Pharmaceutical composition containing polypeptides

Abstract

Providing pharmaceutical compositions containing polypeptides. [Solution] The present invention relates to a pharmaceutical composition comprising a polypeptide, more specifically, to a pharmaceutical composition for preventing or treating obesity, diabetes, or non-alcoholic fatty liver disease. This pharmaceutical composition is safe and free from side effects such as vomiting or nausea, and has the effect of reducing food intake, improving insulin secretion, suppressing gastric emptying, promoting lipolysis, and lowering triglyceride levels. The present invention relates to a pharmaceutical composition comprising a polypeptide and its medical use, for example, in the treatment or prevention of obesity, diabetes, or non-alcoholic fatty liver disease.

Description

[Technical Field]

[0001] This invention relates to a pharmaceutical composition comprising a polypeptide and its medical use, for example, in the treatment or prevention of obesity, diabetes, or non-alcoholic fatty liver disease. This polypeptide has no side effects such as vomiting or nausea, and has the effect of reducing food intake, improving insulin secretion, suppressing gastric emptying, promoting lipolysis, and lowering triglyceride levels. [Background technology]

[0002] In recent years, along with economic development and rapid scientific and technological growth, the elderly population has increased, and lifestyle-related diseases have surged. This is attributed to stress, poor eating habits, excessive calorie intake, and decreased physical activity. Cardiovascular disease and cerebrovascular disease, complications associated with obesity, rank first and second in mortality rates, and obesity is suggested to be a cause of various lifestyle-related diseases such as diabetes and non-alcoholic fatty liver disease.

[0003] Obesity is defined as a condition in which more fat than normal is accumulated, and the most accurate way to assess obesity is to measure body fat mass. However, because accurate measurement of fat mass is costly, it is assessed using indirect methods. The most commonly used indirect methods are measuring the Body Mass Index (BMI) and waist circumference. The World Health Organization (WHO) has published a data-based classification linking BMI to mortality risk. This classification defines normal weight as 18.5–24.9 kg / m². 2 , Overweight: 25~29.9kg / m 2 , and obesity: 30 kg / m² 2 It is based on a weight of or greater than that.

[0004] Obesity is known to be caused by an energy imbalance resulting from excessive calorie intake and relatively reduced physical activity, and the resulting increase in body fat. However, since various risk factors such as dietary habits, lifestyle, age, race, and genetic factors are involved in obesity, it is difficult to attribute it to just one factor.

[0005] Diabetes mellitus is classified into insulin-dependent diabetes mellitus (Type 1 diabetes), non-insulin-dependent diabetes mellitus (Type 2 diabetes), and malnutrition-associated diabetes mellitus (MRDM). Type 2 diabetes mellitus, which accounts for the majority of diabetes cases (more than 90%), is a metabolic disorder characterized by hyperglycemia and is reported to be caused by decreased insulin secretion from pancreatic beta cells or increased insulin resistance in peripheral tissues due to genetic, metabolic, and environmental factors. In this regard, it is well known that increased body fat reduces insulin sensitivity, and that the accumulation of abdominal fat in particular is associated with impaired glucose tolerance. Furthermore, insulin resistance is closely related to obesity in patients with Type 2 diabetes, and it is known that the more severe the obesity, the greater the insulin resistance.

[0006] Non-alcoholic fatty liver disease (NAFLD) refers to a range of diseases including simple steatosis, non-alcoholic steatohepatitis (NASH) with hepatocyte damage (hepatocyte ballooning), inflammation, fibrosis, and, in more advanced cases, cirrhosis, where fat accumulates excessively in liver cells regardless of alcohol consumption. The prevalence of NFLD is rapidly increasing along with the rising prevalence of obesity worldwide. While the prevalence of diabetes varies by country, it accounts for approximately 20-30% of the total population in Western countries, and its incidence reaches about 16% in South Korea.

