Pharmaceutical composition comprising polypeptide

Abstract

To provide polypeptide-containing pharmaceutical compositions.SOLUTION: The invention relates to a pharmaceutical composition comprising a polypeptide, more specifically, a pharmaceutical composition for preventing or treating obesity, diabetes or non-alcoholic fatty liver disease. Th pharmaceutical composition is safe without any side effects such as nausea or vomiting with effects of reducing food intake, enhancing insulin secretion, suppressing gastric emptying, promoting lipolysis, and lowering a level of triglycerides. The invention relates to a polypeptide-containing pharmaceutical composition, medical use of the polypeptide-containing pharmaceutical composition, for example, use in treatment or prevention of obesity, diabetes or non-alcoholic fatty liver disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions containing polypeptides and their medical uses, such as in the treatment or prevention of obesity, diabetes, or non-alcoholic fatty liver disease. The polypeptides have the effects of reducing food intake, improving insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels, without causing side effects such as vomiting or nausea. [Background technology]

[0002] In recent years, with the development of the economy and rapid growth in science and technology, the elderly population has increased and adult diseases have skyrocketed. This is due to stress, poor eating habits, excessive calorie intake, and decreased physical activity. Cardiovascular disease and cerebrovascular disease, which are complications associated with obesity, are the first and second leading causes of death, and obesity has been suggested to be a cause of various adult diseases such as diabetes and non-alcoholic fatty liver disease.

[0003] Obesity refers to the accumulation of more than normal amounts of fat, and the most accurate way to assess obesity is to measure body fat mass. However, accurate measurement of fat mass is expensive, so it is assessed using indirect methods. The most commonly used indirect methods are measuring body mass index (BMI) and waist circumference. The World Health Organization (WHO) has published a classification based on data relating BMI to mortality risk. This is: normal weight: 18.5-24.9 kg / m 2 , Overweight: 25~29.9kg / m 2 , and obesity: 30 kg / m 2 or more body weight, based on

[0004] The cause of obesity is known to be an energy imbalance caused by excessive calorie intake and relatively reduced activity, resulting in increased body fat. However, because obesity is associated with various risk factors, such as dietary habits, lifestyle, age, race, and genetic factors, it is difficult to attribute it to a single factor.

[0005] Diabetes mellitus is classified into insulin-dependent diabetes mellitus (type I diabetes), non-insulin-dependent diabetes mellitus (type II diabetes), and malnutrition-related diabetes mellitus (MRDM). Type II diabetes, the primary cause of more than 90% of diabetic patients, is a metabolic disease characterized by hyperglycemia. It is reported to be caused by reduced insulin secretion from pancreatic beta cells or increased insulin resistance in peripheral tissues due to genetic, metabolic, and environmental factors. In this regard, it is known that increased body fat reduces insulin sensitivity, and abdominal fat accumulation in particular is associated with impaired glucose tolerance. Insulin resistance is also closely related to obesity in patients with type II diabetes, and the more severe the obesity, the greater the insulin resistance.

[0006] Nonalcoholic fatty liver disease (NAFLD) refers to a range of diseases, including simple steatosis, where excess fat accumulates in hepatocytes regardless of alcohol consumption, nonalcoholic steatohepatitis (NASH), which involves hepatocellular damage (hepatocellular ballooning), inflammation, fibrosis, and, in more advanced cases, cirrhosis. The prevalence of NAFLD is rapidly increasing along with the increasing prevalence of obesity worldwide. The prevalence of diabetes varies by country, but accounts for approximately 20-30% of the total population in Western countries, and its prevalence reaches approximately 16% in Korea.

[0007] Non-alcoholic fatty liver disease (NAFLD) is closely related to metabolic syndrome, including obesity, type II diabetes, dyslipidemia, and similar diseases based on insulin resistance. In fact, many prediabetic and type II diabetic patients have been shown to have NAFLD / NAFH, and these patients are known to have a high rate of progression to cirrhosis and liver cancer (i.e., hepatocellular carcinoma). Meanwhile, the prevalence of diabetes is high in patients with NAFLD, which is evident in patients with NAFLD.

[0008] Obese patients are primarily advised to control their weight through healthier diets and physical activity, but if these methods are ineffective, patients may be treated with medication or surgery.

[0009] The current market for anti-obesity drugs is estimated to be worth over $1 billion, growing at approximately 10% annually. The main drugs used as anti-obesity drugs are psychotropic appetite-reducing drugs (such as lorcaserin and phentermine), most of which act on the central nervous system and are known to suppress patients' appetites and help them lose weight, but long-term use can lead to side effects such as abuse and addiction, palpitations, anxiety, and insomnia.

[0010] Xenical is a non-psychotropic anti-obesity drug. Pancreatic lipase is a key enzyme responsible for the breakdown of triglycerides into 2-monoacylglycerol and fatty acids. A representative pancreatic lipase inhibitor is tetrahydrolipstatin (orlistat), a derivative of lipstatin derived from Streptomyces toxitricini. It has high efficacy in blocking the absorption of approximately 30% of ingested fat. While tetrahydrolipstatin (orlistat) is currently available as a drug, it has side effects such as gastrointestinal disorders, anaphylaxis, and cholestasis. Therefore, few therapeutic agents are safe for use in obese patients.

[0011] In the drug therapy for treating non-alcoholic fatty liver disease, drug therapy acts on the mechanisms that aggravate non-alcoholic fatty liver disease, such as insulin resistance, oxidative stress, apoptosis, inflammatory cytokines, etc., and suppresses the progression of non-alcoholic fatty liver disease.Among these, antidiabetic drugs are known to improve the general pathophysiological conditions for the development of fatty liver, thereby improving fatty liver and lowering blood glucose.However, up to now, there is no drug approved for the treatment of fatty liver disease, so there is still an unmet medical need for the development of effective therapeutic agents.

[0012] On the other hand, glucagon derivatives are currently attracting attention. Glucagon is produced in the pancreas when blood glucose levels begin to drop due to drug treatment, disease, hormone or enzyme deficiency, etc. Glucagon stimulates the liver to release glucose by breaking down glycogen, thereby raising blood glucose levels to normal levels. In addition to its blood glucose-raising effect, glucagon has also been reported to suppress appetite and activate hormone-sensitive lipase in adipocytes to promote fat breakdown, thereby exerting anti-obesity effects. One such glucagon derivative, glucagon-like peptide-1 (GLP-1), is still under development as a therapeutic agent for alleviating hyperglycemia in diabetic patients. It functions by promoting insulin synthesis and secretion, inhibiting glucagon secretion, suppressing gastric emptying, promoting glucose utilization, and suppressing food intake. Furthermore, exendin-4, derived from lizard venom and sharing approximately 50% amino acid identity with GLP-1, is known to activate the GLP-1 receptor and alleviate hyperglycemia in diabetic patients. However, GLP-1 receptor agonists for treating obesity or diabetes have been reported to cause side effects such as vomiting and nausea.

[0013] Oxyntomodulin, which can bind to both the GLP-1 and glucagon receptors, has emerged as a potential alternative to GLP-1. Oxyntomodulin, a peptide derived from the glucagon precursor (i.e., preglucagon), suppresses GLP-1-mediated food intake, regulates blood glucose levels by inhibiting hepatic gluconeogenesis, improves satiety, and possesses the lipolytic functions of glucagon. Therefore, oxyntomodulin has great potential as an antidiabetic and antiobesity drug.

