Pharmaceutical compositions comprising polypeptides

Abstract

The present application 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 an effect of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition comprising a polypeptide and its medical use in, for example, the treatment or prevention of obesity, diabetes, or non-alcoholic fatty liver disease. The polypeptide has the effect 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

[0002] Recently, with the rapid economic development and scientific and technological growth, the aging population is growing and adult diseases are rapidly increasing. This is due to stress, poor eating habits, excessive calorie intake, and reduced physical activity. As a complication accompanying obesity, the mortality rates of heart disease and cerebrovascular disease rank first and second, and obesity is considered to be the cause of various adult diseases such as diabetes, non-alcoholic fatty liver disease, etc.

[0003] Obesity refers to a state in which the amount of fat accumulation is higher than the normal amount and the most accurate method for assessing obesity is to measure the amount of body fat. However, it is expensive to accurately measure the amount of fat, and thus an indirect method is used to assess it. The most commonly used indirect method is to measure the body mass index (BMI) and the waist circumference. The World Health Organization (WHO) announced a classification based on data relating BMI to the risk of death: normal weight: 18.5 to 24.9 kg / m 2 , overweight: 25 to 29.9 kg / m 2 , and obesity: 30 kg / m 2 or more.

[0004] The cause of obesity is known to be energy imbalance due to excessive calorie intake and relatively reduced activity, and the resulting increase in body fat. However, it is difficult to attribute only one factor, because various risk factors such as eating habits, lifestyle, age, race, genetic factors, etc. are involved in obesity.

[0005] Diabetes is classified into insulin-dependent diabetes (Type I diabetes), insulin-independent diabetes (Type II diabetes), and malnutrition-related diabetes (MRDM). Type II diabetes, which accounts for more than 90% of diabetes patients, is a metabolic disease characterized by hyperglycemia and is reported to be caused by decreased insulin secretion from pancreatic beta cells or increased insulin resistance of peripheral tissues due to genetic factors, metabolic factors, and environmental factors. In this regard, when body fat increases, insulin sensitivity decreases, and in particular, the accumulation of abdominal fat is known to be associated with impaired glucose tolerance. Also, it is known that insulin resistance is closely related to obesity in patients with Type II diabetes, in which the more severe the obesity, the greater the insulin resistance.

[0006] Non-alcoholic fatty liver disease (NAFLD) refers to a series of diseases including simple steatosis not dependent on alcohol consumption, which comprises excessive accumulation of fat in hepatocytes, non-alcoholic steatohepatitis (NASH) comprising hepatocyte ballooning, inflammation, fibrosis, and more advanced cases, i.e., cirrhosis. The prevalence of non-alcoholic fatty liver disease is rapidly increasing along with the increase in the prevalence of obesity worldwide, and although the prevalence of diabetes varies from country to country, diabetes accounts for about 20 to 30% of the total population in western countries and its incidence reaches about 16% or so in Korea.

[0007] Non-alcoholic fatty liver disease shows a close relationship with metabolic syndrome including obesity, type II diabetes, dyslipidemia, etc. based on insulin resistance. In fact, it is known that many pre-diabetic and type II diabetic patients exhibit non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, and the progression rate of cirrhosis and liver cancer (e.g., hepatocellular carcinoma) is high in these patients. Meanwhile, the prevalence of diabetes is high in non-alcoholic fatty liver disease patients and is significantly high in non-alcoholic steatohepatitis patients.

[0008] It is mainly recommended that obese patients control their weight through a healthier diet and physical activity, but when these methods are ineffective, the patients can be treated with drugs or surgery.

[0009] The current anti-obesity drug market is estimated to reach more than about one billion dollars and to grow by about 10% per year. The drug primarily used as an anti-obesity drug is an anorectic agent (lorcaserine, phentermine, etc.) classified as a psychotropic drug that acts on the central nervous system in most cases. Such a drug is known to exhibit an effect of suppressing the appetite of a patient to reduce body weight, but has side effects such as abuse and addiction, palpitation, anxiety, insomnia, etc. when used for a long period of time.

[0010] Orlistat (Xenical) is one of the drugs used as an anti-obesity drug that is not a psychotropic drug. Pancreatic lipase serves as a key enzyme that decomposes triglyceride into 2-monoacylglycerol and fatty acid. A representative pancreatic lipase inhibitor is a derivative of lipstatin derived from Streptomyces toxitricini and orlistat (tetrahydrolipstatin) having a high level of efficacy in inhibiting the absorption of about 30% of ingested fat. Currently, orlistat is commercially available as a drug, but has side effects such as gastrointestinal disorders, allergic reactions, cholestasis, etc. Therefore, therapeutic agents that can be safely used for the treatment of obese patients are few.

[0011] In drug therapy for treating non-alcoholic fatty liver disease, drugs exacerbate mechanisms of non-alcoholic fatty liver disease such as insulin resistance, oxidative stress, apoptosis, inflammatory cytokines, etc., and inhibit the progression of non-alcoholic fatty liver disease. Among these, anti-diabetic agents are known to have effects of improving fatty liver by improving the pathophysiological state often seen at the onset of fatty liver and effects of lowering blood glucose. However, since no drugs have been approved as an indication for treating fatty liver disease at present, the medical need for developing effective therapeutic agents has not been met.

[0012] Meanwhile, the present focus is on glucagon derivatives. Glucagon is produced in the pancreas when blood glucose levels start to drop due to drug therapy, disease, hormone or enzyme deficiency, etc. Glucagon stimulates the liver to release glucose by breaking down glycogen and is used to raise blood glucose levels to normal levels. In addition to the effect of raising blood glucose, it has been reported that glucagon also inhibits appetite and activates hormone-sensitive lipase in fat cells to promote the degradation of fat, thereby exhibiting an anti-obesity effect. As one of such glucagon derivatives, glucagon-like peptide-1 (GLP-1) is a still-developing substance as a therapeutic agent for alleviating hyperglycemia in diabetic patients and is used to promote insulin synthesis and secretion, inhibit the secretion of glucagon, inhibit gastric emptying, promote the use of glucose, and inhibit food intake. Also, exenatide-4 made from lizard venom, which has about 50% amino acid homology with GLP-1, is known to activate GLP-1 receptors to alleviate hyperglycemia in diabetic patients. However, it has been reported that GLP-1 receptor agonists for treating obesity or diabetes have the problem of causing side effects such as vomiting and nausea.

