the present invention can exhibit an excellent anti-obesity effect even when administered in small amounts. Consequently, the peptide of the present invention is a dual agonist capable of stimulating cAMP formation at both GLP-1 and glucagon receptors, and is expected to have a more effective effect in the treatment of obesity compared to existing glucagon. In this respect, the peptide of the present invention may provide a more attractive option for the treatment of obesity and obesity-related diseases. The peptide of the present invention, being a dual agonist, can combine the effect of GLP-1 on food intake and the effect of glucagon on lipid metabolism, thereby acting synergistically to accelerate the elimination of lipid accumulation and the continuous reduction of body weight. This synergistic effect as a dual agonist can help reduce cardiovascular risk factors, such as high cholesterol and LDL, which may be completely independent of the effect on body weight. Consequently, the peptide of the present invention can be used as a drug to prevent weight gain, promote weight loss, reduce overweight, and treat not only obesity, including morbid obesity (e.g., through the regulation of appetite, feeding, food intake, calorie intake, and / or energy expenditure), but also obesity-related diseases, including, but not limited to, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea, and other health conditions. Furthermore, the peptide of the present invention can be used to treat medical conditions that may be associated with obesity, such as metabolic syndrome, hypertension, arteriosclerosis-inducing dyslipidemia, atherosclerosis, coronary heart disease, stroke, etc.However, in relation to these symptoms, the effect of the peptide of the present invention may be mediated completely or partially through effects related to body weight or may be independent of them. In order to improve the therapeutic effect of the glucagon derivative of the present invention, the glucagon derivative can be modified using a conventional technique in the field, such as polymer modification with polyethylene glycol (PEG), glycan, etc., or fusion with albumin, transferrin, fatty acids, immunoglobulin, etc. For example, at least one amino acid in the side chain of the compound of the present invention can be conjugated with a polymer in vivo to increase solubility, half-life, and / or bioavailability. These modifications are known to reduce the clearance of therapeutic proteins and peptides. Preferably, the polymer may be water-soluble (amphipathic or hydrophilic), non-toxic, and pharmaceutically inactive, and more preferably, may include PEG, a PEG homopolymer or copolymer, a monomethyl-substituted PEG polymer (mPEG), or a poly-amino acid such as poly-lysine, poly-aspartic acid, and poly-glutamic acid. It is obvious to those skilled in the art that glucagon derivatives modified in this way have a more excellent therapeutic effect than native glucagon. Consequently, variants of glucagon derivatives are also included within the scope of the present invention. In another aspect, the present invention provides a polynucleotide that encodes the peptide. As used in this document, the term “homology,” when used with respect to polynucleotides, refers to sequence similarity with a wild-type amino acid sequence and a wild-type nucleotide sequence, and includes gene sequences that share at least 75% with the polynucleotide sequence encoding the polypeptide, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%. These homology comparisons can be performed visually or using readily available comparison software. Commercially available computer programs can calculate homologies between two or more sequences as a percentage. Homology (%) can be calculated for neighboring sequences. The peptide can be obtained in a large quantity by inserting the polynucleotide that encodes the peptide into a vector followed by its expression. In this class of recombinant expression, the polynucleotide of the present invention is generally inserted into an appropriate vector, and forms a cloning or recombinant vector possessing the polynucleotide, and the vector is also included within the scope of the present invention. As used in this dissertation, the term recombinant vector refers to a DNA construct comprising the nucleotide sequence of a polynucleotide encoding the target peptide, which is operatively linked to an appropriate regulatory sequence capable of expressing the target peptide in a suitable host cell. The regulatory sequence may include a promoter capable of initiating transcription, an operator sequence for regulating transcription, a sequence encoding an appropriate ribosomal mRNA-binding domain, and a sequence for regulating transcriptional and translational termination. Once transformed in a suitable host cell, the