[0014] Based on the dual functions of oxyntomodulin peptides, active research is being conducted to develop drugs for treating diabetes and obesity. For example, Korean Patent No. 925017 discloses a pharmaceutical composition for oral, parenteral, mucosal, rectal, subcutaneous, or transdermal administration for treating human overweight, which contains oxyntomodulin as an active ingredient. However, it has been reported that anti-obesity drugs containing oxyntomodulin have a short half-life in vivo and exhibit low therapeutic effects on obesity even when administered at high doses three times a day.

[0015] Meanwhile, efforts are underway to overcome the short in vivo half-life of therapeutic peptides by maintaining a high level of pharmacological efficacy for a long period of time and thereby maximizing the efficacy of therapeutic drugs. U.S. Patent No. 7,141,547 discloses a fusion protein of GLP-1 and its analogs with albumin using recombinant DNA technology, and U.S. Patent No. 8,273,854 discloses a fusion protein of GLP-1 and its analogs with immunoglobulin fragments (Fc). While these technologies partially address the short in vivo half-life of peptides, they do not eliminate the immunogenicity-related issues caused by the administration of proteins that are not native to the human body. As a result, this technology has the disadvantage that the pharmacological efficacy of drugs may decrease with long-term administration. Drug production also poses the additional problem of requiring large-scale cell culture and purification systems. Furthermore, due to the nature of recombinant proteins, drugs may contain impurities derived from host cells, which may not be completely identical in each batch, making it difficult to control the quality of the drug. In addition, when using peptides with disulfide bonds, such as calcitonin, there is a drawback in that the yield may be reduced due to misfolding.Furthermore, when drugs contain unnatural amino acid residues, it is difficult to produce the drugs using recombinant protein production methods.

[0016] On the other hand, U.S. Patent No. 8,110,665 discloses a peptide whose short half-life is improved by preparing a conjugate using a nonpeptidic polymer and an immunoglobulin fragment (Fc). However, this patent describes a complicated manufacturing process that involves separately producing the biologically active peptide, the nonpeptidic polymer, and the immunoglobulin fragment, and then combining the peptide, polymer, and immunoglobulin fragment together, which results in problems such as residual by-products and reduced yield.

[0017] PEGylation of therapeutic peptides and proteins is the most powerful pharmaceutical technology for improving their in vivo half-life. PEGylation of peptides and proteins increases their molecular weight, protects against proteolytic degradation, and masks immunogenic sites, thereby increasing the drug's in vivo half-life and reducing the immunogenicity of peptides and proteins. Therefore, PEGylation technology is effective in enhancing the therapeutic efficacy of peptide drugs by resolving the problems associated with peptide drugs. Due to these advantages, PEGylation of peptides and proteins plays an important role in enhancing the therapeutic efficacy of drug delivery systems.

[0018] However, the PEG-based method has the drawbacks of reducing the activity of peptide drugs and resulting in low yields due to the poor reactivity of PEG with peptides of increasing molecular weight. In this regard, a PEGylation method with a simple manufacturing process and high selectivity is needed.

[0019] Therefore, there is a need for a therapeutic agent for treating obesity, diabetes, or non-alcoholic fatty liver disease that has the effects of reducing food intake, improving insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels, without causing side effects such as vomiting or nausea, and that can be obtained in high yield by optimizing the preparation method.

[0020] [Prior art document]

[0021] [Patent documents]

[0022] Patent Document 1: Korean Patent No. 0925017, entitled "Oxyntomodulin for "Preventing or Treating Excess Weight"

[0023] Patent Document 2: U.S. Patent No. 7,141,547, entitled "Albumin Fusion Proteins Comprising GLP-1 Polypeptides"

[0024] Patent Document 3: U.S. Patent No. 8,273,854, entitled "GLP-1 Analog Fusion Proteins"

[0025] Patent Document 4: U.S. Patent No. 8,110,665, entitled "Pharmaceutical Composition Comprising an Immunoglobulin FC Region as a Carrier"

[0026] Patent Document 5: Korean Patent No. 1665009, titled "Pharmaceutical Composition for Preventing or Treating Non-alcoholic Fatty Liver Diseases" Summary of the Invention [Problem to be solved by the invention]

[0027] In order to solve the above problems, the present inventors have conducted research and efforts to develop a therapeutic agent for treating obesity, diabetes, or non-alcoholic fatty liver disease that is safe, has no side effects such as vomiting or nausea, and has the effects of reducing food intake, improving insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels, and have also developed a method for preparing the therapeutic agent in high yield, preparing a polypeptide having the amino acid sequence represented by the following general formula 1. As a result, the present inventors have confirmed that a composition containing the polypeptide has an excellent effect in preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease, and that a site-specific conjugate of a polypeptide and a non-peptidic polymer has an excellent effect in preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease by increasing the blood half-life of the polypeptide while maintaining the in vivo activity of the polypeptide, thereby completing the present invention.

[0028] [General formula 1]

[0029] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0030] wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

[0031] X1 is deletion, glycine, or aminoisobutyric acid (Aib);

[0032] R2 is EKRAK, EQAAK, or EEAVK; and

[0033] R3 is a deletion, cysteine, lysine, or methionine).

[0034] Therefore, it is an object of the present invention to provide a pharmaceutical composition comprising a polypeptide for preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease. [Means for solving the problem]

[0035] In order to solve the above problems, a pharmaceutical composition according to one exemplary embodiment of the present invention comprises a polypeptide having an amino acid sequence represented by the following general formula 1:

[0036] [General formula 1]

[0037] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0038] (wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxy-imidazo-propionyl, 4-imidazoacetyl, or β-carboxy-imidazo-propionyl; X1 is a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK, EQAAK, or EEAVK; and R3 is a deletion, cysteine, lysine, or methionine).

[0039] The polypeptide may be covalently bound to any one or more selected from the group consisting of non-peptidic polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn-binding substances, saccharides, elastin, heparin, and derivatives thereof, or may form a microsphere.

[0040] R2 includes glutamic acid (E) and lysine (K), which together may form a ring via an amide bond that may contribute to the alpha-helical structure of the polypeptide.

[0041] The non-peptidic polymer may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Derivatives of non-peptidic polymers known in the relevant art and other derivatives that can be readily prepared by those skilled in the art are also within the scope of the present invention.

[0042] Preferably, the non-peptidic polymer may be polyethylene glycol or a derivative thereof.

[0043] The molecular weight of the non-peptidic polymer may be 3,000 to 100,000 Da.

[0044] In this case, the polyethylene glycol derivatives are methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidyl propionate (PEG succinimidyl propionate), methoxypolyethylene glycol succinimidyl propionate (methoxyPEG succinimidyl propionate), acrylate polyethylene glycol succinimidyl propionate (acrylate PEG succinimidyl propionate), thiol polyethylene glycol succinimidyl propionate (thiol PEG succinimidyl propionate), hydroxypropyl polyethylene glycol, ... The copolymer may be at least one selected from the group consisting of roxysuccinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of the derivatives thereof.

[0045] The polyethylene glycol or derivative thereof may be linear or branched.

[0046] The pharmaceutical composition can be used to prevent or treat one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0047] Non-alcoholic fatty liver disease can include one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer.