[0013] As an alternative to GLP-1, oxyntomodulin, which can bind to both GLP-1 and glucagon receptors, has become the focus of attention. Oxyntomodulin is a peptide derived from the precursor of glucagon (i.e., proglucagon), exhibits effects of inhibiting food intake, inhibiting gluconeogenesis in the liver to regulate blood glucose levels, and increasing satiety by GLP-1, and has lipolytic function on glucagon. Therefore, oxyntomodulin is highly likely to be an anti-diabetic and anti-obesity drug.

[0014] Based on the dual function of the oxyntomodulin peptide, research for developing a drug for treating diabetes and obesity is actively being performed. For example, Registered Korean Patent No. 925017 discloses an oral, parenteral, mucosal, rectal, subcutaneous, or transdermal pharmaceutical composition for treating human overweight, which comprises oxyntomodulin as an effective ingredient. However, it is reported that the anti-obesity drug comprising oxyntomodulin has a short in vivo half-life and exhibits a low level of therapeutic effect on obesity even when administered three times a day at a high dose.

[0015] Meanwhile, efforts are continuously made to overcome the problem of a short in vivo half-life of therapeutic peptides to maintain a high level of pharmacological effect for a long period of time and thus maximize the effect of therapeutic agents. US 7,141,547 discloses a fusion protein of GLP-1 and its analogues with albumin using recombinant DNA technology, and US 8,273,854 discloses a fusion protein of GLP-1 and its analogues with an immunoglobulin fragment (Fc). These technologies partially improve the problem of a short in vivo half-life of peptides, but cannot escape the problem of immunogenicity caused by the administration of non-human natural proteins. Therefore, these technologies have the disadvantage that the pharmacological effect of a drug can be reduced when the drug is administered for a long period of time. Also, an additional problem is that a large-scale cell culture and purification system for producing a drug is required, and the quality of the drug is difficult to control because the drug can have impurities derived from host cells in terms of the properties of recombinant proteins and each batch of the drug can not be exactly the same. Also, when a peptide having a disulfide bond such as calcitonin is used, it has the disadvantage that the yield can be reduced due to misfolding. Further, when the peptide has a non-natural amino acid residue, it is difficult to produce a drug through a recombinant protein production method.

[0016] Meanwhile, US 8,110,665 discloses a conjugate prepared by using a non-peptide polymer and an immunoglobulin fragment (Fc) to improve a peptide having a short half-life. However, this patent describes a complex production process that involves separately producing a biologically active peptide, a non-peptide polymer, and an immunoglobulin fragment and binding the peptide, the polymer, and the immunoglobulin fragment together, which introduces problems such as residual by-products and a reduced yield.

[0017] Meanwhile, the pegylation of therapeutic peptides and proteins is the most effective drug technology for improving the in vivo half-life. The pegylation of peptides and proteins is used to increase their molecular weight, protect proteolytic sites, and mask immunogenic sites, thereby resulting in an increase in the in vivo half-life of a drug and a decrease in the immunogenicity of peptides and proteins. Therefore, the pegylation technology is effective in enhancing the therapeutic effect by solving the problems related to peptide drugs. Due to these advantages, the pegylation of peptides and proteins plays an important role in enhancing the therapeutic effect of a drug delivery system.

[0018] However, the method using PEG has a disadvantage in that the pegylation reduces the activity of the peptide drug and the yield is decreased due to poor reactivity of the peptide as the molecular weight of PEG increases. In this regard, there is a need for a method of pegylation by means of a simple production process and a highly selective reaction.

[0019] Accordingly, there is a demand for a therapeutic agent for treating obesity, diabetes, or non-alcoholic fatty liver disease, which has an effect of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and reducing triglyceride levels without any side effects such as vomiting or nausea, and which can be obtained in high yield by optimizing a preparation method.

[0020] [Prior Art Documents]

[0021] [Patent Documents]

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

[0023] Patent Document 2: Registered US Patent No. 7141547 entitled "Albumin Fusion Proteins Comprising GLP-1 Polypeptides"

[0024] Patent Document 3: Registered US Patent No. 8273854 entitled "GLP-1 Analog Fusion Proteins"

[0025] Patent Document 4: Registered US Patent No. 8110665 entitled "Pharmaceutical Composition Comprising an Immunoglobulin FC Region as a Carrier"

[0026] Patent Document 5: Registered Korean Patent No. 1665009 entitled "Pharmaceutical Composition for Preventing or Treating Non-alcoholic Fatty Liver Diseases" SUMMARY

[0027] To solve the above problems, the inventors of the present application have conducted research and endeavored to develop a therapeutic agent for treating obesity, diabetes, or non-alcoholic fatty liver disease, which is safe and also 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, and also developed a method capable of preparing the therapeutic agent at a high yield and a polypeptide having an amino acid sequence represented by the following general formula 1. Accordingly, the inventors of the present application have confirmed that a composition comprising the polypeptide has an excellent effect of preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease, and that a site-specific conjugate of the polypeptide and a non-peptide polymer has an effect of preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease by increasing the half-life of the polypeptide in blood while maintaining the in vivo activity of the polypeptide, thereby completing the present application.

[0028] [General Formula 1]

[0029] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (SEQ ID NOs. 7-9),

[0030] wherein R1 is histidine, desamino-histidyl, N-dimethyl-histidyl, β-imidazolopropionyl, 4-imidazoleacetyl, or β-carboxy-imidazolopropionyl;

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

[0032] R2 is EKRAK (SEQ ID NO. 10), EQAAK (SEQ ID NO. 11), or EEAVK (SEQ ID NO. 12); and

[0033] R3 is absent, cysteine, lysine, or methionine.

[0034] Accordingly, an object of the present application is to provide a pharmaceutical composition comprising a polypeptide for preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease.

[0035] Solution to the problem

[0036] To solve the above problems, the pharmaceutical composition according to one exemplary embodiment of the present application comprises a polypeptide having an amino acid sequence represented by the following general formula 1.