recombinant vector can replicate or function independently of the host genome and can be integrated into the host genome itself. The recombinant vector used in the present invention is not particularly limited, provided it is replicable in a host cell, and can be constructed using any vector known in the art. Examples of conventional vectors that may be used include plasmids, cosmids, viruses, and wild-type or recombinant bacteriophages. For example, a phage vector or cosmid vector such as pWE15, M13, MBL3, MBL4, IXII, Ashii, APII, t10, t11, Charon4A, Charon21A, etc., may be used. As a plasmid vector, a pBR-based, pUC-based, pBluescriptl-based, pGEM-based, pTZ-based, pCL-based, or pET-based plasmid may be used. The vectors for use in the present invention are not particularly limited, but any vector known in the art may be used. The recombinant vector can be used to transform a host cell to produce the peptide of the present invention. Furthermore, as part of the present invention, the transformed cell can be used for the amplification of nucleic acid fragments or replication of the vectors of the present invention, or a cultured cell or cell line can be used for the recombinant production of the peptide of the present invention. As used in this dissertation, the term “transformation” refers to the introduction of a recombinant vector containing a polynucleotide encoding the target protein into a host cell so that the target protein encoded by the polynucleotide can be expressed in the host cell. It does not matter whether the polynucleotide is inserted within or outside the chromosome, as long as the transformed polynucleotide can be expressed in the host cell. Furthermore, the polynucleotide includes DNA and RNA, which encode the target protein. The polynucleotide can be introduced in any form, provided it can be expressed after being introduced into a host cell. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genomic structure that includes all the essential features required for self-expression. The expression cassette typically includes a promoter, which is operatively linked to the polynucleotide, a transcription termination signal, a ribosome-binding domain, and a translation termination signal. The expression cassette can be a self-replicating expression vector. Additionally, the polynucleotide itself can be inserted into a host cell and operatively linked to a sequence required for its expression in the host cell, but this is not limited to this. Furthermore, as used in this document, the term operationally bound refers to a state in which a promoter sequence, which initiates and mediates the transcription of the polynucleotide encoding the target protein, and is functionally linked to the gene sequence. The host cell suitable for the present invention is not particularly limited, provided that the host cell can express the polynucleotide of the present invention. Examples of host cells for use in the present invention may include Escherichia sp. such as E. coli; Bacillus sp. such as Bacillus subtilis; Pseudomonas sp. 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, BSC, etc. In another aspect, the present invention provides a pharmaceutical composition for preventing or treating obesity that contains the peptide as an active ingredient. As used herein, the term “prevention” refers to any action that produces the suppression or delay of the onset of obesity by the administration of the peptide or pharmaceutical composition of the present invention, and the term treatment refers to any measure that produces an improvement in the symptoms of obesity or a beneficial alteration by the administration of the peptide or pharmaceutical composition of the present invention. As used herein, the term “administration” refers to the introduction of a particular substance into a patient in an appropriate manner. The route of administration of the pharmaceutical composition of the present invention, although not particularly limited, may be any of the common routes, provided that the pharmaceutical composition can reach the target tissue in the body, for example, by intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, etc. X As used in this document, the term “obesity” refers to a medical condition in which excess body fat has accumulated, and people are considered obese when their body mass index (BMI; a measurement obtained by dividing a person’s weight in kilograms by the square of their height in meters) is 25 or higher. Obesity is generally induced by an energy imbalance because calorie intake is greater than energy expenditure. Obesity is a metabolic disease that can induce diabetes and hyperlipidemia, increase the risk of sexual dysfunction, arthritis, and cardiovascular disease, and in some cases, is also associated with the development of cancer. