Pharmaceutical composition comprising polypeptide

Abstract

The present invention relates to a pharmaceutical composition comprising a polypeptide, and more particularly, to a pharmaceutical composition for preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease. The pharmaceutical composition is safe without any side effects such as vomiting or nausea and has the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis and reducing triglyceride levels.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511674302.1 (22) Application Date 2019.07.19 (30) Priority Data 10-2018-0083946 2018.07.19 KR 10-2019-0060513 2019.05.23 KR (62) Divisional Application Data 201980047328.X 2019.07.19 (71) Applicant D&D Pharmaceutical Technology Co., Ltd. Address Gyeonggi Province, South Korea (72) Inventors K.C. Lee O.Y. Park H.T. Ahn E.J. Park J.H. Shin S.M. Lim (74) Patent Agency Shenzhen Liujia Intellectual Property Agency Co., Ltd. 44372 Patent Attorney Liu Hui (51) Int.Cl. C07K 14 / 605(2006.01) A61K 38 / 26(2006.01) A61K 47 / 60(2017.01) A61P 3 / 04(2006.01) (54) Invention Title: Pharmaceutical Composition Containing Polypeptides (57) Abstract: This invention relates to a pharmaceutical composition comprising polypeptides, and more specifically, to a pharmaceutical composition for the prevention or treatment of obesity, diabetes, or non-alcoholic fatty liver disease. The pharmaceutical composition is safe without any side effects such as vomiting or nausea and has the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels. Claims 2 pages, Description 18 pages, Drawings 6 pages, CN 121591873 A 2026.03.03 CN 1 21 59 18 73 A 1. A polypeptide having an amino acid sequence represented by the following general formula 1: [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl 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.2. A pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the following general formula 1: [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl, 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. 3. The pharmaceutical composition according to claim 2, wherein the polypeptide is covalently bound to or forms microspheres with any one or more of the group selected from: non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin, and derivatives thereof. 4. The pharmaceutical composition of claim 2, wherein R2 comprises glutamic acid (E) and lysine (K), and the glutamic acid and the lysine form a ring together via an amide bond. 5. The pharmaceutical composition of claim 2, wherein the non-peptide polymer is selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitosan, hyaluronic acid, and combinations thereof. 6. The pharmaceutical composition of claim 3, wherein the non-peptide polymer is polyethylene glycol or a derivative thereof. 7. The pharmaceutical composition of claim 6, wherein the molecular weight of the non-peptide polymer is from 3,000 to 100,000 Da.8. The pharmaceutical composition of claim 6, wherein the polyethylene glycol derivative is at least one selected from the group consisting of: methoxy polyethylene glycol, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, polyethylene glycol propionate succinimide (PEG propionate succinimide), methoxy polyethylene glycol propionate succinimide (methoxyPEG propionate succinimide), polyethylene glycol propionate succinimide acrylate (PEG propionate succinimide), thiol polyethylene glycol. Succinimide propionate (thiol PEG succinimide propionate), hydroxysuccinimide polyethylene glycol (hydroxysuccinimide PEG), methoxy polyethylene glycol succinimide carboxymethyl ester (mPEG succinimide carboxymethyl ester), polyethylene glycol succinimide carboxymethyl ester (PEG succinimide carboxymethyl ester), polyethylene glycol carbonate succinimide (PEG carbonate succinimide), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of derivatives thereof. 9. The pharmaceutical composition according to claim 6, wherein the polyethylene glycol or its derivative is linear or branched. (Claims 1 / 2 page 2 CN 121591873 A) 10. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is used for the prevention or treatment of obesity. Claims 2 / 2 Page 3 CN 121591873 A Pharmaceutical Composition Containing Polypeptides

[0001] This application is a divisional application of Chinese Patent Application No. 201980047328.X, filed on July 19, 2019, entitled "Pharmaceutical Composition Containing Polypeptides". Technical Field

[0002] The present invention relates to a pharmaceutical composition containing polypeptides and its medical use in, for example, treating or preventing obesity, diabetes or non-alcoholic fatty liver disease. Polypeptides have the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis and lowering triglyceride levels, without side effects such as vomiting or nausea. Background Art

[0003] Recently, with economic development and rapid growth in science and technology, the aging population is increasing and adult diseases are rapidly increasing. This is due to stress, poor eating habits, excessive calorie intake and reduced physical activity. As complications accompanying obesity, heart disease and cerebrovascular disease rank first and second in mortality, and obesity is considered a cause of various adult diseases such as diabetes and non-alcoholic fatty liver disease.

[0004] Obesity is a state in which the amount of fat accumulated is higher than normal, and the most accurate method for assessing obesity is to measure body fat mass. However, accurately measuring body fat mass is expensive, and therefore indirect methods are used to assess it.The most commonly used indirect methods are measuring body mass index (BMI) and waist circumference. The World Health Organization (WHO) has announced a classification based on data linking BMI to mortality risk: normal weight: 18.5 to 24.9 kg / m2, overweight: 25 to 29.9 kg / m2, and obese: 30 kg / m2 or more.

[0005] Obesity is said to be caused by an energy imbalance resulting from excessive calorie intake and relatively reduced physical activity, leading to increased body fat. However, it is difficult to specify only one factor, as various risk factors such as dietary habits, lifestyle, age, race, and genetic factors are involved in obesity.

[0006] Diabetes is classified into insulin-dependent diabetes mellitus (Type 1 diabetes), insulin-independent diabetes mellitus (Type 2 diabetes), and malnutrition-associated diabetes mellitus (MRDM). Type 2 diabetes, which accounts for more than 90% of diabetes cases, is a metabolic disease characterized by hyperglycemia and is reported to be caused by decreased insulin secretion from pancreatic β cells or increased insulin resistance in peripheral tissues due to genetic, metabolic, and environmental factors. In this regard, insulin sensitivity decreases when body fat increases, and the accumulation of abdominal fat, in particular, is known to be associated with impaired glucose tolerance. Furthermore, insulin resistance is known to be closely associated with obesity in patients with type 2 diabetes, with greater insulin resistance associated with more severe obesity.

[0007] Non-alcoholic fatty liver disease (NAFLD) refers to a range of diseases including simple steatosis independent of alcohol consumption, involving excessive fat accumulation in hepatocytes, including hepatocyte damage (ballooning degeneration of hepatocytes), inflammation, fibrosis, and in more advanced cases, non-alcoholic steatohepatitis (NASH) with cirrhosis. The prevalence of NAFLD is rapidly increasing with the increasing prevalence of obesity worldwide, and although the prevalence of diabetes varies from country to country, it accounts for approximately 20% to 30% of the total population in Western countries and reaches approximately 16% in South Korea.

[0008] NAFLD exhibits a close association with metabolic syndrome, including obesity, type 2 diabetes, and dyslipidemia, based on insulin resistance. In fact, many patients with prediabetes and type II diabetes are known to present with non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, and the rate of progression of cirrhosis and liver cancer (e.g., hepatocellular carcinoma) is high in these patients. Meanwhile, diabetes is highly prevalent in patients with non-alcoholic fatty liver disease and significantly prevalent in patients with non-alcoholic steatohepatitis.

[0009] It is primarily recommended that obese patients control their weight through a healthier diet and physical activity, but when these methods are ineffective, patients can be treated with medication or surgery.