[0037] [General Formula 1]

[0038] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (SEQ ID NOs. 7-9),

[0039] 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 (SEQ ID NO.10), EQAAK (SEQ ID NO.11), or EEAVK (SEQ ID NO.12); and R3 is a deletion, cysteine, lysine, or methionine.

[0040] 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 its fragments, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin and its derivatives.

[0041] 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.

[0042] 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 with prior art also fall within the scope of this invention.

[0043] Preferably, the non-peptide polymer can be polyethylene glycol or a derivative thereof.

[0044] The molecular weight of this non-peptide polymer can be from 3,000 to 100,000 Da.

[0045] 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 (methoxyPEG propionate succinimide), acrylate polyethylene glycol propionate succinimide (acrylate 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), their derivatives, and their multi-branched forms.

[0046] The polyethylene glycol or its derivatives may be linear or branched.

[0047] This 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.

[0048] Nonalcoholic fatty liver disease can include one or more diseases selected from the group consisting of: nonalcoholic fatty liver, nonalcoholic steatohepatitis, cirrhosis, and liver cancer.

[0049] 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.

[0050] [General Formula 1]

[0051] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3(SEQ ID NOs.7-9),

[0052] 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 (SEQ ID NO.10), EQAAK (SEQ ID NO.11), or EEAVK (SEQ ID NO.12); and R3 is a deletion, cysteine, lysine, or methionine.

[0053] 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 with prior art also fall within the scope of this invention.

[0054] Preferably, the non-peptide polymer can be polyethylene glycol or a derivative thereof.

[0055] 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 (methoxyPEG propionate succinimide), acrylate polyethylene glycol propionate succinimide (acrylate 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), their derivatives, and their multi-branched forms.

[0056] The mixing of the non-peptide polymer with the peptide to react with each other may involve reacting the peptide and the non-peptide polymer in a molar ratio of 1:1 to 1:5.

[0057] The reaction of the non-peptide polymer with the peptide can be carried out at pH 4.0 to 9.0.

[0058] When the non-peptide polymer is mixed with the polypeptide to react with each other, the reaction time can be in the range of 0.5 to 24 hours.

[0059] This 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.

[0060] Nonalcoholic fatty liver disease can include one or more diseases selected from the group consisting of: nonalcoholic fatty liver, nonalcoholic steatohepatitis, cirrhosis, and liver cancer.

[0061] 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 present invention, comprises administering the pharmaceutical composition to a subject.

[0062] Advantages of the invention

[0063] The pharmaceutical compositions according to the invention may contain polypeptides and thus have the effects of reducing food intake, enhancing insulin secretion, inhibiting gastric emptying, promoting lipolysis, and lowering triglyceride levels.

[0064] Furthermore, the pharmaceutical composition according to the invention may contain polypeptides and thus may reduce side effects such as vomiting or nausea.

[0065] Furthermore, the pharmaceutical compositions according to the invention may contain non-peptide polymers that are highly selective and react with peptides, and therefore can be prepared in high yields.

[0066] Furthermore, the pharmaceutical composition according to the invention may comprise conjugates including polypeptides 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. Attached Figure Description

[0067] Figure 1 HPLC results of the conjugates of Example 2, which contain peptides and non-peptide polymers, are shown.

[0068] Figure 2 MALDI-TOF results for the conjugates of Example 2, which contain peptides and non-peptide polymers, are shown.

[0069] Figure 3 The results show the extent of change in long-term mean blood glucose concentration by measuring glycated hemoglobin (HbA1c) levels after treatment with the conjugate of Example 2 (**p<0.01).

[0070] Figure 4 The results show the final body weight of mice after two weeks of treatment with the conjugate of Example 2 at different dosing frequencies (***p<0.001).

[0071] Figure 5 The results show the changes in blood glucose in mice after administration of the conjugates of Examples 2 or 6.

[0072] Figure 6 Results of the intraperitoneal glucose tolerance test (ipGTT) following administration of the conjugates of Example 2 or 6 are shown.

[0073] Figure 7 The results of changes in serum cholesterol measurements after administration of the conjugate of Example 2 are shown.

[0074] Figure 8 The results of the measurement change in liver weight after application of the conjugate of Example 2 are shown.

[0075] Figure 9 The results of observing mouse liver tissue after administration of the conjugate of Example 2 are shown (dark stained areas represent normal liver tissue, and white (bright) stained areas represent lipid droplets).

[0076] Figure 10 The results of changes in serum cholesterol measurements after administration of the conjugate of Example 2 are shown.

[0077] Figure 11 The results of the measurement change in liver weight after application of the conjugate of Example 2 are shown.

[0078] Figure 12 The results of measurements of changes in liver triglyceride levels after administration of the conjugate of Example 2 are shown.

[0079] Figure 13 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) are shown.

[0080] Figure 14 The results of measuring the NAFLD activity score (NAS) after application of the conjugate of Example 2 are shown. Detailed Implementation

[0081] The present invention provides a polypeptide having an amino acid sequence represented by the following general formula 1.

[0082] [General Formula 1]

[0083] R1-X1-QGTFTSDYSKYFD-R2-EFVQWFMNT-R3(SEQ ID NOs.7-9),

[0084] R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl or β-carboxyl-imidazo-propionyl;

[0085] X1 is a deletion, glycine, or aminoisobutyric acid (Aib);

[0086] R2 is EKRAK (SEQ ID NO.10), EQAAK (SEQ ID NO.11), or EEAVK (SEQ ID NO.12); and

[0087] R3 can be a deletion, cysteine, lysine, or methionine.

[0088] 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.

[0089] [General Formula 1]

[0090] R1-X1-QGTFTSDYSKYFD-R2-EFVQWFMNT-R3(SEQ ID NOs.7-9),

[0091] R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl or β-carboxyl-imidazo-propionyl;

[0092] X1 is a deletion, glycine, or aminoisobutyric acid (Aib);

[0093] R2 is EKRAK (SEQ ID NO.10), EQAAK (SEQ ID NO.11), or EEAVK (SEQ ID NO.12); and

[0094] R3 can be a deletion, cysteine, lysine, or methionine.

[0095] The present invention provides a pharmaceutical composition comprising a polypeptide having an amino acid sequence represented by the following general formula 1.