[0007] Non-alcoholic fatty liver disease (NALF) is closely associated with metabolic syndrome, including obesity, type 2 diabetes, dyslipidemia, and similar diseases based on insulin resistance. In fact, many prediabetic and type 2 diabetic patients have been shown to have NALF / non-alcoholic steatohepatitis, and these patients are known to have a high rate of progression to cirrhosis and liver cancer (i.e., hepatocellular carcinoma). On the other hand, the prevalence of diabetes is high among patients with NALF, and this is evident in patients with non-alcoholic steatohepatitis.

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

[0009] The current market for anti-obesity drugs is estimated to be worth over $1 billion and is growing at approximately 10% annually. The drugs primarily used as anti-obesity medications are appetite suppressants (such as lorcaserin and phentermine) classified as psychotropic drugs. Most of these drugs act on the central nervous system and are known to suppress appetite and reduce weight, but long-term use can lead to abuse and addiction, as well as side effects such as palpitations, anxiety, and insomnia.

[0010] Xenical is one of the medications used as an anti-obesity drug that is not a psychotropic drug. Pancreatic lipase acts as an important enzyme that breaks down triglycerides into 2-monoacylglycerol and fatty acids. A representative pancreatic lipase inhibitor is tetrahydrolipustatin (orlistat), a derivative of lipustatin derived from Streptomyces toxitricini, which has a high level of efficacy in inhibiting the absorption of about 30% of ingested fat. Currently, tetrahydrolipustatin (orlistat) is marketed as a drug, but it has side effects such as gastrointestinal disorders, anaphylaxis, and cholestasis. Therefore, there are few treatments that can be safely used in obese patients.

[0011] In drug therapy for non-alcoholic fatty liver disease (NFC), the drug therapy acts on mechanisms that worsen NFC, such as insulin resistance, oxidative stress, apoptosis, and inflammatory cytokines, thereby suppressing the progression of NFC. Among these, antidiabetic drugs are known to improve fatty liver and lower blood glucose levels by improving the general pathophysiological conditions that contribute to the development of fatty liver. However, to date, there are no drugs approved for the treatment of fatty liver disease, indicating an unaddressed medical need for the development of effective therapeutic agents.

[0012] On the other hand, glucagon derivatives are currently attracting attention. Glucagon is produced in the pancreas when blood glucose levels begin to drop due to drug treatment, disease, hormone or enzyme deficiency, etc. Glucagon stimulates the liver to release glucose by breaking down glycogen, thereby raising blood glucose levels to normal levels. In addition to its effect of raising blood glucose, glucagon has also been reported to suppress appetite and promote fat breakdown by activating hormone-sensitive lipase in fat cells, thereby exhibiting an anti-obesity effect. One such glucagon derivative is glucagon-like peptide-1 (GLP-1), which is still under development as a therapeutic agent to alleviate hyperglycemia in diabetic patients. It functions to promote insulin synthesis and secretion, inhibit glucagon secretion, suppress gastric emptying, promote glucose utilization, and suppress food intake. It is also known that exendin-4, made from lizard venom and having approximately 50% amino acid homology with GLP-1, activates the GLP-1 receptor and reduces hyperglycemia in diabetic patients. However, GLP-1 receptor agonists used to treat obesity or diabetes have been reported to have problems causing side effects such as vomiting and nausea.

[0013] Oxintmodulin, which can bind to both the GLP-1 receptor and the glucagon receptor, is attracting attention as a GLP-1 alternative. Oxintmodulin is a peptide derived from the precursor of glucagon (i.e., preglucagon) and exhibits effects such as suppressing food intake by GLP-1, regulating blood glucose levels by inhibiting gluconeogenesis in the liver, and improving satiety, as well as possessing the lipolytic function of glucagon. Therefore, oxintmodulin has high potential as an antidiabetic and anti-obesity drug.

[0014] Based on the dual function of oxyntomodulin peptide, research is actively being conducted to develop drugs to treat diabetes and obesity. For example, registered Korean Patent No. 925017 discloses a pharmaceutical composition for oral, parenteral, mucosal, rectal, subcutaneous, or transdermal administration to treat human excess body weight, which contains oxyntomodulin as the active ingredient. However, anti-obesity drugs containing oxyntomodulin have been reported to have a short half-life in vivo and to show low levels of therapeutic effect against obesity even when administered at high doses three times a day.