[0048] A method for preparing a pharmaceutical composition according to another exemplary embodiment of the present invention includes mixing and reacting a non-peptidic polymer with a polypeptide having an amino acid sequence represented by the following general formula 1:

[0049] [General formula 1]

[0050] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0051] (wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxy-imidazo-propionyl, 4-imidazoacetyl, or β-carboxy-imidazo-propionyl; X1 is a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK, EQAAK, or EEAVK; and R3 is a deletion, cysteine, lysine, or methionine).

[0052] The non-peptidic polymer may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Derivatives of non-peptidic polymers known in the relevant art and other derivatives that can be readily prepared by those skilled in the art are also within the scope of the present invention.

[0053] Preferably, the non-peptidic polymer may be polyethylene glycol or a derivative thereof.

[0054] In this case, the polyethylene glycol derivatives are methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidyl propionate (PEG succinimidyl propionate), methoxypolyethylene glycol succinimidyl propionate (methoxyPEG succinimidyl propionate), acrylate polyethylene glycol succinimidyl propionate (acrylate PEG succinimidyl propionate), thiol polyethylene glycol succinimidyl propionate (thiol PEG succinimidyl propionate), hydroxypropyl polyethylene glycol, ... The copolymer may be at least one selected from the group consisting of roxysuccinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of the derivatives thereof.

[0055] Mixing the non-peptidyl polymer and the polypeptide to react with each other may involve reacting the polypeptide and the non-peptidyl polymer in a molar ratio of 1:1 to 1:5.

[0056] The non-peptidyl polymer and the polypeptide may be mixed at a pH of 4.0 to 9.0 to react with each other.

[0057] In mixing the non-peptidyl polymer and the polypeptide to react with each other, the reaction time may be in the range of 0.5 to 24 hours.

[0058] The pharmaceutical composition can be used to prevent or treat one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0059] Non-alcoholic fatty liver disease can include one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer.

[0060] According to another exemplary embodiment of the present invention, a method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease includes administering a pharmaceutical composition to a subject. [Effects of the Invention]

[0061] The pharmaceutical composition according to the present invention may contain a polypeptide, and therefore has the effects of reducing food intake, improving insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels.

[0062] Furthermore, since the pharmaceutical composition according to the present invention can contain a polypeptide, side effects such as vomiting or nausea can be reduced.

[0063] Furthermore, the pharmaceutical compositions according to the present invention can be prepared in high yields because they can contain non-peptidic polymers that are highly selective and reactive with polypeptides.

[0064] Furthermore, the pharmaceutical composition according to the present invention may contain a conjugate comprising a polypeptide and a non-peptidic polymer, which may have a longer in vivo half-life and may have a high therapeutic effect on obesity even when administered at a low dose, and may also have the effect of lowering blood glucose levels so that blood glucose can be maintained at normal levels, and the effect of effectively lowering triglyceride levels. [Brief explanation of the drawings]

[0065] [Figure 1] FIG. 1 shows the HPLC results of the conjugate of Example 2, which comprises a polypeptide and a non-peptidic polymer.

[0066] [Figure 2] FIG. 2 shows the MALDI-TOF results of the conjugate of Example 2, which comprises a polypeptide and a non-peptidic polymer.

[0067] [Figure 3] FIG. 3 shows the results of measuring glycated hemoglobin levels (HbA1c) after the end of treatment with the conjugate of Example 2 to determine the extent of change in long-term average blood glucose concentrations (**p<0.01).

[0068] [Figure 4] FIG. 4 shows the results of the final body weight of the mice after treatment with the conjugate of Example 2 for 2 weeks at different dosing frequencies (***p<0.001).

[0069] [Figure 5] FIG. 5 shows the results of the change in blood glucose in mice after administration of the conjugates of Examples 2 or 6.

[0070] [Figure 6] FIG. 6 shows the results of an intraperitoneal glucose tolerance test (ipGTT) after administration of the conjugates of Examples 2 or 6.

[0071] [Figure 7] FIG. 7 shows the results of measuring the change in serum cholesterol after administration of the conjugate of Example 2.

[0072] [Figure 8] FIG. 8 shows the results of measuring the change in liver weight after administration of the conjugate of Example 2.

[0073] [Figure 9]FIG. 9 shows the results of observing the liver tissue of mice after administration of the conjugate of Example 2 (darkly stained areas represent normal liver tissue, and white (brightly) stained areas represent lipid droplets).

[0074] [Figure 10] FIG. 10 shows the results of measuring the change in serum cholesterol after administration of the conjugate of Example 2.

[0075] [Figure 11] FIG. 11 shows the results of measuring the change in liver weight after administration of the conjugate of Example 2.

[0076] [Figure 12] FIG. 12 shows the results of measuring the change in liver triglyceride levels after administration of the conjugate of Example 2.

[0077] [Figure 13] FIG. 13 shows the results of observing the liver tissue of mice after administration of the conjugate of Example 2 (darkly stained areas represent normal liver tissue, and white (brightly) stained areas represent lipid droplets).

[0078] [Figure 14] FIG. 14 shows the results of measuring the NAFLD activity score (NAS) after administration of the conjugate of Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention provides a polypeptide having an amino acid sequence represented by the following general formula 1:

[0080] [General formula 1]

[0081] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0082] wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

[0083] X1 is deletion, glycine, or aminoisobutyric acid (Aib);

[0084] R2 is EKRAK, EQAAK, or EEAVK; and

[0085] R3 is a deletion, cysteine, lysine, or methionine).

[0086] The present invention provides a polypeptide having an amino acid sequence represented by the following general formula 1 for use in the prevention or treatment of a disease selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease:

[0087] [General formula 1]

[0088] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0089] wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

[0090] X1 is deletion, glycine, or aminoisobutyric acid (Aib);

[0091] R2 is EKRAK, EQAAK, or EEAVK; and

[0092] R3 is a deletion, cysteine, lysine, or methionine).

[0093] The present invention provides a pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the following general formula 1:

[0094] [General formula 1]

[0095] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3,

[0096] wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

[0097] X1 is deletion, glycine, or aminoisobutyric acid (Aib);

[0098] R2 is EKRAK, EQAAK, or EEAVK; and

[0099] R3 is a deletion, cysteine, lysine, or methionine).

[0100] The present invention provides a pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the above general formula 1 for preventing or treating a disease selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0101] Amino acids referred to herein are abbreviated according to the IUPAC-IUB nomenclature rules as set out in Table 1 below.

[0102] [Table 1]

[0103] In general formula 1, R1 is preferably histidine at the N-terminus of the polypeptide, but the present invention is not limited thereto.

[0104] X1 is preferably glycine or Aib, more preferably Aib, in which case X1 is not particularly limited as long as it can increase the chemical stability of the polypeptide.

[0105] X1 is also preferred as long as it is resistant to dipeptidyl peptidase-4 (DPP-4), thereby increasing the enzyme stability.

[0106] R2 is preferably EQAAK or EEAVK, more preferably EQAAK, although the present invention is not limited thereto.

[0107] R2 includes glutamic acid (E) and lysine (K), which preferably form a ring together via an amide bond, but the present invention is not limited thereto. Therefore, when two residues in the amino acid sequence of a polypeptide form a covalent ring via an amide bond, the covalent ring can increase in vivo stability and improve the ability to bind to glucagon receptors or glucagon derivative receptors. The covalent ring can also contribute to the α-helical structure of the polypeptide.