[0096] [General Formula 1]

[0097] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3(SEQ ID NOs.7-9),

[0098] R1 is histidine, deamino-histyl, N-dimethyl-histyl, β-hydroxy-imidazo-propionyl, 4-imidazo-acetyl or β-carboxyl-imidazo-propionyl;

[0099] X1 is a deletion, glycine, or aminoisobutyric acid (Aib);

[0100] R2 is EKRAK (SEQ ID NO.10), EQAAK (SEQ ID NO.11), or EEAVK (SEQ ID NO.12); and

[0101] R3 can be a deletion, cysteine, lysine, or methionine.

[0102] 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.

[0103] The amino acids mentioned in this article are abbreviated according to the IUPAC-IUB naming rules, as listed in Table 1 below.

[0104] [Table 1]

[0105] Amino acid Abbreviation Amino acid Abbreviation Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamic acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan w Tyrosine Y Valine V

[0106] In general formula 1, R1 is preferably a histidine residue at the N-terminus of the polypeptide, but the present invention is not limited thereto.

[0107] X1 is preferably glycine or Aib, more preferably Aib. In this case, X1 is not particularly limited as long as it enhances the chemical stability of the polypeptide.

[0108] Furthermore, X1 is preferred as long as it can be resistant to dipeptidyl peptidase-4 (DPP-4), thereby enhancing the stability of the enzyme.

[0109] R2 is preferably EQAAK (SEQ ID NO.11) or EEAVK (SEQ ID NO.12), more preferably EQAAK (SEQ ID NO.11), but the present invention is not limited thereto.

[0110] R2 contains glutamic acid (E) and lysine (K), and the glutamic acid and the lysine

[0111] Preferably, the two residues in the amino acid sequence of the polypeptide form a covalent ring 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 its ability to bind to glucagon receptors or glucagon derivative receptors. Furthermore, the covalent ring can contribute to the α-helical structure of the polypeptide.

[0112] R3 is the C-terminus of the polypeptide, which can bind to substances to enhance their in vivo half-life or sustainability. In this case, R3 is preferably cysteine, but the invention is not limited thereto.

[0113] The polypeptide has 70% to 90% sequence homology with the amino acid sequence shown in SEQ ID NO.1 (SEQ ID NO.1:HSQGTFTSDYSKYLDSR-RAQDFVQWLMNT).

[0114] Here, it is 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 that 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.

[0115] In other words, a polypeptide containing an amino acid sequence having 70% to 90% sequence homology with the amino acid sequence described in SEQ ID NO. 1 can 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).

[0116] 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 present invention is not limited thereto.

[0117] 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 available comparison programs. Commercially available computer programs can be used to calculate the homology between two or more sequences as a percentage (%). Homology (%) can be calculated for adjacent sequences. A large number of peptides can be obtained by inserting polynucleotides encoding peptides into a vector and expressing the peptide.

[0118] In this specification, the term "peptide" refers to a compound in which two or more α-amino acids are linked by peptide bonds.

[0119] Simultaneously, 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 its fragments, nucleotides, fibronectin, transferrin, FcRn binding materials, sugars, elastin, heparin and its derivatives.

[0120] Preferably, the non-peptide polymer is covalently bonded to the polypeptide, but the present invention is not limited thereto.

[0121] The polypeptide covalently binds to the above-mentioned substances or forms microspheres with them, and thus has the effects of enhancing blood stability, delaying drug release into the kidneys, and inducing changes in receptor affinity.

[0122] When a peptide covalently binds to a non-peptide polymer, the peptide 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 peptide can vary depending on the functional groups of the non-peptide polymer and the amino acid sequence of the peptide. Preferably, the binding site is not particularly limited as long as the non-peptide polymer polymerizes to the C-terminus of the peptide or can be prepared in high yield due to a high reaction rate.

[0123] When non-peptide polymers bind to peptides, non-peptide polymers with maleimide groups can bind to the peptide using the thiol group (-SH) of the C-terminal cysteine ​​residue of the peptide, or non-peptide polymers with succinimide derivatives can bind to the peptide using the amino group (K) of the lysine residue of the peptide.

[0124] 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 the invention.

[0125] 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 polyethylene glycol succinimide-carboxymethyl ester (mPEG-succinimide-carboxymethyl ester), acrylate 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 polyethylene glycol maleimide, a di-branched methoxy polyethylene glycol maleimide, or a tri-branched methoxy polyethylene glycol maleimide, more preferably a tri-branched methoxy polyethylene glycol maleimide.

[0126] The polyethylene glycol or its derivatives that may be used herein are linear or branched, preferably di-branched or tri-branched, and more preferably tri-branched.

[0127] The molecular weight of the non-peptide polymer can 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 range, the non-peptide polymer can bind to the peptide to enhance the solubility of the resulting conjugate and prolong the in vivo retention time of the conjugate.

[0128] Therefore, the pharmaceutical compositions according to the invention comprise conjugates having non-peptide polymers that bind to polypeptides, and thus can enhance in vivo stability and prolong in vivo half-life.

[0129] Furthermore, the pharmaceutical compositions according to the invention comprise peptides or conjugates comprising peptides and non-peptide polymers and are therefore suitable for use 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.

[0130] Furthermore, 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.

[0131] Diseases caused by a lack of insulin secretion or decreased insulin sensitivity can include type 1 diabetes, type 2 diabetes, and diabetes complications.

[0132] Furthermore, the pharmaceutical compositions according to the present invention comprising polypeptides or conjugates comprising polypeptides 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.

[0133] Furthermore, the pharmaceutical compositions according to the present invention comprising polypeptides or conjugates comprising polypeptides 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.

[0134] When the compositions of the present invention are used as pharmaceuticals, the pharmaceutical compositions containing polypeptides can be formulated into various dosage forms for oral or parenteral administration, and then these dosage forms are administered clinically, but the present invention is not limited thereto.

[0135] 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 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.

[0136] Pharmaceutical compositions containing peptides can be administered parenterally. In this case, parenteral administration is carried out by methods such as subcutaneous injection, intravenous injection, intramuscular injection, nasal spray, administration through mucous membranes to the nasal cavity or intestines, inhalation, or intrapleural injection.