[0010] The current market for anti-obesity drugs is estimated to be over one billion US dollars and is growing at about 10% annually. The main drugs used as anti-obesity medications are anorexia nervosa agents (lorcaserine, phentermine, etc.), which are classified as psychotropic drugs that act primarily on the central nervous system. These drugs are known to suppress appetite and reduce weight, but have side effects such as abuse and addiction, palpitations, anxiety, and insomnia when used for extended periods.

[0011] Orlistat (Xenical) is one of the drugs used as a non-psychotropic anti-obesity medication. Pancreatic lipase acts as a key enzyme in breaking down triglycerides into 2-monoacylglycerols and fatty acids. A representative pancreatic lipase inhibitor is tetrahydrolipstatin (orlistat), a derivative of liposatin derived from Streptomyces toxitricini, which has a high level of efficacy in inhibiting the absorption of approximately 30% of ingested fat. Currently, orlistat is commercially available as a drug, but it has side effects such as gastrointestinal disturbances, allergic reactions, and cholestasis. Therefore, there are few therapeutic agents that can be safely used in obese patients.

[0012] In drug therapy for the treatment of non-alcoholic fatty liver disease, the drug works by mechanisms that exacerbate non-alcoholic fatty liver disease, such as insulin resistance, oxidative stress, apoptosis, and inflammatory cytokines, and inhibits the progression of non-alcoholic fatty liver disease. Among these, antidiabetic agents are known to improve fatty liver by improving the physiological and pathological state common at the onset of fatty liver and to lower blood sugar. However, because no drug has been approved for the treatment of fatty liver disease, the medical need to develop effective therapeutic agents has not yet been met.

[0013] Meanwhile, glucagon derivatives are currently of interest. Glucagon is produced in the pancreas when blood sugar levels begin to decline due to drug treatment, disease, hormones, or enzyme deficiencies. Glucagon stimulates the liver to release glucose by breaking down glycogen and is used to raise blood sugar levels to normal levels. In addition to raising blood sugar, glucagon has been reported to suppress appetite and activate hormone-sensitive lipases in fat cells to promote fat degradation, thus exhibiting an anti-obesity effect. As one such glucagon derivative, glucagon-like peptide-1 (GLP-1) is a substance still under development as a therapeutic agent to alleviate hyperglycemia in diabetic patients. It is used to promote insulin synthesis and secretion, inhibit glucagon secretion, suppress gastric emptying, promote glucose utilization, and suppress food intake.Moreover, exenatide-4, derived from lizard venom and possessing approximately 50% amino acid homology with GLP-1, is known to be used to activate GLP-1 receptors to alleviate hyperglycemia in diabetic patients. However, it has been reported that GLP-1 receptor agonists used to treat obesity or diabetes have side effects such as vomiting and nausea.

[0014] As an alternative to GLP-1, gastrin, which can bind to both GLP-1 and glucagon receptors, has become a focus of attention. Gastrin is a peptide derived from the precursor of glucagon (i.e., proglucagon) and exhibits the effects of inhibiting food intake through GLP-1, inhibiting gluconeogenesis in the liver to regulate blood glucose levels, and increasing satiety, as well as having lipolytic function against glucagon. Therefore, gastrin has high potential as an anti-diabetic and anti-obesity drug.

[0015] Based on the dual function of the gastrin peptide, research is being actively pursued to develop drugs for the treatment of diabetes and obesity. For example, Korean Patent No. 925017 discloses a pharmaceutical composition for treating human overweight, administered orally, parenterally, mucosally, rectally, subcutaneously, or transdermally, comprising a gastrin as an active ingredient. However, it has been reported that anti-obesity drugs containing gastrin have short in vivo half-lives and exhibit low levels of therapeutic efficacy against obesity, even when administered at high doses three times daily.

[0016] Meanwhile, ongoing efforts are being made to overcome the problem of short in vivo half-lives of therapeutic peptides, to preserve high levels of pharmacological effects over a longer period of time and thus maximize the efficacy of the therapeutic agent. US 7,141,547 discloses a fusion protein of GLP-1 and its albumin analogue using recombinant DNA technology, and US 8,273,854 discloses a fusion protein of GLP-1 and its immunoglobulin fragment (Fc) analogue. These technologies partially improve the problem of short in vivo half-life of peptides, but they cannot eliminate the immunogenicity-related problems caused by the administration of non-human natural proteins. Therefore, a drawback of these technologies is that the pharmacological efficacy of drugs may decrease when administered over long periods. Furthermore, another problem is the need for large-scale cell culture and purification systems for drug production, and the difficulty in controlling drug quality because, due to the nature of recombinant proteins, drugs may contain impurities derived from host cells, and each batch may not be entirely identical. Moreover, when using peptides with disulfide bonds, such as calcitonin, the disadvantage is that misfolding may reduce yield. Furthermore, when the peptide contains non-natural amino acid residues, it is difficult to produce drugs using recombinant protein production methods.

[0017] Meanwhile, US 8,110,665 discloses a method for improving the half-life of peptides by preparing conjugates using non-peptide polymers and immunoglobulin fragments (Fc). However, this patent describes a complex manufacturing process involving the separate production of bioactive peptides, non-peptide polymers, and immunoglobulin fragments, as well as the combination of the peptides, polymers, and immunoglobulin fragments, which introduces problems such as residual byproducts and reduced yield.

[0018] Meanwhile, the PEGylation of therapeutic peptides and proteins is the most effective pharmaceutical technique for improving in vivo half-life. PEGylation of peptides and proteins increases their molecular weight, protects protein hydrolysis sites, and masks immunogenic sites, thereby increasing the in vivo half-life of the drug and reducing the immunogenicity of the peptides and proteins. Therefore, PEGylation technology is effective in enhancing therapeutic efficacy by addressing problems related to peptide drugs. Due to these advantages, PEGylation of peptides and proteins plays an important role in enhancing the therapeutic efficacy of drug delivery systems.

[0019] However, a disadvantage of using the PEG method is that PEGylation reduces the activity of the peptide drug and decreases the yield due to the decreased reactivity of the peptide with increasing PEG molecular weight. In this regard, there is a need for a PEGylation method that utilizes a simple production process and a highly selective reaction.

[0020] Therefore, there is a need for therapeutic agents for treating obesity, diabetes, or non-alcoholic fatty liver disease, which have the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels, without any side effects such as vomiting or nausea, and can be obtained in high yields through optimized preparation methods.

[0021] [Prior Art Documents] [Patent Documents] Patent Document 1: Korean Patent No. 0925017 entitled "Oxyntomodulin for Preventing or Treating Excess Weight" Patent Document 2: US Patent No. 7141547 entitled "Albumin Fusion Proteins Comprising GLP-1 Polypeptides" Patent Document 3: US Patent No. 8273854 entitled "GLP-1 Analog Fusion Proteins" Patent Document 4: US Patent No. 8110665 entitled "Pharmaceutical Composition Comprising an Immunoglobulin FC Region as a Carrier" Patent Document 5: Pharmaceutical Composition for Preventing or Treating Nonalcoholic Fatty Liver Disease The invention relates to Korean Patent No. 1665009, registered in China, page 3 / 18, document number 6, titled "Composition for Preventing or Treating Non-alcoholic Fatty Liver Diseases," published in CN 121591873 A. The invention aims to address the aforementioned problems by developing a therapeutic agent for treating obesity, diabetes, or non-alcoholic fatty liver disease. This therapeutic agent is safe and has the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels without any side effects such as vomiting or nausea. Furthermore, the inventors have developed a method for preparing the therapeutic agent in high yield and for preparing polypeptides having an amino acid sequence represented by the following general formula 1. Therefore, the inventors have demonstrated that compositions containing polypeptides have excellent effects in preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease, and that site-specific conjugates with polypeptides and non-peptide polymers prevent or treat obesity, diabetes, or non-alcoholic fatty liver disease by increasing the half-life of the polypeptide in the blood while maintaining its in vivo activity.