[0015] On the other hand, attempts are being made to overcome the short in vivo half-life of therapeutic peptides, maintain high levels of pharmacological efficacy over long periods, and thereby maximize the effectiveness of therapeutic drugs. U.S. Patent No. 7,141,547 discloses a fusion protein of GLP-1 and its analogues with albumin using recombinant DNA technology, and U.S. Patent No. 8,273,854 discloses a fusion protein of GLP-1 and its analogues with immunoglobulin fragments (Fc). While these technologies have partially improved the short in vivo half-life of peptides, they do not eliminate problems associated with immunogenicity caused by the administration of proteins that are not inherent to the human body. As a result, this technology has the drawback that the pharmacological efficacy of the drug may decrease with long-term administration. In addition, there are the additional problems of requiring large-scale cell culture and purification systems for drug manufacturing, and furthermore, because drugs may contain impurities originating from host cells due to the nature of recombinant proteins, and may not be exactly the same from batch to batch, it can be difficult to control the quality of the drug. Furthermore, when using peptides containing disulfide bonds, such as calcitonin, there is a drawback that yield may decrease due to misfolding. In addition, if the drug contains non-natural amino acid residues, it is difficult to manufacture the drug using recombinant protein manufacturing methods.

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

[0017] On the other hand, PEGylation of therapeutic peptides and proteins is the most powerful pharmaceutical technology for improving their in vivo half-life. PEGylation of peptides and proteins increases their molecular weight, protects proteolytic sites, and masks immunogenic sites, resulting in an increased in vivo half-life of the drug and reduced immunogenicity of the peptides and proteins. Therefore, PEGylation technology is effective in enhancing therapeutic efficacy by solving problems related to peptide drugs. Due to these advantages, PEGylation of peptides and proteins plays a crucial role in improving the therapeutic effect of drug delivery systems.

[0018] However, the method using PEG has drawbacks: PEGylation reduces the activity of peptide drugs, and the increased molecular weight of PEG leads to poor reactivity with peptides, resulting in low yields. Therefore, a simple manufacturing process and a highly selective PEGylation method are needed.

[0019] Therefore, there is a need for therapeutic drugs to treat obesity, diabetes, or non-alcoholic fatty liver disease that do not have side effects such as vomiting or nausea, and that have the effect of reducing food intake, improving insulin secretion, suppressing gastric emptying, promoting lipolysis, and lowering triglyceride levels, and that can be obtained in high yield by optimizing the preparation method.

[0020] [Prior art document]

[0021] [Patent]

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

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

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

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

[0026] Patent Document 5: Korean Patent No. 1,665,009, title "Pharmaceutical Composition for Preventing or Treating Non-alcoholic Fatty Liver Diseases" SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

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

[0028] [General Formula 1]

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

[0030] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

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

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

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

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

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

[0036] [General formula 1]

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

[0038] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK, EQAAK, or EEAVK; and R3 is deletion, cysteine, lysine, or methionine).

[0039] Polypeptides may be covalently bonded with one or more substances selected from the group consisting of non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and its fragments, nucleotides, fibronectin, transferrin, FcRn-binding substances, sugars, elastin, heparin, and derivatives thereof, or they may form microspheres.

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

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

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

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

[0044] In this case, polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidylpropionate (PEG succinimidylpropionate), methoxypolyethylene glycol succinimidylpropionate (methoxyPEG succinimidylpropionate), acrylate polyethylene glycol succinimidylpropionate (acrylatePEG succinimidylpropionate), thiol polyethylene glycol succinimidylpropionate (thiol PEG succinimidylpropionate), and hydroxyl At least one selected from the group consisting of hydroxysuccinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), its derivatives, and multi-branched forms of its derivatives.

[0045] Polyethylene glycol or its derivatives may be linear or branched.