[0108] R3 is the C-terminus of the polypeptide to which a substance may be attached to improve in vivo half-life or in vivo sustainability, in which case R3 is preferably cysteine, although the present invention is not limited thereto.

[0109] The polypeptide may have 70% to 90% sequence homology to the amino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 1: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT).

[0110] The amino acid sequence of SEQ ID NO: 1 is partially or entirely identical to the amino acid sequence of native glucagon, which has been reported to promote the degradation of glycogen and insulin and to have anti-obesity effects. However, native glucagon has low solubility and precipitates at neutral pH, limiting its use as a therapeutic agent.

[0111] That is, a polypeptide comprising an amino acid sequence having 70% to 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 can be a glucagon derivative or an oxyntomodulin derivative. In this case, the oxyntomodulin derivative is a peptide produced from a glucagon precursor (e.g., preglucagon).

[0112] Preferably, the polypeptide has 73% to 90%, more preferably 75% to 90% sequence homology to the amino acid sequence set forth in SEQ ID NO: 1, although the present invention is not limited thereto.

[0113] As used herein, the term "homology" refers to the degree of similarity between wild-type amino acid sequences and wild-type nucleic acid sequences. In this case, homology comparison between these sequences is performed using available comparison programs. The homology between two or more sequences can be calculated as a percentage (%) using commercially available computer programs. The homology (%) can be calculated for adjacent sequences. A polynucleotide encoding a peptide can be inserted into a vector and the peptide can be expressed to obtain a large amount of the peptide.

[0114] As used herein, the term "peptide" refers to a compound in which two or more α-amino acids are joined via a peptide bond.

[0115] Alternatively, the polypeptide may be covalently bound to one or more selected from the group consisting of non-peptidic polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn-binding substances, saccharides, elastin, heparin, and derivatives thereof, or may form a microsphere.

[0116] Preferably, the non-peptidic polymer is covalently attached to the polypeptide, although the invention is not so limited.

[0117] The polypeptides are covalently bonded to the aforementioned substances or form microspheres, which have the effects of increasing stability in the blood, delaying drug release to the kidney, and inducing changes in affinity for receptors.

[0118] A polypeptide can improve its in vivo half-life and extend its in vivo retention time when covalently bound to a non-peptidic polymer. In this case, the binding site between the non-peptidic polymer and the polypeptide can vary depending on the functional group of the non-peptidic polymer and the amino acid sequence of the polypeptide. Preferably, the binding site is not particularly limited, as long as the non-peptidic polymer is polymerized at the C-terminus of the polypeptide or can be prepared in high yield due to its high reaction rate.

[0119] When a non-peptidic polymer is attached to a polypeptide, a non-peptidic polymer having a maleimide group may be attached to the polypeptide using the sulfhydryl (-SH) group of the C-terminal cysteine ​​of the polypeptide, or a non-peptidic polymer having a succinimide derivative may be attached to the polypeptide using the amine group of a lysine (K) of the polypeptide.

[0120] The non-peptidic polymer may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Preferably, the non-peptidic polymer is polyethylene glycol or a derivative thereof, but the present invention is not limited thereto. Derivatives of non-peptidic polymers known in the relevant art and other derivatives that can be easily prepared by those skilled in the art are also within the scope of the present invention.

[0121] Polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidyl propionate (PEG succinimidyl propionate), methoxypolyethylene glycol succinimidyl propionate (methoxyPEG succinimidyl propionate), acrylate polyethylene glycol succinimidyl propionate (acrylate PEG succinimidyl propionate), thiol polyethylene glycol succinimidyl propionate (thiol PEG succinimidyl propionate), hydroxy The polyethylene glycol derivative may be at least one selected from the group consisting of succinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of the derivatives thereof. Preferably, the polyethylene glycol derivative is linear methoxypolyethylene glycol maleimide, bi-branched methoxypolyethylene glycol maleimide, or tri-branched methoxypolyethylene glycol maleimide, more preferably tri-branched methoxypolyethylene glycol maleimide.

[0122] The polyethylene glycol or derivatives thereof that may be used herein are linear or branched, preferably bi- or tri-branched, more preferably tri-branched.

[0123] The molecular weight of the nonpeptidic polymer may be 3,000 to 100,000 Da, preferably 20,000 to 70,000 Da, and more preferably 40,000 to 60,000 Da. When the molecular weight of the nonpeptidic polymer is within this range, the nonpeptidic polymer can be attached to a polypeptide to increase the solubility of the resulting conjugate and extend the in vivo retention time of the conjugate.

[0124] Therefore, the pharmaceutical composition according to the present invention contains a conjugate having a non-peptidic polymer bound to a polypeptide, thereby increasing in vivo stability and extending in vivo half-life.

[0125] Furthermore, since the pharmaceutical composition according to the present invention comprises a conjugate comprising a polypeptide or a conjugate comprising a polypeptide and a non-peptidic polymer, it can be used in a pharmaceutical composition for the purpose of preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0126] Furthermore, the pharmaceutical composition according to the present invention comprising the polypeptide or the conjugate comprising the polypeptide and the non-peptidyl polymer can be used in a pharmaceutical composition for the purpose of preventing or treating a disease caused by a deficiency in insulin secretion or a decrease in insulin sensitivity.

[0127] Diseases caused by a deficiency in insulin secretion or a decrease in insulin sensitivity can include type I diabetes, type II diabetes, and diabetic complications.

[0128] Furthermore, the pharmaceutical composition according to the present invention, which comprises a polypeptide or a conjugate comprising a polypeptide and a non-peptidyl polymer, can be used as a pharmaceutical composition for the purpose of preventing, ameliorating, or treating diseases such as hyperlipidemia, cardiovascular disease, arteriosclerosis, and lipid-related metabolic syndrome.

[0129] Furthermore, the pharmaceutical composition according to the present invention comprising a conjugate comprising a polypeptide or a polypeptide and a non-peptidic polymer can be used as a pharmaceutical composition for the purpose of preventing, ameliorating, or treating liver diseases such as liver cancer, liver cirrhosis, non-alcoholic steatohepatitis, and non-alcoholic fatty liver.

[0130] When the composition of the present invention is used as a pharmaceutical, the pharmaceutical composition containing the polypeptide can be formulated into the following various dosage forms for oral or parenteral administration, and then clinically administered, but the present invention is not limited thereto.

[0131] Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, troches, etc. In addition to the active ingredient, these formulations contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and may optionally contain disintegrating agents such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures, and / or absorbents, colorants, flavorings, and sweeteners.

[0132] Pharmaceutical compositions containing polypeptides can be administered parenterally, such as by subcutaneous injection, intravenous injection, intramuscular injection, nasal spray, nasal or intestinal administration via mucous membranes, inhalation, or intrapleural injection.

[0133] In this case, to enable preparation into a formulation for parenteral administration, the polypeptide can be mixed with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in a unit dosage form in an ampule or vial. The composition may be sterile and / or may contain adjuvants such as preservatives, stabilizers, wetting agents or emulsion promoters, salts and / or buffers for regulating osmotic pressure, and other therapeutically useful substances. In this case, the composition may be formulated according to conventional methods such as mixing, granulating, or coating methods.