[0022] [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-imidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK, EQAAK, or EEAVK; and R3 is a deletion, cysteine, lysine, or methionine.

[0023] Therefore, the object of the present invention is to provide a pharmaceutical composition comprising a polypeptide for the prevention or treatment of obesity, diabetes, or non-alcoholic fatty liver disease.

[0024] Solution to the problem In order to solve the above problems, a pharmaceutical composition according to an exemplary embodiment of the present invention comprises a polypeptide having an amino acid sequence represented by the following general formula 1.

[0025] [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl, 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.

[0026] The polypeptide can be covalently bound to or can form microspheres with any one or more of the following groups: non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin, and derivatives thereof.

[0027] R2 contains glutamic acid (E) and lysine (K), and the glutamic acid and lysine can form a ring together via an amide bond, which may contribute to the α-helical structure of the polypeptide.

[0028] The non-peptide polymer can be selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Derivatives of non-peptide polymers known in the relevant art and other derivatives that can be readily prepared at the level of the prior art also fall within the scope of the present invention.

[0029] Preferably, the non-peptide polymer can be polyethylene glycol or a derivative thereof.

[0030] The molecular weight of the non-peptide polymer can be from 3,000 to 100,000 Da.

[0031] In this case, the polyethylene glycol derivative may be at least one selected from the group consisting of: methoxy polyethylene glycol, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, polyethylene glycol propionate succinimide (PEG propionate succinimide), methoxy polyethylene glycol propionate succinimide (methoxy PEG propionate succinimide), acrylate polyethylene glycol propionate succinimide (acrylate PEG propionate succinimide), thiol polyethylene glycol propionate succinimide (thiol PEG propionate succinimide) Hydroxysuccinimide polyethylene glycol (hydroxysuccinimide PEG), methoxy polyethylene glycol succinimide carboxymethyl ester (mPEG succinimide carboxymethyl ester), polyethylene glycol succinimide carboxymethyl ester (PEG succinimide carboxymethyl ester), polyethylene glycol carbonate succinimide ester (PEG carbonate succinimide ester), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of derivatives thereof.

[0032] The polyethylene glycol or its derivatives may be linear or branched.

[0033] The pharmaceutical composition may be used for the prevention or treatment of one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0034] The non-alcoholic fatty liver disease may include one or more diseases selected from the group consisting of: non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer.

[0035] A method for preparing a pharmaceutical composition according to another exemplary embodiment of the present invention comprises mixing a non-peptide polymer with a polypeptide having an amino acid sequence represented by the following general formula 1 to react with each other.

[0036] [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl 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.

[0037] The non-peptide polymer may be selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Derivatives of non-peptide polymers known in the relevant art and other derivatives that can be readily prepared at the level of the prior art also fall within the scope of the present invention.

[0038] Preferably, the non-peptide polymer can be polyethylene glycol or a derivative thereof.

[0039] In this case, the polyethylene glycol derivative can be at least one selected from the group consisting of: methoxy polyethylene glycol, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, polyethylene glycol propionate succinimide (PEG propionate succinimide), methoxy polyethylene glycol propionate succinimide (methoxyPEG propionate succinimide), acrylate polyethylene glycol propionate succinimide (PEG propionate succinimide), thiol polyethylene glycol propionate succinimide Esters (thiol PEG propionate succinimide ester), hydroxysuccinimide-based polyethylene glycol (hydroxysuccinimide-based PEG), methoxy polyethylene glycol succinimide-based carboxymethyl ester (mPEG succinimide-based carboxymethyl ester), polyethylene glycol succinimide-based carboxymethyl ester (PEG succinimide-based carboxymethyl ester), polyethylene glycol carbonate succinimide ester (PEG carbonate succinimide ester), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of derivatives thereof.

[0040] The reaction of the non-peptide polymer with the peptide may involve reacting the peptide and the non-peptide polymer at a molar ratio of 1:1 to 1:5.

[0041] The reaction of the non-peptide polymer with the peptide may be carried out at pH 4.0 to 9.0.

[0042] When the non-peptide polymer is mixed with the polypeptide to react with each other, the reaction time can be within the range of 0.5 to 24 hours.

[0043] 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.

[0044] The 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.

[0045] A method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease according to another exemplary embodiment of the invention comprises administering the pharmaceutical composition to a subject.

[0046] Advantageous effects of the invention The pharmaceutical composition according to the invention can contain polypeptides and therefore has the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and reducing triglyceride levels.

[0047] Moreover, the pharmaceutical composition according to the invention can contain polypeptides and therefore can reduce side effects such as vomiting or nausea.

[0048] In addition, the pharmaceutical compositions according to the invention may contain non-peptide polymers that are highly selective and react with peptides and can therefore be prepared in high yields.

[0049] Further, the pharmaceutical composition according to the invention may comprise conjugates including peptides and non-peptide polymers and thus may have a long in vivo half-life and high therapeutic efficacy for obesity even when administered at low doses, and may also have the effect of lowering blood glucose levels so as to maintain blood glucose at normal levels and effectively lowering triglyceride levels. Brief Description of the Drawings

[0050] Figure 1 shows the HPLC results of the conjugate of Example 2 comprising peptides and non-peptide polymers.

[0051] Figure 2 shows the MALDI-TOF results of the conjugate of Example 2 comprising peptides and non-peptide polymers.

[0052] Figure 3 shows the results of measuring glycated hemoglobin (HbA1c) levels after treatment with the conjugate of Example 2 to determine the extent of change in long-term average blood glucose concentration (**p<0.01).

[0053] Figure 4 shows the results of the final body weight of mice after treatment with the conjugate of Example 2 at different dosing frequencies for two weeks (***p<0.001).

[0054] Figure 5 shows the results of changes in blood glucose in mice after administration of the conjugate of Example 2 or 6.

[0055] Figure 6 shows the results of the intraperitoneal glucose tolerance test (ipGTT) after administration of the conjugate of Example 2 or 6.

[0056] Figure 7 shows the results of changes in serum cholesterol measurements after administration of the conjugate of Example 2.

[0057] Figure 8 shows the results of changes in liver weight measurements after administration of the conjugate of Example 2.

[0058] Figure 9 shows the results of observing mouse liver tissue after administration of the conjugate of Example 2 (dark stained areas represent normal liver tissue, and white (bright) stained areas represent lipid droplets).

[0059] Figure 10 shows the results of changes in serum cholesterol measurements after administration of the conjugate of Example 2.

[0060] Figure 11 shows the results of changes in liver weight measurements after administration of the conjugate of Example 2.

[0061] Figure 12 shows the results of changes in liver triglyceride levels after administration of the conjugate of Example 2.

[0062] Figure 13 shows the results of observing mouse liver tissue after administration of the conjugate of Example 2 (dark areas represent normal liver tissue, and white (bright) areas represent lipid droplets).