[0137] In this case, to allow for formulation for parenteral administration, the peptide can be mixed with a stabilizer or buffer to prepare a solution or suspension, which can then be formulated 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 emulsification promoters, salts or buffers for osmotic adjustment, and other therapeutically useful substances. In this case, the composition can be formulated using conventional methods such as mixing, granulation, or coating.

[0138] The amount of the polypeptide-containing pharmaceutical composition to be applied to the human body according to the present invention can vary depending on the patient's age, weight, sex, administration method, health condition, and disease severity. For example, the pharmaceutical composition can be administered orally or parenterally at a dose of 0.001 to 200 mg / kg / day, as determined by a physician or pharmacist.

[0139] Furthermore, the present invention provides a method for preparing a pharmaceutical composition comprising a conjugate comprising a polypeptide and a non-peptide polymer.

[0140] First, in the method for 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 both polypeptides and non-peptide polymers will be omitted.

[0141] Specifically, the method for preparing the pharmaceutical composition includes mixing a non-peptide polymer with a peptide to react with each other. In this case, the peptide and non-peptide polymer can react in a molar ratio of 1:1 to 1:5, such that the peptide and non-peptide polymer can bind to each other in a 1:1 molar ratio. In this case, a molar ratio of 1:1 to 1:2 is preferred, and more preferably a molar ratio of 1:1.2 is preferred, but the invention is not limited thereto. When mixing is performed within this molar ratio range, conjugates can be obtained in high yields, which makes it possible to prepare high-purity conjugates containing peptides and non-peptide polymers.

[0142] According to an exemplary embodiment of the present invention, the conjugate can also be prepared by covalently linking a non-peptide polymer to the C-terminus of a polypeptide. For example, the conjugate can also be prepared by using a methoxy polyethylene glycol having a maleimide group as the non-peptide polymer and using the polypeptide having a cysteine ​​residue at the C-terminus as the polypeptide, and thus the conjugate can have a high yield and a prolonged blood half-life.

[0143] Mixing non-peptide polymers with peptides to react with each other can be performed at pH 4.0 to 9.0, preferably pH 5.5 to 7.5, but the invention is not limited thereto. When mixing is performed outside this pH range, the yield will decrease. For example, when using methoxy polyethylene glycol with maleimide groups as the non-peptide polymer and mixing it with a peptide having a cysteine ​​residue at its C-terminus as the peptide, mixing is preferably performed at pH 6 to 8. When the peptide 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 peptide.

[0144] Because the yield of conjugates when non-peptide polymers are mixed with peptides to react with each other is 85% to 95%, the process will be economically viable and highly renewable due to the high yield. Therefore, this process will be effectively used in the preparation of pharmaceuticals.

[0145] When non-peptide polymers are mixed with peptides to react with each other, the reaction time can range from 0.5 to 24 hours or from 1 to 24 hours, and is 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 peptides may be degraded or the economic benefits may decrease due to the long processing time.

[0146] Furthermore, when mixing non-peptide polymers with peptides to react with each other, the temperature can be in the range of 0 to 100°C, preferably 4 to 40°C, but the present invention is not limited thereto. Moreover, there are no particular limitations on the temperature as long as the peptides or non-peptide polymers do not undergo chemical changes.

[0147] When a non-peptide polymer is mixed with a peptide to react with each other, each of the peptide and non-peptide polymers 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 readily available in the relevant fields.

[0148] 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 polypeptide to a subject.

[0149] Furthermore, 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 polypeptide to a subject other than a human.

[0150] Invention Model

[0151] The invention will now be described in detail with reference to the accompanying drawings, enabling those skilled in the art to readily practice it. However, it should be understood that the invention may be embodied in various forms and is not intended to be limited to this context. Throughout the specification, similar reference numerals refer to similar elements.

[0152] Example 1

[0153] The polypeptide in which cysteine ​​is introduced (molecular weight: 3,509 Da; SEQ ID NO.2: H(Aib)QGTFTSDYSKYLD) E QAA K EFVQWLMNTC).

[0154] Here, underlined residues in the amino acid sequence of SEQ ID NO.2, highlighted in bold, indicate that a covalent ring is formed between the residues.

[0155] Preparative Example 1: Synthesis of conjugates containing peptides and non-peptide polymers

[0156] To prepare conjugates comprising peptides and non-peptide polymers, a peptide (molecular weight: 3,509 Da; SEQ ID NO.2: H(Aib)QGTFTSDYSKYLD) with cysteine ​​introduced into the C-terminal region (position 30) was used. E QAA K EFVQWLMNTC) is used as a polypeptide.

[0157] Meanwhile, as listed in Table 2 below, maleimide-activated monomethoxy PEG (mPEG-MAL, NOF (Japan)) is used as a non-peptide polymer.

[0158] To prepare the conjugates of Examples 2 to 7, peptides as listed in Table 2 below were prepared. In this case, each peptide was dissolved in dimethyl sulfoxide (DMSO), and mPEG-MAL was dissolved in 50 mM phosphate-buffered saline (pH 6).

[0159] [Table 2]

[0160]

[0161]

[0162] In the amino acid sequences SEQ ID NO.2 to 4 listed in Table 2, the two residues that are underlined and highlighted in bold refer to residues that have a covalent ring formed between the residues.

[0163] 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 PEGylated 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 PEGylated peptide (see [link to HPLC]). Figure 1 Next, the molecular weight of the PEGylated peptide was measured using a MALDI-TOF mass spectrometer (see [link to MALDI-TOF mass spectrometry]). Figure 2 Furthermore, based on the chromatograms obtained during the chromatographic separation process, the yields of the conjugates in Examples 2 to 7 were calculated as the area ratio of the conjugate to the peptide. The results are listed in Table 3.

[0164] [Table 3]

[0165] Item Yield Example 2 90％ Example 3 92％ Example 4 91％ Example 5 89％ Example 6 91％ Example 7 90％

[0166] As shown in Table 3, the preparation of conjugates was confirmed in yields of 90% or higher. Therefore, the method for preparing the pharmaceutical compositions 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 because the preparation process is simple, economically feasible, and highly renewable.

[0167] Experimental Example 1: Measurement of in vitro activity in Example 2

[0168] To examine the preventive or therapeutic effects of the conjugate in 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).