[0134] The amount of a pharmaceutical composition containing a polypeptide according to the present invention administered to a human body may vary depending on the patient's age, weight, sex, mode of administration, health condition, and severity of the disease. For example, the pharmaceutical composition may be administered orally or parenterally at a dose of 0.001 to 200 mg / kg / day, according to the judgment of a physician or pharmacist.

[0135] The present invention also provides a method for preparing a pharmaceutical composition comprising a conjugate comprising a polypeptide and a non-peptidyl polymer.

[0136] First, in the method for preparing a pharmaceutical composition, the polypeptide has the amino acid sequence represented by General Formula 1 as described above. Furthermore, since the non-peptidic polymer is as described above, detailed descriptions of the polypeptide and non-peptidic polymer will be omitted.

[0137] Specifically, the method for preparing a pharmaceutical composition includes mixing and reacting a nonpeptidic polymer and a polypeptide. In this case, the polypeptide and the nonpeptidic polymer can be reacted at a molar ratio of 1:1 to 1:5, so that the polypeptide and the nonpeptidic polymer can be conjugated to each other at a molar ratio of 1:1. In this case, the mixing is preferably carried out at a molar ratio of 1:1 to 1:2, more preferably at a molar ratio of 1:1.2, although the present invention is not limited thereto. When the mixing is carried out within this molar ratio range, the conjugate can be obtained in high yield, thereby enabling the preparation of a highly pure conjugate comprising a polypeptide and a nonpeptidic polymer.

[0138] According to one exemplary embodiment of the present invention, the conjugate can also be prepared by covalently binding a non-peptidic polymer to the C-terminus of a polypeptide. For example, the conjugate can be prepared with high reactivity by using methoxypolyethylene glycol having a maleimide group as the non-peptidic polymer and a polypeptide having a cysteine ​​at its C-terminus as the polypeptide, thereby achieving high yield and extending the blood half-life.

[0139] Mixing to react the non-peptidic polymer and polypeptide can be carried out at a pH of 4.0 to 9.0, preferably 5.5 to 7.5, although the present invention is not limited thereto. Mixing outside this pH range results in a lower yield. For example, when methoxypolyethylene glycol having a maleimide group is used as the non-peptidic polymer and a polypeptide having a cysteine ​​at its C-terminus is used as the polypeptide, mixing is preferably carried out at a pH of 6 to 8. Reacting the polypeptide and methoxypolyethylene glycol within this pH range can suppress side reactions such as maleimide ring-opening without causing side reactions due to the amine groups of the polypeptide.

[0140] Conjugates can be produced in 85-95% yield when non-peptidic polymers and polypeptides are mixed and reacted with each other. This process is economically feasible and highly reproducible due to its extremely high yield. Therefore, this process may be effectively used to prepare pharmaceuticals.

[0141] In the reaction of the non-peptidic polymer and the polypeptide, the reaction time can be in the range of 0.5 to 24 hours, or 1 to 24 hours, preferably 2 hours, but the present invention is not limited thereto. If the reaction time is less than 0.5 hours, the yield will be low and the purity will decrease. On the other hand, if the reaction time exceeds 24 hours, the polypeptide may be decomposed, and the economic efficiency may decrease due to the long processing time.

[0142] Furthermore, when mixing the non-peptidyl polymer and the polypeptide to react with each other, the temperature may be in the range of 0 to 100° C., preferably 4 to 40° C., but the present invention is not limited thereto. Furthermore, the temperature is not particularly limited as long as there is no chemical change in the polypeptide or the non-peptidyl polymer.

[0143] In the reaction of the non-peptidic polymer and the polypeptide, the polypeptide and the non-peptidic polymer can be dissolved in the same or different solvents. Preferably, the solvent is a buffer solution, alcohol, dimethyl sulfoxide (DMSO), or a mixture thereof, but the present invention is not limited thereto. The solvent may also be any solvent that can be easily used in the related art.

[0144] The present invention provides a method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease, comprising administering to a subject a pharmaceutical composition comprising a polypeptide.

[0145] The present invention also provides a method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease, comprising administering to a non-human subject a pharmaceutical composition comprising the polypeptide. [Example]

[0146] The present invention will now be described in detail with reference to the accompanying drawings so that those skilled in the art can easily practice the present invention. It should be understood, however, that the present invention may be embodied in various forms and is not intended to be limited in this context. Like reference numerals refer to like elements throughout the specification.

[0147] Example 1

[0148] Cysteine-introduced polypeptide (molecular weight: 3,509 Da; SEQ ID NO: 2) [ka] ).

[0149] Here, in the amino acid sequence of SEQ ID NO: 2, residues that are underlined and highlighted in bold indicate that a covalent bond ring is formed between those residues.

[0150] Preparation Example 1: Synthesis of a conjugate containing a polypeptide and a non-peptidic polymer

[0151] To prepare a conjugate comprising a polypeptide and a non-peptidic polymer, a polypeptide (molecular weight: 3,509 Da; SEQ ID NO: 2) was prepared in which a cysteine ​​was introduced into the C-terminal region (position 30). [ka] ) was used as the polypeptide.

[0152] On the other hand, maleimide-activated monomethoxy PEG (mPEG-MAL, NOF Corp. (Japan)) was used as a non-peptidic polymer, as shown in Table 2 below.

[0153] To prepare the conjugates of Examples 2 to 7, the polypeptides listed in Table 2 below were prepared by dissolving each polypeptide in dimethyl sulfoxide (DMSO) and mPEG-MAL in 50 mM phosphate buffered saline (pH 6).

[0154] [Table 2]

[0155] In the amino acid sequences of SEQ ID NOs: 2 to 4 listed in Table 2, the two underlined and bolded residues indicate residues between which a covalent bond ring is formed.

[0156] The polypeptide and nonpeptide conjugate were mixed in a molar ratio of 1:1.2 and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was separated by ion exchange chromatography using a TSK SP-5PW column (7.5 x 75 mm, Tosoh, Japan) at a flow rate of 0.8 mL / min. The separation was monitored at a UV wavelength of 280 nm. The PEGylated polypeptide was separated using a linear gradient method with 20 mM acetate buffer (pH 4) (mobile phase A) and 1 M sodium chloride solution (in 20 mM acetate buffer (pH 4)) (mobile phase B) as the mobile phase. HPLC was performed to evaluate the purity of the PEGylated polypeptide (see Figure 1). The molecular weight of the PEGylated polypeptide was then measured using a MALDI-TOF mass spectrometer (see Figure 2). The yields of the conjugates of Examples 2 to 7 were calculated as the area ratio of the conjugate to the polypeptide from the chromatograms obtained during the chromatographic separation process. The results are shown in Table 3.

[0157] [Table 3]

[0158] As shown in Table 3, it was confirmed that the conjugates were prepared in a yield of 90% or higher. Therefore, the method for preparing a pharmaceutical composition according to the present invention has the advantage that the conjugates can be obtained in high yield due to their high reactivity with peptides, and therefore can be effectively used to prepare therapeutic drugs. Furthermore, due to the simple preparation process, this method has the advantage that it is economically feasible and highly reproducible.

[0159] Experimental Example 1: Measurement of in vitro activity of Example 2

[0160] To investigate the preventive or therapeutic effects of the conjugate of Example 2 on obesity, diabetes, and non-alcoholic fatty liver disease, this experiment was carried out using a cell line expressing the GLP-1 (glucagon derivative) receptor and the glucagon receptor (GCGR).