[0063] Figure 14 shows the results of measuring the NAFLD activity score (NAS) after administration of the conjugate of Example 2. Specification 6 / 18 pages 9 CN 121591873 A Detailed Description

[0064] The present invention provides a polypeptide having an amino acid sequence represented by the following general formula 1.

[0065] [General Formula 1] R1-X1-QGTFTSDYSKYFD-R2-EFVQWFMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl, 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.

[0066] 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 diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0067] [General Formula 1] R1-X1-QGTFTSDYSKYFD-R2-EFVQWFMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl, 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.

[0068] The present invention provides a pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the following general formula 1.

[0069] [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, wherein R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl, 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.

[0070] The present invention provides a pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the above General Formula 1 for the prevention or treatment of diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0071] The amino acids mentioned herein are abbreviated according to the IUPAC-IUB nomenclature rules, as listed in Table 1 below.

[0072] [Table 1] Specification 7 / 18 pages 10 CN 121591873 A

[0073] In general formula 1, R1 is preferably histidine at the N-terminus of the polypeptide, but the invention is not limited thereto.

[0074] X1 is preferably glycine or Aib, more preferably Aib. In this case, X1 is not particularly limited as long as it can enhance the chemical stability of the polypeptide.

[0075] Moreover, X1 is preferred as long as it can be resistant to dipeptidyl peptidase-4 (DPP-4), thereby enhancing the stability of the enzyme.

[0076] R2 is preferably EQAAK or EEAVK, more preferably EQAAK, but the invention is not limited thereto.

[0077] R2 contains glutamic acid (E) and lysine (K), and the glutamic acid and lysine preferably form a ring together via an amide bond, but the invention is not limited thereto. Thus, when two residues in the amino acid sequence of the polypeptide form a covalent ring via an amide bond, the covalent ring can enhance in vivo stability and improve the ability to bind to glucagon receptors or glucagon derivative receptors. Moreover, the covalent ring can contribute to the α-helical structure of the polypeptide.

[0078] R3 is the C-terminus of the polypeptide, which can bind to substances to enhance the in vivo half-life or in vivo sustainability. In this case, R3 is preferably cysteine, but the invention is not limited thereto.

[0079] The polypeptide may have 70% to 90% sequence homology with the amino acid sequence shown in SEQ ID NO. 1 (SEQ ID NO. 1: HSQGTFTSDYSKYLDSR-RAQDFVQWLMNT).

[0080] Here, it has been reported that the amino acid sequence described in SEQ ID NO. 1 is partially or entirely identical to the amino acid sequence of natural glucagon, and natural glucagon promotes the degradation of glycogen and insulin and exhibits anti-obesity effects. However, the use of natural glucagon as a therapeutic agent is limited due to its low solubility at neutral pH and its precipitation.

[0081] That is, a polypeptide containing an amino acid sequence having 70% to 90% sequence homology with the amino acid sequence described in SEQ ID NO. 1 may be a glucagon derivative or a gastrin derivative. In this case, the gastrin derivative is a peptide made from a glucagon precursor (e.g., proglucagon).

[0082] Preferably, the polypeptide may have 73% to 90%, more preferably 75% to 90%, sequence homology with the amino acid sequence described in SEQ ID NO. 1, but the invention is not limited thereto.

[0083] In this specification, the term "homology" refers to the degree of similarity to wild-type amino acid sequences and wild-type nucleic acid sequences. In this case, a comparison of homology between these sequences is performed using an available comparison program. Commercially available computer programs can be used to calculate the homology between two or more sequences in the form of a percentage (%). Homology (%) can be calculated for adjacent sequences. A large number of peptides can be obtained by inserting the polynucleotide encoding the peptide into a vector and expressing the peptide.

[0084] In this specification, the term "peptide" refers to a compound in which two or more α-amino acids are linked by peptide bonds.

[0085] Meanwhile, the polypeptide can covalently bind to or form microspheres with any one or more of the following groups: non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin and derivatives thereof.

[0086] Preferably, the non-peptide polymer is covalently bound to the polypeptide, but the invention is not limited thereto.

[0087] The polypeptide covalently binds to or forms microspheres with the above-mentioned substances, and thus has the effects of enhancing blood stability, delaying drug release into the kidneys, and inducing changes in receptor affinity.

[0088] When the polypeptide is covalently bound to a non-peptide polymer, the polypeptide can enhance its in vivo half-life and prolong its in vivo retention time. In this case, the binding site between the non-peptide polymer and the polypeptide can vary depending on the functional groups of the non-peptide polymer and the amino acid sequence of the polypeptide. Preferably, the binding site is not particularly limited as long as the non-peptide polymer polymerizes to the C-terminus of the polypeptide or can be prepared in high yield due to a high reaction rate.

[0089] When the non-peptide polymer binds to the polypeptide, the non-peptide polymer having a maleimide group can bind to the polypeptide using the thiol group (-SH) of the C-terminal cysteine ​​of the polypeptide, or the non-peptide polymer having a succinimide derivative can bind to the polypeptide using the amino group of the lysine (K) of the polypeptide.

[0090] The non-peptide polymer can be selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. Preferably, the non-peptide polymer is polyethylene glycol or a derivative thereof, but the invention is not limited thereto. Derivatives of non-peptide polymers known in the relevant art and other derivatives that can be readily prepared with prior art also fall within the scope of this invention.

[0091] The polyethylene glycol derivative may be at least one selected from the group consisting of: methoxy polyethylene glycol, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, polyethylene glycol propionate succinimide (PEG propionate succinimide), methoxy polyethylene glycol propionate succinimide (methoxyPEG propionate succinimide), acrylate polyethylene glycol propionate succinimide (acrylate PEG propionate succinimide), thiol polyethylene glycol propionate succinimide (thiol... The polyethylene glycol derivatives include: PEG-succinimide ester, hydroxysuccinimide-based polyethylene glycol (hydroxysuccinimide-PEG), methoxy-based polyethylene glycol succinimide-carboxymethyl ester (mPEG-succinimide-carboxymethyl ester), acrylate-based polyethylene glycol succinimide-carboxymethyl ester (PEG-succinimide-carboxymethyl ester), polyethylene glycol carbonate succinimide ester (PEG-succinimide-carboxymethyl ester), polyethylene glycol propionaldehyde (PEG-propionaldehyde), polyethylene glycol butyraldehyde (PEG-butyraldehyde), and their derivatives and multi-branched forms. Preferably, the polyethylene glycol derivative is a linear methoxy-based polyethylene glycol maleimide, a di-branched methoxy-based polyethylene glycol maleimide, or a tri-branched methoxy-based polyethylene glycol maleimide, more preferably a tri-branched methoxy-based polyethylene glycol maleimide.

[0092] The polyethylene glycol or its derivatives that may be used herein are linear or branched, preferably di- or tri-branched, and more preferably tri-branched.

[0093] The molecular weight of the non-peptide polymer may be from 3,000 to 100,000 Da, preferably from 20,000 to 70,000 Da, and more preferably from 40,000 to 60,000 Da. When the molecular weight of the non-peptide polymer is within this molecular weight range, the non-peptide polymer may bind to the polypeptide to enhance the solubility of the resulting conjugate and prolong the in vivo retention time of the conjugate.