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

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

[0048] Another exemplary embodiment of the present invention involves a method for preparing a pharmaceutical composition, which includes mixing a non-peptide polymer with a polypeptide having an amino acid sequence represented by the following general formula 1 and reacting them together.

[0049] [General formula 1]

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

[0051] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK, EQAAK, or EEAVK; and R3 is deletion, cysteine, lysine, or methionine).

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

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

[0054] In this case, polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidylpropionate (PEG succinimidylpropionate), methoxypolyethylene glycol succinimidylpropionate (methoxyPEG succinimidylpropionate), acrylate polyethylene glycol succinimidylpropionate (acrylatePEG succinimidylpropionate), thiol polyethylene glycol succinimidylpropionate (thiol PEG succinimidylpropionate), and hydroxyl At least one selected from the group consisting of hydroxysuccinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), its derivatives, and multi-branched forms of its derivatives.

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

[0056] The mixing of non-peptide polymers and polypeptides for mutual reaction may be carried out at a pH of 4.0 to 9.0.

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

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

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

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

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

[0062] Furthermore, since the pharmaceutical composition according to the present invention can contain polypeptides, it can reduce side effects such as vomiting or nausea.

[0063] Furthermore, since the pharmaceutical composition according to the present invention may contain a non-peptidic polymer that is highly selective and reactive with polypeptides, it can be prepared in high yield.

[0064] Furthermore, since the pharmaceutical composition according to the present invention may contain a conjugate comprising polypeptides and non-peptide polymers, it may have a longer in vivo half-life and potentially have a high therapeutic effect on obesity even when administered at low doses. It may also have the effect of lowering blood glucose levels to maintain blood glucose at normal levels, and effectively lowering triglyceride levels. [Brief explanation of the drawing]

[0065] [Figure 1] Figure 1 shows the HPLC results of the conjugate of Example 2, which contains polypeptides and non-peptidic polymers.

[0066] [Figure 2] Figure 2 shows the MALDI-TOF results for the conjugate of Example 2, which includes polypeptides and non-peptidic polymers.

[0067] [Figure 3] Figure 3 shows the measurement results of glycated hemoglobin levels (HbA1c) after the completion of treatment with the conjugate in Example 2, in order to determine the degree of change in the long-term average blood glucose concentration (**p<0.01).

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

[0069] [Figure 5] Figure 5 shows the results of changes in blood glucose levels in mice after administration of the conjugates in Example 2 or 6.

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

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

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

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

[0074] [Figure 10] Figure 10 shows the measurement results of changes in serum cholesterol after administration of the conjugate in Example 2.

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

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

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

[0078] [Figure 14] Figure 14 shows the measurement results of the NAFLD activity score (NAS) after administration of the conjugate in Example 2. [Modes for carrying out the invention]

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

[0080] [General formula 1]

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

[0082] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

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

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

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

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

[0087] [General formula 1]

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

[0089] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

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

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

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

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

[0094] [General formula 1]

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

[0096] (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl;

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

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

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

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

[0101] The amino acids mentioned herein have been abbreviated according to IUPAC-IUB nomenclature rules, as shown in Table 1 below.

[0102] [Table 1]

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

[0104] 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.

[0105] Furthermore, X1 is preferable insofar as it is resistant to dipeptidyl peptidase-4 (DPP-4), thereby enhancing enzyme stability.

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

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

[0108] R3 is the C-terminus of a polypeptide that can bind to a substance to improve its in vivo half-life or sustainability. In this case, R3 is preferably cysteine, but the present invention is not limited thereto.

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

[0110] The amino acid sequence described in Sequence ID No. 1 is partially or entirely identical to that of natural glucagon, which has been reported to promote the breakdown of glycogen and insulin and exhibit anti-obesity effects. However, natural glucagon has low solubility and precipitates at neutral pH, limiting its use as a therapeutic agent.

[0111] In other words, a polypeptide containing an amino acid sequence having 70% to 90% sequence homology to the amino acid sequence described in Sequence ID No. 1 may be a glucagon derivative or an oxytomodulin derivative. In this case, the oxytomodulin derivative is a peptide prepared from a glucagon precursor (e.g., preglucagon).