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient, or diluent. As used in this document, the term pharmaceutically acceptable refers to a sufficient quantity that can exhibit a therapeutic effect but not incur any adverse reactions, and can be easily determined by experts in the art according to factors known in the medical field, such as the type of diseases to be treated, age, weight, sex of the patient, sensitivity to drugs, routes of administration, number of administrations, drugs to be combined or used concurrently, etc. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a coloring agent, and a fragrance. For injectable administration, a buffering agent, a preservative, an analgesic, a solubilizer, an isotonic agent, and a stabilizer may be mixed together for use. For topical administration, the pharmaceutically acceptable carrier may include a base, an excipient, a lubricant, a preservative, etc. The pharmaceutical composition of the present invention can be formulated in various dosage forms in combination with a pharmaceutically acceptable carrier. For example, for oral administration, the pharmaceutical composition can be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injectable administration, the pharmaceutical composition can be formulated in an ampoule as a unit-dose form or for multiple-dose administration. The pharmaceutical composition can also be formulated as solutions, suspensions, tablets, lozenges, capsules, and extended-release preparations. On the other hand, examples of suitable vehicle, excipient, and diluent for the pharmaceutical composition of the present invention may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oils, etc. Furthermore, the pharmaceutical composition of the present invention may also include fillers, anticoagulants, lubricants, humectants, perfumes, antiseptics, etc. Additionally, the pharmaceutical composition can be formulated in one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid medicine for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations, and suppositories. Additionally, the pharmaceutical composition can be formulated in a formulation suitable for unit administration into a patient's body according to the conventional procedure, preferably in a formulation type useful for the administration of a peptide drug, and is administered orally or parenterally by means of subcutaneous, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intrapulmonary, intradermal, subcutaneous, intraperitoneal, intranasal, local intragastric, sublingual, intravaginal or intrarectal injection according to the conventional procedure, but is not limited to them. Additionally, the peptide can be used by mixing it with various vehicles, such as saline solution or organic solvents, which are accepted as drugs. To increase stability or absorption capacity, the peptide can be used in conjunction with carbohydrates such as glucose, sucrose, or dextran, or antioxidants such as glutathione, chelating agents, low molecular weight proteins, or other stabilizers, etc. The quantity and number of administrations of the pharmaceutical composition can be determined according to the types of drugs as active ingredients, along with other factors of the present invention, such as the diseases to be treated, routes of administration, age, sex, weight of the patient, severity of the disease, etc. Yo The total effective dose of the composition of the present invention can be administered to a patient as a single dose or as multiple doses over a long period according to a fractionated treatment protocol. The pharmaceutical composition of the present invention may have a different content of the active ingredient depending on the severity of the disease. Preferably, the total dose of the peptide of the present invention can be from approximately 0.0001 pg to 500 mg per 1 kg of the patient's body weight. However, with respect to the peptide dose, the effective dose is determined by taking into consideration various factors such as the patient's age, weight, health conditions, sex, severity of the disease, diet, and excretion rate, etc. Those skilled in the art can determine the appropriate effective dose according to the particular use of the composition of the present invention. The formulations, routes of administration, and procedures for administering the pharmaceutical composition of the present invention may not be particularly limited, provided that the pharmaceutical composition can exhibit the effect of the present invention. Because the pharmaceutical composition of the present invention has an excellent duration and in vivo titer, the number and frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced. The pharmaceutical composition may be administered alone or in combination with other pharmaceutical formulations that exhibit a preventive or therapeutic