[0094] Therefore, the pharmaceutical composition according to the invention comprises a conjugate having a non-peptide polymer bound to a polypeptide, and thus can enhance in vivo stability and prolong in vivo half-life.

[0095] Moreover, the pharmaceutical composition according to the invention comprises a polypeptide or a conjugate comprising a polypeptide and a non-peptide polymer and can therefore be used in pharmaceutical compositions to prevent or treat one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

[0096] Moreover, the pharmaceutical compositions according to the present invention comprising polypeptides or conjugates comprising polypeptides and non-peptide polymers can be used in pharmaceutical compositions to prevent or treat diseases caused by insulin deficiency or decreased insulin sensitivity.

[0097] Diseases caused by insulin deficiency or decreased insulin sensitivity may include type 1 diabetes, type 2 diabetes, and diabetic complications.

[0098] Moreover, the pharmaceutical compositions according to the present invention comprising peptides or conjugates comprising peptides and non-peptide polymers can be used as pharmaceutical compositions to prevent, improve or treat diseases such as hyperlipidemia, cardiovascular disease, arteriosclerosis and lipid-related metabolic syndrome.

[0099] Moreover, the pharmaceutical compositions according to the present invention comprising peptides or conjugates comprising peptides and non-peptide polymers can be used as pharmaceutical compositions to prevent, improve or treat liver diseases such as liver cancer, cirrhosis, non-alcoholic steatohepatitis and non-alcoholic fatty liver disease.

[0100] When the compositions of the present invention are used as pharmaceutical products, the pharmaceutical compositions comprising peptides can be formulated into various dosage forms for oral or parenteral administration, and then these dosage forms are clinically administered, but the present invention is not limited thereto.

[0101] Orally administered formulations include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, lozenges, etc. In addition to the active ingredient, these formulations also contain diluents (e.g., lactose, dextran, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). These tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone, and may optionally contain disintegrants such as starch, agar, alginate, or its sodium salt, or effervescent mixtures and / or absorbents, colorants, flavorings, and sweeteners.

[0102] Pharmaceutical compositions containing polypeptides can be administered parenterally. In this case, parenteral administration is carried out by methods such as subcutaneous injection, intravenous injection, intramuscular injection, intranasal spray, administration through mucous membranes to the nasal cavity or intestine, inhalation, or intrapleural injection.

[0103] In this case, to allow for the preparation of 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 into unit dosage forms such as ampoules or vials. The composition can be sterile and / or contain adjuvants such as preservatives, stabilizers, wetting agents or emulsifying agents, salts or buffers for osmotic regulation, and other therapeutically useful substances. In this case, the composition can be formulated according to conventional methods such as mixing, granulation, or coating methods.

[0104] The amount of the polypeptide-containing pharmaceutical composition to be administered to the human body according to the invention can vary depending on the patient's age, weight, sex, administration mode, health condition, and severity of disease. For example, the pharmaceutical composition can be administered orally or parenterally at a dose of 0.001 to 200 mg / kg / day, depending on the judgment of a physician or pharmacist.

[0105] Moreover, the invention provides a method for preparing a pharmaceutical composition comprising a conjugate containing a polypeptide and a non-peptide polymer.

[0106] First, in the method of preparing the pharmaceutical composition, the polypeptide has an amino acid sequence represented by general formula 1 as described above. Furthermore, the non-peptide polymer is as described above, and therefore a detailed description of the polypeptide and non-peptide polymer will be omitted.

[0107] Specifically, the method of preparing the pharmaceutical composition includes mixing the non-peptide polymer with the polypeptide to react with each other. In this case, the polypeptide and non-peptide polymer can react in a molar ratio of 1:1 to 1:5, such that the polypeptide and non-peptide polymer can bind to each other in a molar ratio of 1:1. In this case, it is preferable to mix in a molar ratio of 1:1 to 1:2, and more preferably in a molar ratio of 1:1.2, but the invention is not limited thereto. When mixing is performed within this molar ratio range, conjugates can be obtained in high yield (see specification 10 / 18 pages 13 CN 121591873 A), which makes it possible to prepare high-purity conjugates containing both polypeptides and non-peptide polymers.

[0108] According to an exemplary embodiment of the invention, the conjugate can also be prepared by covalently linking the non-peptide polymer to the C-terminus of the polypeptide. For example, a conjugate can be prepared by using methoxy polyethylene glycol with maleimide groups as a non-peptide polymer and using the polypeptide with a cysteine ​​residue at its C-terminus as the polypeptide, and thus the conjugate can have a high yield and a prolonged blood half-life.

[0109] The mixing of the non-peptide polymer and the polypeptide to react with each other can be carried out at pH 4.0 to 9.0, preferably at pH 5.5 to 7.5, but the invention is not limited thereto. When mixing is carried out outside this pH range, the yield will decrease. For example, when methoxy polyethylene glycol with maleimide groups is used as a non-peptide polymer and a polypeptide with a cysteine ​​residue at its C-terminus is mixed as a polypeptide, the mixing is preferably carried out at pH 6 to 8. When the polypeptide and methoxy polyethylene glycol react within this pH range, side reactions such as ring-opening of maleimide can be suppressed without causing side reactions caused by the amino groups of the polypeptide.

[0110] Because the yield of the conjugate when the non-peptide polymer is mixed with the peptide to react with each other is 85% to 95%, the process will be economically feasible and highly renewable due to the high yield. Therefore, the process will be effectively used to prepare pharmaceuticals.

[0111] When the non-peptide polymer is mixed with the peptide to react with each other, the reaction time can be in the range of 0.5 to 24 hours or 1 to 24 hours, and preferably 2 hours, but the invention is not limited thereto. When the reaction time is less than 0.5 hours, the yield will decrease and the purity will decrease. On the other hand, when the reaction time exceeds 24 hours, the peptide may be degraded or the economic efficiency will decrease due to the long processing time.

[0112] Moreover, when the non-peptide polymer is mixed with the peptide to react with each other, the temperature can be in the range of 0 to 100°C, preferably 4 to 40°C, but the invention is not limited thereto.Moreover, there are no particular limitations on temperature as long as the peptide or non-peptide polymer does not undergo a chemical change.

[0113] When mixing the non-peptide polymer with the peptide to react with each other, each of the peptide and non-peptide polymer can be dissolved using the same or different solvents. Preferably, the solvent is a buffer solution, ethanol, dimethyl sulfoxide (DMSO) or a mixture thereof, but the invention is not limited thereto. Moreover, the solvent includes solvents that can be readily used in the relevant fields.

[0114] 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, the method comprising administering a pharmaceutical composition comprising a peptide to a subject.

[0115] Moreover, 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, the method comprising administering a pharmaceutical composition comprising a peptide to a subject other than a human.

[0116] Mode of Invention Hereinafter, the invention will be described in detail with reference to the accompanying drawings, which will enable those skilled in the art to readily practice the invention. However, it should be understood that the invention may be embodied in various forms but is not intended to be limited to this context. Throughout the specification, similar reference numerals refer to similar elements.

[0117] Example 1: A polypeptide in which cysteine ​​is introduced (molecular weight: 3,509 Da; SEQ ID NO. 2: H(Aib) QGTFTSDYSKYLDEQAAKEFVQWLMNTC).

[0118] Here, underlined residues in the amino acid sequence of SEQ ID NO. 2 are highlighted in bold to indicate that a covalent ring is formed between the residues.