[0112] Preferably, the polypeptide may have 73% to 90%, more preferably 75% to 90%, sequence homology to the amino acid sequence described in SEQ ID NO: 1, but the present invention is not limited thereto.

[0113] In this specification, the term "homology" refers to the degree of similarity between a sequence and a wild-type amino acid sequence and a wild-type nucleic acid sequence. In this case, homology comparisons between these sequences are 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. Large quantities of peptides can be obtained by inserting a polynucleotide encoding a peptide into a vector and expressing the peptide.

[0114] In this specification, the term "peptide" refers to a compound in which two or more α-amino acids are linked together via peptide bonds.

[0115] On the other hand, polypeptides may be covalently bonded with one or more substances selected from the group consisting of non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and its fragments, nucleotides, fibronectin, transferrin, FcRn-binding substances, sugars, elastin, heparin, and derivatives thereof, or they may form microspheres.

[0116] The non-peptidic polymer is preferably covalently bonded to the polypeptide, but the present invention is not limited thereto.

[0117] Polypeptides either covalently bond with the aforementioned substances or form microspheres, thus enhancing blood stability, delaying drug release to the kidneys, and inducing changes in receptor affinity.

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

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

[0120] The non-peptide polymer may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic acid-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 present invention is not limited thereto. Derivatives of non-peptide polymers known in the related art, and other derivatives that can be readily prepared at the level of the prior art, are also within the scope of the present invention.

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

[0122] Polyethylene glycol or its derivatives that may be used herein are linear or branched, preferably bibranched or tribranched, and more preferably tribranched.

[0123] The molecular weight of the non-peptidic polymer may be 3,000 to 100,000 Da, preferably 20,000 to 70,000 Da, and more preferably 40,000 to 60,000 Da. When the molecular weight of the non-peptidic polymer is within this molecular weight range, the non-peptidic polymer can bind to the polypeptide, thereby increasing the solubility of the resulting conjugate and extending the in vivo retention time of the conjugate.

[0124] Therefore, because the pharmaceutical composition according to the present invention contains a conjugate having a non-peptide polymer bound to a polypeptide, it can enhance in vivo stability and extend the in vivo half-life.

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

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

[0127] Diseases caused by insulin deficiency or decreased insulin sensitivity may include type 1 diabetes, type 2 diabetes, and diabetic complications.

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

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

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

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

[0132] Pharmaceutical compositions containing polypeptides can be administered parenterally. In this case, parenteral administration may be carried out by methods such as subcutaneous injection, intravenous injection, intramuscular injection, intranasal spray, administration via the mucous membrane into the nasal cavity or intestine, inhalation, or intrapleural injection.

[0133] In this case, to enable preparation of formulations for parenteral administration, the polypeptide can be mixed with a stabilizer or buffer to prepare a solution or suspension, which can then be prepared into unit dosage forms of ampoules or vials. The composition may be sterile and / or may contain adjuvants such as preservatives, stabilizers, wetting agents or emulsification promoters, salts and / or buffers for osmotic pressure regulation, and other therapeutically useful substances. In this case, the composition may be formulated according to conventional methods such as mixing, granulation, or coating methods.

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

[0135] Furthermore, the present invention provides a method for preparing a pharmaceutical composition comprising a conjugate containing a polypeptide and a non-peptide polymer.

[0136] 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, since the non-peptidic polymer is as described above, a detailed explanation of the polypeptide and non-peptidic polymer is omitted.

[0137] Specifically, the method for preparing a pharmaceutical composition involves mixing a non-peptide polymer and a polypeptide and reacting them with each other. In this case, the polypeptide and the non-peptide polymer can react in a molar ratio of 1:1 to 1:5, and as a result, the polypeptide and the non-peptide polymer can be bound to each other in a molar ratio of 1:1. In this case, the mixing is preferably carried out in a molar ratio of 1:1 to 1:2, more preferably 1:1.2, but the present invention is not limited thereto. When mixing is carried out within this molar ratio range, the conjugate can be obtained in high yield, thereby making it possible to prepare a high-purity conjugate containing polypeptides and non-peptide polymers.