effect against obesity. The pharmaceutical formulation exhibiting a preventive or therapeutic effect against obesity may include, but is not limited to, a GLP-1 receptor agonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a methicillin-concentrating hormone (MCH) receptor antagonist, a Y2 / 3 receptor agonist, an MC3 / 4 receptor agonist, a gastric / pancreatic lipase inhibitor, a 5HT2c agonist, a 3A receptor agonist, an amylin receptor agonist, a ghrelin antagonist, and / or a ghrelin receptor antagonist, etc. In another aspect, the present invention provides the procedure for preventing or treating obesity that includes administering the peptide or a pharmaceutical composition containing the peptide to a subject. As used herein, the term "subject" refers to a subject suspected of having obesity or at risk of developing obesity, and specifically, to mammals, including humans, rats, and cattle, but the subject may be any object treatable with the peptide of the present invention, without limitation. Administration of a pharmaceutical composition containing the peptide of the present invention can effectively treat a subject suspected of having obesity, and the obesity is the same as described above. The therapeutic procedure of the present invention may include the administration of a pharmaceutically effective amount of the peptide-containing pharmaceutical composition. The total daily dose of the composition may be determined by a physician using appropriate medical judgment, and the composition may be administered once or in a few divided doses.However, in view of the purpose of the present invention, the therapeutically effective dose of the composition for any particular patient may vary according to several factors well known in the medical field, including the class and degree of responses to be achieved, specific compositions according to whether or not other agents are used with the same, the patient's age, body weight, health conditions, sex and diet, time and route of administration, the discharge rate of the composition, the duration of treatment, other drugs used in combination or simultaneously with the composition of the present invention, and other factors known in the medical field. In yet another aspect, the present invention provides a use of the peptide to prepare a drug to prevent or treat obesity. [Mode for the invention] From this point forward, the present invention will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples. Example 1: Production of cell lines for in vivo activation <1-1 > Production of cell lines that show cAMP response to GLP-1 The PCR reaction was performed using the open reading frame (ORF) cDNA (OriGene Technologies, Inc., USA.) of a human GLP-1 receptor gene as a template along with the forward and reverse primers represented by the SEC ID NOS: 15 and 16, which include the restriction sites for Hindlll and EcoRI, respectively. Specifically, the PCR reaction (denaturation at 95 °C for 60 seconds, annealing at 55 °C for 60 seconds, and elongation at °C for 30 seconds) was performed for 30 cycles. The PCR product was subjected to electrophoresis on a 1.0% agarose gel, and a 405 bp fragment was obtained by elution. Direct primer: 5'-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3' Reverse primer: 5'-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3' The PCR product was cloned into a known animal cell expression vector, xOGC / dhfr (Korean Patent No. 10-0880509, the same hereafter), to construct a recombinant vector, xOGC / GLPIR. The recombinant vector constructed in this way, xOGC / GLP1R, was transformed into CHO DG44 Chinese hamster ovary cell line cells, which were cultured in DMEM / F12 medium containing 10% FBS using Lipofectamine (Invitrogene, USA), and selected and cultured in a selective medium containing G418 (1 mg / ml) and Methotrexate (10 nM). Monoclone cell lines were selected from these, and from among them, cell lines showing excellent cAMP responses to GLP-1 in a dose-dependent manner were finally selected. <1-2> Production of cell lines that show cAMP response to glucagon A PCR reaction was performed using the cDNA open reading frame (ORF) (OriGene Technologies, Inc., USA) of a human glucagon receptor gene as a template, along with the forward and reverse primers represented by SEC ID NOSr: 17 and 18, which include restriction sites for EcoRI and Xhol, respectively. Specifically, the PCR reaction (denaturation at 95 °C for 60 seconds, annealing at 55 °C for 60 seconds, and elongation at 68 °C for 30 seconds) was performed for 30 cycles. The PCR product was subjected to electrophoresis on a 1.0% agarose gel, and a 435 bp fragment was obtained by elution. Direct primer 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' Reverse primer 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3' The PCR