[0119] Preparative Example 1: Synthesis of a conjugate containing a polypeptide and a non-peptide polymer To prepare a conjugate containing a polypeptide and a non-peptide polymer, a polypeptide (molecular weight: 3,509 Da; SEQ ID NO. 2: H(Aib) QGTFTSDYSKYLDEQAAKEFVQWLMNTC) in which cysteine ​​is introduced into the C-terminal region (position 30) was used as the polypeptide. (See specification page 11 / 18, CN 121591873 A QGTFTSDYSKYLDEQAAKEFVQWLMNTC)

[0120] Meanwhile, as listed in Table 2 below, maleimide-activated monomethoxy PEG (mPEG-MAL, NOF (Japan)) was used as a non-peptide polymer.

[0121] In order to prepare the conjugates of Examples 2 to 7, polypeptides as listed in Table 2 below were prepared. In this case, each polypeptide was dissolved in dimethyl sulfoxide (DMSO), and mPEG-MAL was dissolved in 50 mM phosphate buffered saline (pH 6).

[0122] [Table 2]

[0123] In the amino acid sequences SEQ ID NO. 2 to 4 listed in Table 2, the two residues underlined and highlighted in bold refer to residues having a covalent ring formed between the residues.

[0124] The peptide and non-peptide conjugate were mixed at 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 at a flow rate of 0.8 mL / min using a TSK SP-5PW column (7.5 × 75 mm, Tosoh, Japan). The separation was monitored at a UV wavelength of 280 nm. The polyethylene glycol-modified peptide 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 polyethylene glycol-modified peptide (see Figure 1). Next, the molecular weight of the polyethylene glycol-modified peptides was measured using a MALDI-TOF mass spectrometer (see Figure 2). Furthermore, the yields of the conjugates in Examples 2 to 7 were calculated as the area ratio of the conjugate to the peptide based on the chromatograms obtained during the chromatographic separation process. The results are listed in Table 3.

[0125] [Table 3] Specification 12 / 18 pages 15 CN 121591873 A

[0126] As listed in Table 3, it was confirmed that the conjugates were prepared in yields of 90% or higher. Therefore, the method for preparing the pharmaceutical composition according to the invention has the advantage that it can be effectively used to prepare therapeutic agents because the conjugates are obtained in high yields due to their high reactivity with peptides, and the method is economically feasible and highly renewable due to the simplicity of the preparation process.

[0127] Experimental Example 1: Measurement of in vitro activity of Example 2 To examine the preventive or therapeutic effects of the conjugate of Example 2 on obesity, diabetes, and non-alcoholic fatty liver disease, this experiment was performed using cell lines expressing GLP-1 (glucagon derivative) receptor and glucagon receptor (GCGR).

[0128] To determine the activity against the GLP-1 receptor, HEK293 / CRE-Luc cells expressing human glucagon GLP-1 receptor were purchased from GenScript and used.Cells were seeded at 5 x 10⁴ cells / well in 96-well plates and then the wells were treated with the following: the peptide of Example 1 (0.001 to 300 nM), the conjugate of Example 2 (0.001 to 300 nM), natural glucagon (SEQ ID NO.1: HSQGTFTS-DYSKYLDSRRAQDFVQWLMNT, 0.013 to 300 nM), and GLP-1 (SEQ ID NO.6: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, 0.001 to 300 nM). The cells were then incubated in a CO₂ incubator at 37°C for 4 hours. Subsequently, the generated cAMP (luciferase reporter gene) was measured using a One-Glo™ luciferase assay system (Promega) to calculate the EC50 value relative to the GLP-1 receptor. The results are listed in Table 4 below.

[0129] Next, to determine the activity against the glucagon receptor (GCGR), the cAMP Hunter™ eXpress GCGR CHO-K1 GPCR kit from DiscoverX was used. CHO-K1 cells expressing the human glucagon receptor were seeded in 96-well plates at a density of 3 x 10⁴ cells / well. Each well was then treated with the following: the peptide from Example 1 (0.013 to 300 nM), the conjugate from Example 2 (0.013 to 300 nM), native glucagon (0.015 to 33.33 nM), and GLP-1 (0.001 to 30.00 nM), and the cells were then incubated in a CO₂ incubator at 37°C for 30 minutes. The amount of cAMP generated was then measured to calculate the EC50 value relative to the glucagon receptor (GCGR). The results are listed in Table 4 below.

[0130] [Table 4]

[0131] As listed in Table 4, GLP-1 exhibits high activity against the GLP-1 receptor but very low activity against the glucagon receptor. On the other hand, natural glucagon exhibits very high activity against the glucagon receptor but low activity against the GLP-1 receptor. Based on these results, the experimental method is confirmed to have high selectivity.

[0132] Meanwhile, it was confirmed that the EC50 value of the peptide of Example 1 on the GLP-1 receptor was 0.13, which is almost the same as the value of GLP-1 as described on pages 13 / 18 of the specification, CN 121591873 A. This indicates that the peptide of Example 1 exhibits high activity against the GLP-1 receptor and also has activity against the glucagon receptor. Moreover, animal experiments confirmed that the peptide of Example 1 has an anti-obesity effect.

[0133] Moreover, it was confirmed that the conjugate of Example 2 retained similar activity on the GLP-1 receptor and glucagon receptor compared to the peptide of Example 1. It was confirmed that when a non-peptide polymer (e.g., PEG) is bound to the peptide, the activity of the peptide on the receptor is generally significantly reduced compared to before conjugation, while even when a non-peptide polymer is bound to the peptide, the activity of the conjugate of Example 2 is reduced less, indicating that the conjugate of Example 2 exhibits an extended in vivo half-life while maintaining high activity.

[0134] Therefore, it was confirmed that the pharmaceutical composition according to the invention has excellent activity on the glucagon receptor and GLP-1 receptor, and thus has anti-diabetic and anti-obesity effects and triglyceride-lowering effects by suppressing appetite, enhancing insulin secretion and promoting lipolysis in adipocytes.

[0135] Experimental Example 2: Measurement of in vivo activity of Example 2 1 To examine 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 to measure changes in food intake, blood glucose, and body weight. The results are listed in Table 5.

[0136] First, an animal model of obesity was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the average weight of the mice to approximately 50 g. Thereafter, the conjugate of Example 2 was administered subcutaneously at a dose of 20 nmol / kg every other day for 2 weeks. As a positive control, the GLP-1 agonist liraglutide was also administered subcutaneously at a dose of 100 nmol / kg once daily for 2 weeks. During the 2 weeks of drug administration, food intake, blood glucose, and body weight were measured every other day at given time points. The results are listed in Table 5 below.

[0137] In this case, body weight and blood glucose are expressed as percentages (%), with 100% as of before administration (day 0).

[0138] [Table 5]

[0139] In this case, the untreated group refers to a group of mice that were administered PBS instead of the conjugate of Example 2.

[0140] As listed in Table 5, the untreated group consumed approximately 40 g of food over 2 weeks, while the group of mice treated with the conjugate of Example 2 consumed approximately 18 g of food. This represents a more than 50% reduction in cumulative food intake compared to the untreated group. Food intake was similar in the groups of mice treated with the positive control and the conjugate of Example 2.