[0161] To determine the activity against the GLP-1 receptor, HEK293 / CRE-Luc cells expressing the human glucagon GLP-1 receptor were purchased from GenScript and used. The cells were plated in a 96-well plate at 5 × 10 4 Cells were seeded at 1000 kJ / well, and then each well was treated with the polypeptide of Example 1 (0.001-300 nM), the conjugate of Example 2 (0.001-300 nM), native glucagon (SEQ ID NO: 1: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, 0.013-300 nM), and GLP-1 (SEQ ID NO: 6: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, 0.001-300 nM). The cells were then incubated at 37°C in a CO2 incubator for 4 hours. The amount of cAMP (luciferase reporter) produced was then measured using the One-Glo™ Luciferase Assay System (Promega) to determine the EC20 activity for the GLP-1 receptor. 50 The values ​​were calculated and the results are shown in Table 4 below.

[0162] Next, to determine activity against the glucagon receptor (GCGR), we used the DiscoverX cAMP Hunter™ eXpress GCGR CHO-K1 GPCR Assay Kit. CHO-K1 cells expressing the human glucagon receptor were cultured at 3 × 10 in a 96-well plate. 4 The cells were seeded at a density of 1000 cells / well. Then, each well was treated with the polypeptide of Example 1 (0.013 to 300 nM), the conjugate of Example 2 (0.013 to 300 nM), native glucagon (0.015 to 33.33 nM), and GLP-1 (0.001 to 30.00 nM). The cells were then incubated at 37°C in a CO2 incubator for 30 minutes. The amount of cAMP produced was then measured to determine the EC20 activity of the glucagon receptor (GCGR). 50 The values ​​were calculated and the results are shown in Table 4 below.

[0163] [Table 4]

[0164] As shown in Table 4, GLP-1 showed high activity at the GLP-1 receptor, but its activity at the glucagon receptor was so low that it could not be measured. On the other hand, native glucagon showed very high activity at the glucagon receptor, but showed low activity at the GLP-1 receptor. Based on these results, this experimental method was confirmed to be highly selective.

[0165] On the other hand, the polypeptide of Example 1 had an EC value of 0.13 for the GLP-1 receptor. 50 It was confirmed that the polypeptide of Example 1 had a value, which was almost identical to the value of GLP-1. This indicates that the polypeptide of Example 1 exhibited very high activity on the GLP-1 receptor, and also had activity on the glucagon receptor. Furthermore, using animal experiments, it was confirmed that the polypeptide of Example 1 has an anti-obesity effect.

[0166] Furthermore, it was confirmed that the conjugate of Example 2 retained similar activity against the GLP-1 receptor and the glucagon receptor compared to the polypeptide of Example 1. In general, when a nonpeptidic polymer (e.g., PEG) is attached to a polypeptide, the activity of the polypeptide against the receptor is significantly reduced compared to before attachment. On the other hand, it was confirmed that the conjugate of Example 2 showed little reduction in activity against the receptor, even when a nonpeptidic polymer was attached to the polypeptide. This indicates that the conjugate of Example 2 maintained high activity while simultaneously exhibiting an extended in vivo half-life.

[0167] Therefore, it was confirmed that the pharmaceutical composition according to the present invention has excellent activity against glucagon receptors and GLP-1 receptors, and therefore has anti-diabetic and anti-obesity effects, as well as the effect of suppressing appetite, promoting insulin secretion, and lowering triglycerides by promoting lipolysis in adipocytes.

[0168] Experimental Example 2: Measurement of in vivo activity of Example 2 1

[0169] To investigate the preventive or therapeutic effects of the conjugate of Example 2 on obesity or diabetes, the conjugate of Example 2 was administered to C57BL / 6 mice, and the changes in food intake, blood glucose, and body weight were measured. The results are shown in Table 5.

[0170] First, an obese animal model was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the mice's body weight to an average of approximately 50 g. The conjugate of Example 2 was then administered by subcutaneous injection at a dose of 20 nmol / kg every other day for two weeks. As a positive control, the GLP-1 agonist "liraglutide" was also administered by subcutaneous injection at a dose of 100 nmol / kg every other day for two weeks. Food intake, blood glucose, and body weight were measured every other day at predetermined time points during the two-week drug administration period. The results are shown in Table 5 below.

[0171] In this case, the body weight and blood glucose level were expressed as a percentage (%) based on 100% before administration (day 0).

[0172] [Table 5]

[0173] In this case, the untreated group refers to a group of mice administered PBS instead of the conjugate of Example 2.

[0174] As shown in Table 5, the untreated group ate approximately 40 g of food over two weeks, while the group of mice administered the conjugate of Example 2 ate approximately 18 g of food, a reduction in cumulative food intake of more than half compared to the untreated group. Food intake was similar in the positive control and the groups of mice administered the conjugate of Example 2.

[0175] On the other hand, when the pattern of body weight change over time was examined, no change in body weight was observed in the untreated group compared to the body weight observed at the time of administration (day 0), and in the positive control, body weight decreased by about 15% compared to the body weight observed before administration, indicating that the preventive or therapeutic effect of liraglutide on obesity was insufficient.On the other hand, in the group of mice administered with the conjugate of Example 2, body weight decreased significantly by 61% compared to the body weight measured before administration.

[0176] Furthermore, when the pattern of blood glucose changes over time was examined, it was revealed that the blood glucose levels had decreased by approximately 80% compared to the blood glucose levels measured before administration, indicating that the conjugate of Example 2 had the effect of lowering blood glucose levels, but that the positive control had an insufficient effect of lowering blood glucose levels.

[0177] Based on these results, it can be seen that the effect of the conjugate of Example 2 on weight loss was due to the increase in energy metabolism in the body as well as a simple reduction in food intake. The pharmaceutical composition according to the present invention had the effects of reducing food intake, inhibiting gastric emptying, and promoting lipolysis.

[0178] Experimental Example 3: Measurement of in vivo activity of Example 2

[0179] This experiment was carried out in the same manner as in Experimental Example 2, and then glucose tolerance was assessed in the mouse model using an intraperitoneal glucose tolerance test (ipGTT).

[0180] After two weeks of drug administration in the same manner as in Experimental Example 2, 2 g / kg of glucose was intraperitoneally administered, and blood glucose changes over time (0, 15, 30, 60, 90, and 120 minutes) were measured. The results are shown in Table 6 below.

[0181] [Table 6]

[0182] As shown in Table 6, the results obtained after two weeks of drug administration confirmed that blood glucose levels in the untreated group rose sharply due to glucose administration and then declined, whereas the rise in blood glucose was significantly reduced in the group of mice administered with the conjugate of Example 2. Therefore, it was confirmed that the glucose tolerance of the conjugate of Example 2 was increased compared to the untreated group. It was also revealed that the rise in blood glucose was smaller in the group of mice administered with the conjugate of Example 2 compared to the positive control.

[0183] Experimental Example 4: Measurement of in vivo activity of Example 2 3

[0184] To evaluate the preventive or therapeutic effect of the conjugate of Example 2 on diabetes, the conjugate of Example 2 was administered to approximately 7-week-old BKS.Cg-+Lepr db / +Lepr db / OlaHsd mice (db / db mice) were administered the drug, and changes in blood glucose and body weight were measured over time.