[0169] To determine activity against the GLP-1 receptor, HEK293 / CRE-Luc cells expressing the human glucagon GLP-1 receptor were purchased and used from GenScript. Cells were cultured at 5 x 10⁻⁶ cells / year. 4 Cells were seeded per 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 cells were analyzed using One-Globe technology. TM The luciferase assay system (Promega) measures the generated cAMP (luciferase reporter gene) to calculate EC50 relative to the GLP-1 receptor. 50 Values. The results are listed in Table 4 below.

[0170] Secondly, to determine the activity against glucagon receptor (GCGR), a cAMPHunter from DiscoverX was used. TM eXpress GCGR CHO-K1 GPCR kit. Used in 3x10 kits. 4 CHO-K1 cells expressing human glucagon receptor were seeded at a density of 1 cell / well in 96-well plates. Each well was then treated with the following: a peptide from Example 1 (0.013 to 300 nM), a 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 relative to glucagon receptor (GCGR). 50 Values. The results are listed in Table 4 below.

[0171] [Table 4]

[0172]

[0173] As shown in Table 4, GLP-1 exhibits high activity against the GLP-1 receptor but very low, unmeasurable activity against the glucagon receptor. Conversely, natural glucagon exhibits very high activity against the glucagon receptor but low activity against the GLP-1 receptor. These results confirm the high selectivity of the experimental method.

[0174] Simultaneously, the EC50 of the peptide in Example 1 on the GLP-1 receptor was confirmed. 50 The value was 0.13, almost identical to that of GLP-1, indicating that the peptide of Example 1 exhibited high activity against the GLP-1 receptor, and also showed activity against the glucagon receptor. Furthermore, animal experiments confirmed that the peptide of Example 1 had an anti-obesity effect.

[0175] Furthermore, 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 also confirmed that when a non-peptide polymer (e.g., PEG) is bound to the peptide, the peptide's receptor activity is generally significantly reduced compared to before conjugation. However, even when a non-peptide polymer is bound to the peptide, the conjugate of Example 2 showed less reduction in receptor activity, indicating that the conjugate of Example 2 exhibits a prolonged in vivo half-life while maintaining high activity.

[0176] Therefore, the pharmaceutical composition according to the invention has been shown to have excellent activity against glucagon receptors and GLP-1 receptors, and thus has anti-diabetic and anti-obesity effects as well as triglyceride-lowering effects by inhibiting appetite, enhancing insulin secretion and promoting lipolysis in adipocytes.

[0177] Experimental Example 2: Measurement of in vivo activity in Example 2 1

[0178] 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.

[0179] First, an obesity animal model was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing their average body weight to approximately 50 g. Subsequently, the conjugate from 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.

[0180] In this case, body weight and blood glucose are expressed as percentages (%), calculated as 100% before administration (day 0).

[0181] [Table 5]

[0182]

[0183] In this case, the untreated group refers to a group of mice that were given PBS instead of the conjugate of Example 2.

[0184] As shown in Table 5, the untreated group consumed approximately 40g of food over 2 weeks, while the group of mice treated with the conjugate of Example 2 consumed approximately 18g of food. Compared to the untreated group, the cumulative food intake of the mice in the latter group was reduced by more than half. The food intake was similar in the groups of mice treated with the positive control and the conjugate of Example 2.

[0185] 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 administration point (day 0), and in the positive control group, weight decreased by approximately 15% compared to the weight observed before administration, indicating that liraglutide is poorly effective in the prevention or treatment of obesity. On the other hand, in the same group of mice administered the conjugate of Example 2, weight decreased significantly by 61% compared to the weight measured before administration.

[0186] Furthermore, referencing the pattern of blood glucose changes over time revealed that blood glucose levels were reduced by approximately 80% compared to blood glucose levels measured before application, indicating that the conjugate of Example 2 had a blood glucose-lowering effect, while the positive control had a very poor blood glucose-lowering effect.

[0187] Based on these results, it can be seen that the weight loss effect of the conjugate in Example 2 is achieved through an increase in energy metabolism and a simple reduction in food intake. The pharmaceutical composition according to the invention can reduce food intake, inhibit gastric emptying, and promote lipolysis.

[0188] Experimental Example 3: Measurement of in vivo activity in Example 2

[0189] This experiment was performed in the same manner as in Experiment 2, and glucose tolerance was then assessed in a mouse model using the intraperitoneal glucose tolerance test (ipGTT).

[0190] 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.

[0191] [Table 6]

[0192] Time (min) Example 2 Positive control Untreated group 0 40 109 118 15 216 283 600 30 172 202 522 60 61 145 336 90 48 124 236 120 44 103 146

[0193] As shown in Table 6, based on results obtained after two weeks of continuous 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 same 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. Furthermore, 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.

[0194] Experimental Example 4: Measurement of in vivo activity in Example 2 3

[0195] To evaluate the preventive or therapeutic effects of the conjugate in Example 2 on diabetes, approximately 7-week-old BKS.Cg-+Lepr were tested. db / +Lepr db / 01aHsd mice (db / db mice) were administered the conjugate of Example 2, and changes in blood glucose and body weight over time were measured.

[0196] 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 administration, following administration. The results are listed in Table 7 below.

[0197] Next, to perform the intraperitoneal glucose tolerance test (ipGTT), 2 g / kg glucose was administered intraperitoneally 12 days after drug administration, and blood glucose levels were measured over time (0, 15, 30, 60, 90, and 120 minutes). The results are listed in Table 8. To determine the extent of long-term variation in mean blood glucose levels, glycated hemoglobin (HbA1c) levels were also measured after drug administration. Results are... Figure 3 As shown in the image.

[0198] [Table 7]

[0199]

[0200] [Table 8]

[0201] Time (min) Example 2 Untreated group 0 63 244 15 190 564 30 246 600 60 341 600 90 356 600 120 396 600

[0202] As shown in Table 7, weight loss was observed in the mouse group administered the conjugate of Example 2 compared to the untreated group. Furthermore, 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.

[0203] As shown in Table 8, it was also revealed that the mice treated with the conjugate of Example 2 exhibited higher glucose tolerance compared to the untreated group.

[0204] Moreover, such as Figure 3 As shown in the figure, the application 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 applying the conjugate of Example 2.