[0141] Meanwhile, referring to the pattern of weight change over time, no weight change was observed in the untreated group compared to the weight change observed at the application point (day 0), and the positive control group showed a weight reduction of approximately 15% compared to the weight observed before application, indicating poor efficacy of liraglutide in preventing or treating obesity.On the other hand, in the mouse group that was administered the conjugate of Example 2, the body weight decreased significantly by 61% compared to the body weight measured before administration.

[0142] Moreover, referring to the pattern of blood glucose changes over time, it was revealed that the blood glucose level decreased by approximately 80% compared to the blood glucose level measured before administration, indicating that the conjugate of Example 2 has a blood glucose-lowering effect, but the positive control had a very poor blood glucose-lowering effect.

[0143] Based on these results, it can be seen that the effect of the conjugate of Example 2 on weight loss is produced by an increase in energy metabolism in the body and a simple reduction in food intake. The pharmaceutical composition according to the invention is capable of reducing food intake, inhibiting gastric emptying, and promoting lipolysis.

[0144] Experimental Example 3: Measurement of In Vivo Activity of Example 2 2 This experiment was performed in the same manner as Experimental Example 2, and glucose tolerance was then assessed in a mouse model using an intraperitoneal glucose tolerance test (ipGTT).

[0145] After completing the 2-week drug administration in the same manner as in Experimental Example 2, 2 g / kg glucose was injected intraperitoneally to measure changes in blood glucose over time (0, 15, 30, 60, 90, and 120 minutes). The results are listed in Table 6 below.

[0146] [Table 6]

[0147] As listed in Table 6, based on the results obtained after 2 weeks of drug administration, it was confirmed that blood glucose levels in the untreated group rose sharply and then fell due to the administered glucose, but in the mouse group administered the conjugate of Example 2, the increase in blood glucose was significantly reduced. Therefore, it was confirmed that the glucose tolerance of the conjugate of Example 2 was improved compared to the untreated group. Moreover, it was revealed that the increase in blood glucose in the mouse group administered the conjugate of Example 2 was smaller compared to the positive control group.

[0148] Experimental Example 4: Measurement of In Vivo Activity of Example 2 3 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−+ Leprdb / + Leprdb / 01aHsd mice (db / db mice), and changes in blood glucose and body weight over time were measured.

[0149] 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. Changes in blood glucose and body weight were measured every other day during the 12 days of drug administration. The results are listed in Table 7 below.

[0150] Next, to perform an intraperitoneal glucose tolerance test (ipGTT), 2 g / kg glucose was administered intraperitoneally 12 days after drug administration, and changes in blood glucose over time (0, 15, 30, 60, 90, and 120 minutes) were measured. The results are listed 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.

[0151] [Table 7] Specification 15 / 18 pages 18 CN 121591873 A

[0152] [Table 8]

[0153] As listed in Table 7, weight loss was observed in the mouse group administered the conjugate of Example 2 compared to the untreated group. Moreover, it was confirmed that high blood glucose levels were maintained for 2 weeks in the untreated group, while blood glucose levels were reduced in the mouse group administered the conjugate of Example 2.

[0154] As listed in Table 8, it was also revealed that the mouse group administered the conjugate of Example 2 exhibited higher glucose tolerance compared to the untreated group.

[0155] Moreover, as shown in Figure 3, it was revealed that administration of the conjugate of Example 2 significantly reduced the level of glycated hemoglobin, indicating that blood glucose was stably maintained at a low level by administration of the conjugate of Example 2.

[0156] Experimental Example 5: Measurement of the in vivo activity of Example 2 4 To examine the effect of the concentration and frequency of administration of the conjugate of Example 2 on the prevention or treatment of obesity, the conjugate of Example 2 was administered to C57BL / 6 mice, and changes in food intake and body weight over time were measured.

[0157] First, an animal model of obesity was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks and increasing the average weight of the mice to approximately 50 g. Subsequently, the conjugate of Example 2 was administered to each group in this group for 2 weeks, as listed in Table 9 below. After 2 weeks, the final body weight of the mice was measured. The results are shown in Figure 4.

[0158] [Table 9]

[0159] As shown in Figure 4, it is revealed that the conjugate of Example 2 has a more significant effect on weight loss when administered at increasingly higher doses, and even when administered at a low dose of 20 nmol / kg, the conjugate of Example 2 has a significant effect on weight loss. Therefore, it was confirmed that the conjugate of Example 2 exhibited a dose-dependent response. Furthermore, it was confirmed that even when the interval between administrations of the conjugate of Example 2 was extended to once a week, the conjugate of Example 2 maintained the same effect on weight loss. Therefore, the pharmaceutical composition for the prevention or treatment of obesity according to the present invention has a long in vivo half-life, and as specified on pages 16 / 18 of the specification (CN 121591873 A), it can exhibit a high level of therapeutic effect on obesity even when administered at high doses three times daily.

[0160] Experimental Example 6: Measurement of In Vivo Activity of Examples 2 and 6 5 To examine the preventive or therapeutic effects of conjugates of Examples 2 and 6 with different amino acid sequences on obesity or diabetes, an animal model of obesity was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the average weight of the mice to approximately 50 g. Subsequently, each conjugate of Example 2 or 6 was administered subcutaneously at a dose of 20 nmol / kg every other day for 2 weeks. PBS was administered instead of the conjugates of the examples as a control. Blood glucose levels were measured over time during the 2 weeks of administration of the conjugates of Example 2 or 6. The results are shown in Figure 5. Furthermore, to perform an intraperitoneal glucose tolerance test (ipGTT), 2 g / kg glucose was administered intraperitoneally 2 weeks after drug administration, and blood glucose levels were measured over time (0, 15, 30, 60, 90, and 120 minutes). The results are shown in Figure 6.

[0161] As shown in Figure 5, it was revealed that the blood glucose levels of mice administered the conjugates of Example 2 or 6 were lower than those of the control group, but the blood glucose levels of the control group administered PBS were higher.

[0162] As shown in Figure 6, it was revealed that the blood glucose levels in the control group rose sharply and then fell due to the administered glucose, but the increase in blood glucose was significantly reduced in mice administered the conjugates of Example 2 or 6 for 2 weeks compared to the control group. Based on these results, it was confirmed that the glucose tolerance of the conjugates of Example 2 and 6 was improved compared to that of the control group.

[0163] Therefore, it was confirmed that the pharmaceutical composition comprising peptides according to the present invention has the preventive or therapeutic effects on obesity and diabetes as shown in Figures 5 and 6.

[0164] Experimental Example 7: Measurement of in vivo activity of Example 2 6 To examine the preventive or therapeutic effects of the conjugates of Example 2 on non-alcoholic fatty liver disease, the conjugates of Example 2 were 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.

[0165] Specifically, a laboratory animal model of non-alcoholic fatty liver disease was first established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the average weight of the mice to approximately 50 g. Subsequently, the conjugate of Example 2 was 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 daily at a dose of 100 nmol / kg for 2 weeks. Two weeks after drug administration, blood was collected from the mice to measure serum cholesterol concentrations, and the livers were extracted, weighed, paraffin-embedded, and then sectioned. Liver biopsies were then performed using hematoxylin and eosin (H&E).

[0166] Referring to Figures 7 and 8, which measure serum cholesterol levels and liver weight, it was confirmed that the mice administered the conjugate of Example 2 had significantly lower serum cholesterol levels and liver weight compared to the untreated group administered PBS, and also lower serum cholesterol levels and liver weight compared to the positive control group (i.e., the liraglutide treatment group). Moreover, referring to the liver biopsy results shown in Figure 9, it can be seen that the mice administered the conjugate of Example 2 had significantly lower hepatic steatosis compared to the untreated group administered PBS, and also lower hepatic steatosis compared to the positive control group (i.e., the liraglutide treatment group).