[0185] First, the conjugate of Example 2 was administered subcutaneously to 7-week-old db / db mice at a dose of 20 nmol / kg every other day for 12 days. Blood glucose and body weight changes were measured every other day after drug administration for 12 days. The results are shown in Table 7 below.

[0186] Next, to perform an intraperitoneal glucose tolerance test (ipGTT), 2 g / kg of glucose was administered intraperitoneally after 12 days of drug administration, and changes in blood glucose over time (0, 15, 30, 60, 90, and 120 minutes) were measured. The results are shown in Table 8. To determine the extent of long-term changes in average blood glucose levels, glycated hemoglobin (HbA1c) levels were also measured after drug administration. The results are shown in Figure 3.

[0187] [Table 7]

[0188] [Table 8]

[0189] As shown in Table 7, it was confirmed that weight loss was observed in the group of mice administered with the conjugate of Example 2 compared to the untreated group. In addition, it was confirmed that high blood glucose levels were maintained for 2 weeks in the untreated group, whereas blood glucose levels decreased in the group of mice administered with the conjugate of Example 2.

[0190] As shown in Table 8, it was also revealed that the group of mice administered with the conjugate of Example 2 exhibited higher glucose tolerance than the untreated group.

[0191] Furthermore, as shown in Figure 3, it was also revealed that the administration of the conjugate of Example 2 significantly reduced the glycated hemoglobin level, indicating that the administration of the conjugate of Example 2 steadily maintained blood glucose at a low level.

[0192] Experimental Example 5: Measurement of in vivo activity of Example 2 4

[0193] To examine the preventive or therapeutic effects of the administration concentration and frequency of the conjugate of Example 2 on obesity, the conjugate of Example 2 was administered to C57BL / 6 mice, and changes in food intake and body weight were measured over time.

[0194] First, an obese animal model was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, allowing the mice to gain an average weight of approximately 50 g. Then, the conjugate of Example 2 was administered to each group for two weeks, as shown in Table 9 below. After two weeks, the mice's final weights were measured. The results are shown in Figure 4.

[0195] [Table 9]

[0196] As shown in Figure 4, the effect of the conjugate of Example 2 on weight loss became more pronounced as the dose of the conjugate of Example 2 was increased, and even when the conjugate of Example 2 was administered at a low dose of 20 nmol / kg, the effect on weight loss was significant. Therefore, it was confirmed that the conjugate of Example 2 exhibited a dose-dependent response. Furthermore, it was confirmed that the conjugate of Example 2 had the same effect on weight loss even when the administration interval was extended to once a week. Therefore, the pharmaceutical composition for preventing or treating obesity according to the present invention had a long in vivo half-life and was able to exhibit a high level of therapeutic effect on obesity even when administered at a low dose.

[0197] Experimental Example 6: Measurement of in vivo activity of Examples 2 and 6

[0198] To investigate the preventive or therapeutic effects of the conjugates of Examples 2 and 6, which have different amino acid sequences, on obesity or diabetes, an obese animal model was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the mice's body weight to an average of approximately 50 g. Subsequently, each of the conjugates of Examples 2 or 6 was administered subcutaneously at a dose of 20 nmol / kg every other day for two weeks. As a control, PBS was administered instead of the conjugates of Examples 2 or 6. Blood glucose changes were measured over time during the two-week administration of the conjugates of Examples 2 or 6. The results are shown in Figure 5. Additionally, to perform an intraperitoneal glucose tolerance test (ipGTT), 2 g / kg of glucose was intraperitoneally administered after two weeks of drug administration, and blood glucose changes were measured over time (0, 15, 30, 60, 90, and 120 minutes). The results are shown in Figure 6.

[0199] As shown in Figure 5, it was revealed that the blood glucose levels were reduced in mice administered with the conjugates of Examples 2 or 6 compared to the control, whereas the blood glucose levels were elevated in the PBS-administered control.

[0200] As shown in Figure 6, in the control, blood glucose levels rose sharply due to the administered glucose and then fell, but in the mice administered with the conjugate of Example 2 or 6 for two weeks, the rise in blood glucose was significantly reduced compared to the control. Based on these results, it was confirmed that the glucose tolerance of the conjugates of Examples 2 and 6 was increased compared to the glucose tolerance of the control.

[0201] Therefore, as shown in Figures 5 and 6, it was confirmed that the pharmaceutical composition containing the polypeptide according to the present invention has a preventive or therapeutic effect on obesity and diabetes.

[0202] Experimental Example 7: Measurement of in vivo activity of Example 2 6

[0203] To investigate the preventive or therapeutic effect of the conjugate of Example 2 on non-alcoholic fatty liver disease, the conjugate of Example 2 was administered to an animal model of non-alcoholic fatty liver disease, and changes in serum cholesterol levels and liver weight were examined, and liver biopsies were performed. The results are shown in Figures 7 to 9.

[0204] Specifically, an experimental animal model of NAFLD was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, allowing the mice to gain an average body weight of approximately 50 g. The conjugate of Example 2 was then administered subcutaneously at a dose of 20 nmol / kg every other day for 2 weeks. As a control, the GLP-1 agonist liraglutide was also administered subcutaneously at a dose of 100 nmol / kg once daily for 2 weeks. After 2 weeks of drug administration, blood was collected from the mice to measure serum cholesterol levels, and the livers were excised, weighed, embedded in paraffin, and thinly sectioned using a microtome. Liver biopsies were then performed using hematoxylin and eosin (H&E).

[0205] 7 and 8, which show the measurement of serum cholesterol levels and liver weights, it was confirmed that the serum cholesterol levels and liver weights of mice administered with the conjugate of Example 2 were significantly reduced compared to the untreated group administered with PBS, and also compared to the positive control (i.e., the liraglutide-treated group). Furthermore, referring to the liver biopsy results shown in Figure 9, it can be seen that the group of mice administered with the conjugate of Example 2 had significantly reduced hepatic steatosis compared to the untreated group administered with PBS, and also compared to the positive control (i.e., the liraglutide-treated group).

[0206] Therefore, it was revealed that the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention reduces liver weight, serum cholesterol levels, and hepatic steatosis in an animal model of non-alcoholic fatty liver disease, and is therefore effective in preventing and treating non-alcoholic fatty liver disease.

[0207] Experimental Example 8: Measurement of in vivo activity of Example 2 7

[0208] To investigate the preventive or therapeutic effect of the conjugate of Example 2 on non-alcoholic fatty liver disease, the conjugate of Example 2 was administered to an animal model of non-alcoholic fatty liver disease, and changes in serum cholesterol levels, liver weight, and liver triglycerides were measured. The results are shown in Figures 10 to 12.

[0209] First, an experimental animal model of nonalcoholic fatty liver disease was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a high-trans fatty acid diet containing 40% high fat, 20% fructose, and 2% cholesterol for approximately 16 weeks. The conjugate of Example 2 was then administered subcutaneously at a dose of 20 nmol / kg once every three days for four weeks. As a positive control, the GLP-1 agonist liraglutide was also administered subcutaneously at a dose of 53 nmol / kg once daily for four weeks. After the four-week experiment, serum cholesterol levels, liver weight, and liver triglycerides (liver TG) were measured.