[0205] Experimental Example 5: Measurement of in vivo activity in Example 2 4

[0206] To examine the effects of the concentration and frequency of application 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.

[0207] First, an obesity animal model was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for approximately 24 weeks, increasing the mice's average body weight to approximately 50 g. Subsequently, each group in this group was administered the conjugate of Example 2 for 2 weeks, as listed in Table 9 below. After 2 weeks, the final body weight of the mice was measured. Results were presented in... Figure 4 As shown in the image.

[0208] [Table 9]

[0209] Item Method of administration: Group 1 Example 2 was injected subcutaneously at 20 nmol / kg once every other day Group 2 Example 2 was injected subcutaneously at 40 nmol / kg once every other day Group 3 Example 2 was injected subcutaneously at 40 nmol / kg once a week Untreated group PBS was injected subcutaneously once every other day

[0210] like Figure 4 As shown, it was revealed that the conjugate of Example 2 had 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 still had a significant effect on weight loss. Therefore, it was confirmed that the conjugate of Example 2 exhibits 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 according to the invention for the prevention or treatment of obesity has a long in vivo half-life and exhibits a high level of therapeutic effect on obesity even when administered at high doses three times daily.

[0211] Experimental Example 6: Measurement of in vivo activity of Examples 2 and 6 5

[0212] To examine the preventive or therapeutic effects of conjugates with different amino acid sequences from Examples 2 and 6 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 mice's average body weight to approximately 50 g. Subsequently, each conjugate from Examples 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 from the examples as a control. Blood glucose levels were measured over time during the 2 weeks of administration of the conjugates from Examples 2 or 6. Results were presented in... Figure 5 The results are shown in the diagram. Furthermore, to perform the 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 As shown in the image.

[0213] like Figure 5 As shown, it was revealed that mice given the conjugates of Example 2 or 6 had lower blood glucose levels compared to the control group, but mice given PBS had higher blood glucose levels.

[0214] like Figure 6 As shown, the study revealed that blood glucose levels in the control group rose sharply and then fell due to the administration of 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 conjugates of Example 2 and 6 improved glucose tolerance compared to the control group.

[0215] Therefore, it has been confirmed that the pharmaceutical composition comprising peptides according to the present invention has the following properties: Figure 5 and 6 The effects shown are in the prevention or treatment of obesity and diabetes.

[0216] Experimental Example 7: Measurement of in vivo activity in Example 2 6

[0217] To examine the preventive or therapeutic effects of the conjugate of Example 2 on non-alcoholic fatty liver disease (NAFLD), the conjugate of Example 2 was administered to an animal model of NAFLD, and changes in serum cholesterol levels and liver weight were examined, along with liver biopsies. Results were... Figures 7 to 9 As shown in the image.

[0218] 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 mice's average weight to approximately 50 g. Subsequently, the conjugate from 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).

[0219] Reference measurements of serum cholesterol levels and liver weight Figure 7 and 8 This confirmed that mice treated with the conjugate of Example 2 had significantly lower serum cholesterol levels and liver weight compared to the untreated group treated with PBS, and also lower serum cholesterol levels and liver weight compared to the positive control group (i.e., the liraglutide-treated group). Furthermore, reference... Figure 9 The liver biopsy results shown in the figure indicate that the liver steatosis in the mice treated with the conjugate of Example 2 was significantly reduced compared with the untreated group treated with PBS, and was also reduced compared with the positive control group (i.e., the liraglutide-treated group).

[0220] Thus, it is revealed that the pharmaceutical composition according to the present 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.

[0221] Experimental Example 8: Measurement of in vivo activity in Example 2 7

[0222] To examine the preventive or therapeutic effects of the conjugate from Example 2 on non-alcoholic fatty liver disease (NAFLD), the conjugate from Example 2 was administered to an animal model of NAFLD, and changes in serum cholesterol levels, liver weight, and liver triglycerides were measured. Results were... Figures 10 to 12 As shown in the image.

[0223] First, a laboratory animal model of non-alcoholic fatty liver disease was established by feeding normal C57BL / 6 mice (approximately 6 weeks old) a diet high in trans fats (40% high fat, 20% fructose, and 2% cholesterol) for approximately 16 weeks. Subsequently, the conjugate from Example 2 was administered subcutaneously every three days at a dose of 20 nmol / kg 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, serum cholesterol levels, liver weight, and liver triglycerides (liver TG) were measured.

[0224] Reference measurements include serum cholesterol levels, liver weight, and liver triglycerides. Figures 10 to 12 As can be seen, compared with the untreated group treated with saline, the mice treated with the conjugate of Example 2 showed significantly lower serum cholesterol levels, liver weight, and liver triglycerides, and also showed lower serum cholesterol levels, liver weight, and liver triglycerides compared with 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 preventing and treating 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.

[0225] Experimental Example 9: Measurement of in vivo activity in Example 2 8

[0226] 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 effects on non-alcoholic fatty liver disease. Four weeks after administration of the conjugate from Example 2, the experiment was completed in the same manner as in Experimental Example 8, involving the extraction, paraffin embedding, and thin sectioning of mouse livers. Subsequently, hematoxylin and eosin (H&E) staining and Oil Red O staining were performed.

[0227] Therefore, as Figure 13 and 14 As shown, liver histology and NAS results after 4 weeks of administration indicated that the mice administered the conjugate of Example 2 showed 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 preventing and treating 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.

[0228] 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.