[0167] Thus, it is revealed that the pharmaceutical composition according to the invention for the prevention or treatment of non-alcoholic fatty liver disease is effective in the prevention and treatment of non-alcoholic fatty liver disease because the pharmaceutical composition reduces liver weight, serum cholesterol levels, and hepatic steatosis in animal models of non-alcoholic fatty liver disease.

[0168] Experimental Example 8: Measurement of In Vivo Activity of Example 2 7 To examine 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 (NAFLD) as per the instructions for use (pages 17 / 18, CN 121591873 A), and changes in serum cholesterol levels, liver weight, and liver triglycerides were measured. The results are shown in Figures 10 to 12.

[0169] First, a laboratory animal model of NAFLD was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a diet high in trans fat containing 40% high fat, 20% fructose, and 2% cholesterol for approximately 16 weeks. Subsequently, the conjugate of Example 2 was administered subcutaneously at a dose of 20 nmol / kg every three days for 4 weeks. As a positive control, the GLP-1 agonist liraglutide was also administered subcutaneously daily at a dose of 53 nmol / kg for 4 weeks. After the 4-week experiment was completed, serum cholesterol levels, liver weight, and liver triglycerides (liver TG) were measured.

[0170] Referring to Figures 10 to 12, which measure serum cholesterol levels, liver weight, and liver TG, it can be seen that the mice administered the conjugate of Example 2 had significantly lower serum cholesterol levels, liver weight, and liver triglycerides compared to the untreated group administered saline, and also lower than the positive control group (i.e., the liraglutide treatment group). Thus, it is revealed that the pharmaceutical composition according to the invention for the prevention or treatment of non-alcoholic fatty liver disease is effective in the prevention and treatment of non-alcoholic fatty liver disease because the pharmaceutical composition reduces liver weight, serum cholesterol levels, and liver triglycerides in animal models of non-alcoholic fatty liver disease.

[0171] Experimental Example 9: Measurement of In Vivo Activity of Example 2 8 After completing the experiment in the same manner as in Experimental Example 8, liver biopsies were performed and NAFLD activity scores (NAS) were measured to examine the preventive or therapeutic effect on non-alcoholic fatty liver disease. After administration of the conjugate of Example 2 for 4 weeks, the experiment was completed in the same manner as in Experimental Example 8, with mouse livers extracted, paraffin-embedded, and then cut into thin sections. Subsequently, hematoxylin and eosin (H&E) staining and Oil Red O staining were performed.

[0172] Thus, as shown in Figures 13 and 14, the liver histological and NAS results after 4 weeks of administration showed that the mice administered the conjugate of Example 2 had significantly reduced hepatic steatosis and NAS compared to the untreated group and the positive control group (i.e., the liraglutide treatment group) administered saline. Thus, it is revealed that the pharmaceutical composition according to the invention for the prevention or treatment of non-alcoholic fatty liver disease is effective in the prevention and treatment of non-alcoholic fatty liver disease because the pharmaceutical composition reduces hepatic steatosis and has reduced NAS in animal models of non-alcoholic fatty liver disease.

[0173] Although preferred embodiments of the invention have been described in detail above, it should be understood that many variations and / or modifications of the basic inventive concepts taught herein, which may be obvious to those skilled in the art, still fall within the scope of the invention as defined in the appended claims.

[0174] Industrial Applicability The polypeptide-containing pharmaceutical compositions according to the invention can be safely used for the prevention or treatment of obesity, diabetes, or non-alcoholic fatty liver disease because the pharmaceutical compositions have the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels without any side effects such as vomiting or nausea.Instruction Manual 18 / 18 Page 21 CN 121591873 A Figure 1 Figure 2 Instruction Manual Appendix 1 / 6 Page 22 CN 121591873 A Figure 3 Figure 4 Instruction Manual Appendix 2 / 6 Page 23 CN 121591873 A Figure 5 Figure 6 Instruction Manual Appendix 3 / 6 Page 24 CN 121591873 A Figure 7 Figure 8 Instruction Manual Appendix 4 / 6 Page 25 CN 121591873 A Figure 9 Figure 10 Figure 11 Instruction Manual Appendix 5 / 6 Page 26 CN 121591873 A Figure 12 Figure 13 Figure 14 Instruction Manual Appendix 6 / 6 Page 27 CN 121591873 A Abstract Abdominal ultrasound examination method, system and device IRN: 538625 Title: PHARMACEUTICAL COMPOSITION COMPRISING POLYPEPTIDE Abstract: The present invention relates to a pharmaceutical composition including a polypeptide, and more particularly, to a pharmaceutical composition for preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease. The pharmaceutical composition is safe without any side effects such as vomiting or nausea, and has effects of reducing food intake, enhancing insulin secretion, suppressing gastric emptying, promoting lipolysis, and lowering a level of triglycerides.

Claims

1. A polypeptide having an amino acid sequence represented by the following general formula 1: [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl or β-carboxyl-imidazo-propionyl; X1 is a deletion, glycine or aminoisobutyric acid (Aib). R2 is EKRAK, EQAAK, or EEAVK; and R3 can be a deletion, cysteine, lysine, or methionine.

2. A pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the following general formula 1: [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl or β-carboxyl-imidazo-propionyl; X1 is a deletion, glycine or aminoisobutyric acid (Aib). R2 is EKRAK, EQAAK, or EEAVK; and R3 can be a deletion, cysteine, lysine, or methionine.

3. The pharmaceutical composition of claim 2, wherein the polypeptide is covalently bound to or forms microspheres with any one or more of the group selected from: non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin and derivatives thereof.

4. The pharmaceutical composition according to 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.

5. The pharmaceutical composition according to claim 2, wherein the non-peptide polymer is selected from the group consisting of: polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, chitosan, hyaluronic acid, and combinations thereof.

6. The pharmaceutical composition according to claim 3, wherein the non-peptide polymer is polyethylene glycol or a derivative thereof.

7. The pharmaceutical composition of claim 6, wherein the non-peptide polymer has a molecular weight of 3,000 to 100,000 Da.

8. The pharmaceutical composition of claim 6, wherein the polyethylene glycol derivative is at least one selected from the group consisting of: methoxy polyethylene glycol, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide, polyethylene glycol propionate succinimide (PEG propionate succinimide), methoxy polyethylene glycol propionate succinimide (methoxyPEG propionate succinimide), polyethylene glycol propionate succinimide acrylate (PEG propionate succinimide acrylate), thiol polyethylene glycol. Succinimide propionate (thiol PEG succinimide propionate), hydroxysuccinimide polyethylene glycol (hydroxysuccinimide PEG), methoxy polyethylene glycol succinimide carboxymethyl ester (mPEG succinimide carboxymethyl ester), polyethylene glycol succinimide carboxymethyl ester (PEG succinimide carboxymethyl ester), polyethylene glycol carbonate succinimide (PEG carbonate succinimide), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), their derivatives, and their multi-branched forms.

9. The pharmaceutical composition according to claim 6, wherein the polyethylene glycol or its derivative is linear or branched.

10. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is used for the prevention or treatment of obesity.

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