[0210] 10 to 12, which show the measurement of serum cholesterol levels, liver weight, and liver TG, it can be seen that the serum cholesterol levels, liver weight, and liver triglycerides were significantly reduced in mice administered with the conjugate of Example 2 compared to the untreated group administered with saline, and also compared to the positive control (i.e., the liraglutide-treated group). Therefore, it was revealed that the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention reduces liver weight, serum cholesterol, and liver triglycerides in an animal model of non-alcoholic fatty liver disease, and is therefore effective in preventing and treating non-alcoholic fatty liver disease.

[0211] Experimental Example 9: Measurement of in vivo activity of Example 2

[0212] After conducting the same experiment as in Experimental Example 8, liver biopsies were performed to measure the NAFLD activity score (NAS) and examine the preventive or therapeutic effect on nonalcoholic fatty liver disease. After administering the conjugate of Example 2 for 4 weeks, the mouse livers were excised, embedded in paraffin, and sliced ​​using a microtome. Hematoxylin and eosin (H&E) staining and oil red O staining were then performed.

[0213] 13 and 14, the liver tissue images and NAS results after 4 weeks of administration showed that hepatic steatosis and NAS were significantly reduced in mice administered with the conjugate of Example 2, compared to the untreated group administered with saline and the positive control (i.e., the liraglutide-treated group). Therefore, it was revealed that the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention reduces hepatic steatosis and decreases NAS in an animal model of non-alcoholic fatty liver disease, and is therefore effective in preventing or treating non-alcoholic fatty liver disease.

[0214] While preferred embodiments of the present invention have been described in detail hereinabove, it should be understood that many variations and / or modifications of the basic inventive concepts taught herein that are apparent to those skilled in the relevant art will still fall within the scope of the present invention as defined in the appended claims. [Industrial Applicability]

[0215] A pharmaceutical composition comprising the polypeptide according to the present invention has the effects of reducing food intake, improving insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels without causing side effects such as vomiting or nausea, and is therefore useful for the safe prevention or treatment of obesity, diabetes, or non-alcoholic fatty liver disease. In particular embodiments, for example, the following items are provided: (Item 1) General Formula 1 below: [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (SEQ ID NOs: 7 to 9), wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxy-imidazo-propionyl, 4-imidazoacetyl, or β-carboxy-imidazo-propionyl; X1 is deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (SEQ ID NO: 10), EQAAK (SEQ ID NO: 11), or EEAVK (SEQ ID NO: 12); and R3 is a deletion, cysteine, lysine, or methionine) A polypeptide having an amino acid sequence represented by the formula: (Item 2) General Formula 1 below: [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (SEQ ID NOs: 7 to 9), wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxy-imidazo-propionyl, 4-imidazoacetyl, or β-carboxy-imidazo-propionyl; X1 is deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (SEQ ID NO: 10), EQAAK (SEQ ID NO: 11), or EEAVK (SEQ ID NO: 12); and R3 is a deletion, cysteine, lysine, or methionine) A pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the formula: (Item 3) 3. The pharmaceutical composition of item 2, wherein the polypeptide is covalently bound to or forms a microsphere with any one or more selected from the group consisting of non-peptidic polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn-binding substances, saccharides, elastin, heparin, and derivatives thereof. (Item 4) 3. The pharmaceutical composition of claim 2, wherein R2 comprises glutamic acid (E) and lysine (K), and the glutamic acid and the lysine together form a ring via an amide bond. (Item 5) 3. The pharmaceutical composition according to item 2, wherein the non-peptidic polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. (Item 6) 4. The pharmaceutical composition according to item 3, wherein the non-peptidic polymer is polyethylene glycol or a derivative thereof. (Item 7) 7. The pharmaceutical composition according to item 6, wherein the molecular weight of the nonpeptidic polymer is 3,000 to 100,000 Da. (Item 8) The polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidyl propionate (PEG succinimidyl propionate), methoxypolyethylene glycol succinimidyl propionate (methoxyPEG succinimidyl propionate), acrylate polyethylene glycol succinimidyl propionate (acrylate PEG succinimidyl propionate), thiol polyethylene glycol succinimidyl propionate (thiol PEG succinimidyl propionate), hydroxysuccinimide, 7. The pharmaceutical composition according to item 6, wherein the succinimidyl polyethylene glycol is at least one selected from the group consisting of succinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multibranched forms of derivatives thereof. (Item 9) 7. The pharmaceutical composition according to item 6, wherein the polyethylene glycol or derivative thereof is linear or branched. (Item 10) 3. The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used for preventing or treating obesity. (Item 11) 3. The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used for preventing or treating diabetes. (Item 12) Item 3. The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used for preventing or treating non-alcoholic fatty liver disease. (Item 13) Item 13. The pharmaceutical composition of item 12, wherein the non-alcoholic fatty liver disease comprises one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer. (Item 14) 1. A method for preparing a pharmaceutical composition, the pharmaceutical composition comprising: A polypeptide having an amino acid sequence represented by the following general formula 1: Conjugates containing non-peptidic polymers wherein the method comprises: mixing said non-peptidyl polymer and said polypeptide to react with each other; [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (SEQ ID NOs: 7 to 9), wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-hydroxy-imidazo-propionyl, 4-imidazoacetyl, or β-carboxy-imidazo-propionyl; X1 is deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (SEQ ID NO: 10), EQAAK (SEQ ID NO: 11), or EEAVK (SEQ ID NO: 12); and R3 is a deletion, cysteine, lysine, or methionine). (Item 15) Item 15. The method of claim 14, wherein R2 comprises glutamic acid (E) and lysine (K), and the glutamic acid and the lysine together form a ring via an amide bond. (Item 16) Item 15. The method of item 14, wherein the non-peptidic polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. (Item 17) Item 15. The method according to item 14, wherein the non-peptidic polymer is polyethylene glycol or a derivative thereof. (Item 18) The polyethylene glycol derivatives may be methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidyl propionate (PEG succinimidyl propionate), methoxypolyethylene glycol succinimidyl propionate (methoxyPEG succinimidyl propionate), acrylate polyethylene glycol succinimidyl propionate (acrylate PEG succinimidyl propionate), thiol polyethylene glycol succinimidyl propionate (thiol PEG succinimidyl propionate), hydroxysuccinimide, 18. The method of claim 17, wherein the compound is at least one selected from the group consisting of succinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of derivatives thereof. (Item 19) Item 15. The method according to Item 14, wherein the mixing of the non-peptidyl polymer and the polypeptide to react with each other comprises reacting the polypeptide and the non-peptidyl polymer in a molar ratio of 1:1 to 1:5. (Item 20) Item 15. The method according to item 14, wherein the mixing of the nonpeptidyl polymer and the polypeptide to react with each other is carried out at a pH of 4.0 to 9.0. (Item 21) Item 15. The method according to Item 14, wherein the reaction time in the mixing for reacting the nonpeptidyl polymer and the polypeptide with each other is within the range of 0.5 to 24 hours. (Item 22) 15. The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat obesity. (Item 23) Item 15. The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat diabetes. (Item 24) Item 15. The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat non-alcoholic fatty liver disease. (Item 25) 25. The method of item 24, wherein the non-alcoholic fatty liver disease comprises one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer. (Item 26) 1. A method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease, comprising: A method comprising administering to a subject the pharmaceutical composition defined in item 2.

Claims

[Claim 1] The invention as set forth in the drawings.

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