[0229] Industrial applicability

[0230] The pharmaceutical composition comprising polypeptides according to the present invention can be safely used to prevent or treat obesity, diabetes or non-alcoholic fatty liver disease because the pharmaceutical composition 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. sequence list <110> D&D Pharmaceutical Technology Co., Ltd. Lee, Kang Choon Park, Og Yi An, Hyoung Tae Park, Eun Ji Shin, Jae Hee Lim, Sung Mook <120> Pharmaceutical compositions including peptides <130> DDP 100 PCT <140> PCT / KR2019 / 008918 <141> 2019-07-19 <150> KR 10-2018-0083946 <151> 2018-07-19 <150> KR 10-2019-0060513 <151> 2019-05-23 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> PRT <213> Unknown <220> <223> glucagon <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <220> <221> Xaa = Aminoisobutyric acid (Aib) <222> (2)..(2) <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 3 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 3 His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <220> <221> Xaa = Aminoisobutyric acid (Aib) <222> (2)..(2) <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Glu Ala Val Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 5 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 5 His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Glu Ala Val Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25 30 <210> 6 <211> 30 <212> PRT <213> Unknown <220> <223> GLP-1 <400> 6 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 <210> 7 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 7 Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Lys Arg 1 5 10 15 Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 8 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 8 Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Gln Ala 1 5 10 15 Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 9 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 9 Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Glu Ala 1 5 10 15 Val Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 10 Glu Lys Arg Ala Lys 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 11 Glu Gln Ala Ala Lys 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 12 Glu Glu Ala Val Lys 1 5

Claims

1. A polypeptide comprising an amino acid sequence represented by the following general formula 1: [General Formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3, Where R1 is histidine; X1 is aminoisobutyric acid (Aib); R2 is EQAAK (SEQ ID NO. 11); and R3 stands for cysteine.

2. A polypeptide conjugate comprising the polypeptide according to claim 1, and a non-peptide polymer, wherein the non-peptide polymer is covalently bonded to the polypeptide.

3. A pharmaceutical composition comprising the polypeptide according to claim 1.

4. A pharmaceutical composition comprising the conjugate according to claim 2.

5. The pharmaceutical composition according to claim 3, wherein the polypeptide is covalently bound to one or more compounds selected from: non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and fragments thereof, nucleotides, fibronectin, transferrin, FcRn conjugates, sugars, elastin, and heparin.

6. The pharmaceutical composition according to claim 3 or 4, wherein R2 of general formula 1 comprises glutamic acid (E) and lysine (K), and the glutamic acid and the lysine together form a ring via an amide bond.

7. The pharmaceutical composition of claim 4, 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, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, and combinations thereof.

8. The pharmaceutical composition according to claim 4, wherein the non-peptide polymer is selected from dextran, chitin, or hyaluronic acid.

9. The pharmaceutical composition according to claim 4, wherein the non-peptide polymer is polyethylene glycol.

10. The pharmaceutical composition of claim 9, wherein the non-peptide polymer has a molecular weight of 3,000 to 100,000 Da.

11. The pharmaceutical composition of claim 4, wherein the non-peptide polymer is a polyethylene glycol derivative, the polyethylene glycol derivative being 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 acrylate succinimide). Succinimide propionate), thiol polyethylene glycol succinimide propionate (thiol PEG succinimide propionate), hydroxysuccinimide polyethylene glycol (hydroxysuccinimide PEG), methoxy polyethylene glycol succinimide carboxymethyl ester (mPEG succinimide carboxymethyl ester), acrylate polyethylene glycol succinimide carboxymethyl ester (acrylate PEG succinimide carboxymethyl ester), polyethylene glycol carbonate succinimide (PEG carbonate succinimide), polyethylene glycol propionaldehyde (PEG propionaldehyde), and polyethylene glycol butyraldehyde (PEG butyraldehyde).

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

13. The pharmaceutical composition according to claim 4, wherein the polypeptide is composed of H-Aib-QGTFTSDYSKYLDEQAAKEFV-QWLMNTC (SEQ ID NO: 2), wherein the non-peptide polymer is maleimide-activated monomethoxy PEG.

14. Use of the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of obesity.

15. Use of the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of diabetes.

16. Use of the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

17. The application according to claim 16, wherein the non-alcoholic fatty liver disease includes one or more diseases selected from the group consisting of: non-alcoholic fatty liver and non-alcoholic steatohepatitis.

18. A method for preparing a pharmaceutical composition, said pharmaceutical composition comprising: The polypeptide according to claim 1; as well as Non-peptide polymers The method includes: The non-peptide polymer is mixed with the polypeptide to react with each other.

19. The method of claim 18, wherein R2 of the general formula 1 comprises glutamic acid (E) and lysine (K), the glutamic acid and the lysine forming a ring together via an amide bond.

20. The method of claim 18, 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, polyvinyl ether, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), lipid polymers, and combinations thereof.

21. The method of claim 18, wherein the non-peptide polymer is selected from dextran, chitin, or hyaluronic acid.

22. The method of claim 18, wherein the non-peptide polymer is polyethylene glycol.

23. The method of claim 18, wherein the non-peptide polymer is a polyethylene glycol derivative, the polyethylene glycol derivative being 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 acrylate) The following are listed: succinimide ester, thiol polyethylene glycol propionate succinimide ester (thiol PEG propionate succinimide ester), hydroxysuccinimide-based polyethylene glycol (hydroxysuccinimide-based PEG), methoxy polyethylene glycol succinimide-based carboxymethyl ester (mPEGsuccinimide-based carboxymethyl ester), acrylate polyethylene glycol succinimide-based carboxymethyl ester (acrylate PEGsuccinimide-based carboxymethyl ester), polyethylene glycol carbonate succinimide ester (PEG carbonate succinimide ester), polyethylene glycol propionaldehyde (PEG propionaldehyde), and polyethylene glycol butyraldehyde (PEG butyraldehyde).

24. The method of claim 18, wherein mixing the non-peptide polymer with the peptide to react with each other comprises reacting the peptide and the non-peptide polymer at a molar ratio of 1:1 to 1:

5.

25. The method of claim 18, wherein the mixing of the non-peptide polymer with the polypeptide to react with each other is performed at pH 4.0 to 9.

0.

26. The method of claim 18, wherein when the non-peptide polymer is mixed with the polypeptide to react with each other, the reaction time is in the range of 0.5 to 24 hours.

27. Use of the pharmaceutical composition prepared according to claim 18 in the preparation of a medicament for the prevention or treatment of obesity.

28. Use of the pharmaceutical composition prepared by the method according to claim 18 in the preparation of a medicament for the prevention or treatment of diabetes.

29. The use of the pharmaceutical composition prepared according to claim 18 in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

30. The application according to claim 29, wherein the non-alcoholic fatty liver disease includes one or more diseases selected from the group consisting of: non-alcoholic fatty liver and non-alcoholic steatohepatitis.

31. The use of the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of one or more diseases, wherein the one or more diseases are selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease.

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