product was cloned into the known animal cell expression vector, xOGC / dhfr, to construct a recombinant vector, xOGC / GCCR. The recombinant vector constructed in this way, xOGC / GCCR, was transformed into Chinese hamster ovary cell line CHO DG44 cells, which were cultured in DMEM / F12 medium containing 10% FBS, using Lipofectamine (Invitrogene, USA), and selected and cultured in a selective medium containing G418 (1 mg / ml) and Methotrexate (10 nM). Monoclone cell lines were selected from these, and among them, cell lines showing excellent cAMP responses to GLP-1 in a dose-dependent manner were finally selected. Example 2: Synthesis of glucagon derivatives In order to develop a glucagon derivative that has an excellent effect on GLP-1 receptors and glucagon receptors, the amino acid sequence of native glucagon represented by SEC ID NO: 1 was replaced with an amino acid sequence that has an ability to bind to GLP-1 receptors, and glucagon derivatives were synthesized as shown in Table 1 below. [Table 1] SEQ ID NO Amino acid sequence SEQ ID NO: 1 HSQGTFTSDYSKILDSRRAQDFVQWLMNT SEQ ID NO: 2 HXQGTFTSDYSKILDEKCAKEFIQWLVNT SEQ ID NO: 3 HXQGTFTSDYSKILDEKCVKLFIQWLVNT SEC ID NO: 4 HXQGTFTSDYSKILDEKCAKEFVEWLVNT SEQ ID NO: 5 HXQGTFTSDYSKILDEKCAHEFVEWLVNT SEC ID NO: 6 HXQGTFTSDYSKILDSKCAHEFVEWLVNT SEC ID NO: 7 HXQGTFTSDYSKILDSKCVHEFIEWLKNT SEC ID NO: 8 HXQGTFTSDYSKILDSKCAHEFIEWLKNK SEC ID NO: 9 HXQGTFTSDYSKILDSECAH EFI EWLKQG SEQ ID NO: 10 HXQGTFTSDYSKILDDKCAHEFVEWLVNT SEC ID NO: 11 HXQGTFTSDYSKILDEECAKEFIRWLKKG SEC ID NO: 12 HXQGTFTSDYSKILCEKRAKEFVQWLMNT SEC ID NO: 13 HXQGTFTSDYSKILDECRAKEFVQWLMNT SEC ID NO: 14 HXQGTFTSDYSKILDEKCAKEFVQWLMNT In Table 1 above, the amino acid indicated as “X” in the sequences of SEC ID NOS: 2 to 14 represents α-methyl-glutamic acid, which is a non-native amino acid, and the lysine residue in these sequences can form a ring with a glutamic acid residue. Example 3: In vitro measurement of glucagon derivatives activity In order to measure the anti-obesity activities of the glucagon derivatives synthesized in Example 2, the in vitro cellular activities of the glucagon derivatives were measured using the transformed cell lines prepared in Examples 1-1 and 1-2. The transformed cell lines were prepared such that the human GLP-1 receptor gene and the human glucagon receptor gene can be expressed in CHO cells, respectively, and are suitable for measuring GLP-1 and glucagon activities. Accordingly, the activities of the glucagon derivatives synthesized according to the present invention were measured using the transformed cell lines, respectively. Specifically, the transformed cell lines were subcultured two or three times per week, each aliquoted into a 96-well plate with 1 x 105 cells / well, and cultured for 24 hours, respectively. The cultured cells were washed with Krebs-Ringer bicarbonate buffer (KRB) solution, suspended in 40 ml of KRB buffer solution containing 1 mM 3-isobutyl-1-methylxanthine (IBMX), and placed at room temperature for 5 minutes. Native glucagon (SEC ID NO: 1) or glucagon derivatives (representatively, peptides of SEC ID NOS: 12 to 14) according to the present invention were subjected to serial dilution at 5-fold intervals ranging from 1,000 nM to 0.02 nM, 40 µL of the above cells were added thereto, and cultured in a CO2 incubator at 37 °C for 1 hour. Next, 20 ml of cell lysis buffer were added to the respective samples, and the cell lysates were applied to a cAMP assay kit (Molecular Device, USA) to measure the cAMP concentration, and EC50 values ​​were calculated and compared. The results are shown in Table 2 below. [Table 2] Test Material EC50 (nM) hGLP-IR hGCGR GLP-1 0.36 >1,000 glucagon >1,000 1.48 SEC ID NO: 12 0.96 1.38 SEC ID NO: 13 0.27 0.23 SEC ID NO: 14 0.17 0.38 As shown in Table 2 above, the glucagon derivatives according to the present invention showed excellent effects on GLP-1 receptors and glucagon receptors, compared to natural glucagon represented by SEC ID NO: 1. Glucagon is known to have a therapeutic effect on obesity by activating GLP-1 and glucagon receptors, thereby suppressing appetite, improving satiety, and promoting fat cell lysis. Since glucagon derivatives according to the present invention have been shown to have excellent in vitro effects on GLP-1 and glucagon receptors compared to natural glucagon, these glucagon derivatives can be used as a more effective agent for the treatment of obesity than existing glucagon. Although preferred embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions, and substitutions are possible without departing from the scope and spirit of the invention as described in the appended claims.

Claims

1. An isolated peptide, characterized in that it comprises an amino acid sequence selected from the group consisting of the amino acid sequences SEC ID NOS: 12 to 14. 4 Claims follow