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

[0139] The mixing of non-peptidic polymers and polypeptides for mutual reaction can be carried out at a pH of 4.0 to 9.0, preferably 5.5 to 7.5, but the present invention is not limited to this. Mixing outside this pH range will result in a decrease in yield. For example, when methoxypolyethylene glycol having a maleimide group is used as the non-peptidic polymer and a polypeptide having cysteine ​​at its C-terminus is used as the polypeptide, it is preferable to mix at a pH of 6 to 8. Reacting the polypeptide and methoxypolyethylene glycol within this pH range suppresses side reactions such as ring-opening of maleimide without causing side reactions due to the amine group of the polypeptide.

[0140] Since the conjugate process involves mixing non-peptide polymers and polypeptides and reacting them with each other, yielding 85-95%, this process is economically feasible and highly reproducible due to its very high yield. Therefore, this process would be effectively used for the preparation of pharmaceuticals.

[0141] In the mixing of non-peptide polymers and polypeptides reacting with each other, the reaction time may be in the range of 0.5 to 24 hours, or 1 to 24 hours, preferably 2 hours, but the present invention is not limited thereto. If the reaction time is less than 0.5 hours, the yield will be low and the purity will decrease. On the other hand, if the reaction time exceeds 24 hours, the polypeptide may decompose, and the economic effect may decrease due to the long processing time.

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

[0143] In a mixture in which a non-peptide polymer and a polypeptide react with each other, each of the polypeptide and the non-peptide polymer can be dissolved using the same or different solvent. Preferably, the solvent is a buffer, alcohol, dimethyl sulfoxide (DMSO), or a mixture thereof, but the present invention is not limited thereto. The solvent may also include solvents readily available in related art.

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

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

[0146] The present invention will be described in detail below with reference to the accompanying drawings so that a person with common sense in the art to which the invention pertains can easily implement it. However, it should be understood that the present invention can be embodied in various forms but is not intended to be limited to this context. Throughout this specification, similar reference figures refer to similar elements.

[0147] Example 1

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

[0149] In the amino acid sequence of Sequence ID No. 2, the underlined and bolded residues indicate that a covalent ring is formed between them.

[0150] Preparation Example 1: Synthesis of a conjugate containing polypeptides and non-peptide polymers

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

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

[0153] To prepare the conjugates of Examples 2 to 7, the polypeptides 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).

[0154] [Table 2]

[0155] In the amino acid sequences of Sequence IDs 2-4 listed in Table 2, the two residues underlined and highlighted in bold indicate residues that form a covalent ring between them.

[0156] Polypeptides and non-peptide conjugates were mixed in a molar ratio of 1:1.2 and reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was separated by ion-exchange chromatography using a TSK SP-5PW column (7.5 × 75 mm, Tosoh, Japan) at a flow rate of 0.8 mL / min. The separation was monitored at a UV wavelength of 280 nm. The PEGylated polypeptides were 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). The purity of the PEGylated polypeptides was evaluated by HPLC (see Figure 1). Next, the molecular weight of the PEGylated polypeptides was measured using a MALDI-TOF mass spectrometer (see Figure 2). In addition, the yield of the conjugates in Examples 2-7 was calculated from the chromatograms obtained during the chromatographic separation process as the area ratio of the conjugate to the polypeptide. The results are shown in Table 3.

[0157] [Table 3]

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

[0159] Experimental Example 1: Measurement of In Vitro Activity of Example 2

[0160] To investigate the preventive or therapeutic effects of the Example 2 conjugate 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).

[0161] To determine the activity against the GLP-1 receptor, HEK293 / CRE-Luc cells expressing the human glucagon GLP-1 receptor were purchased from GenScript and used. 5 × 10⁶ cells were placed in a 96-well plate. 4 Cells were seeded in wells, and then each well was treated with the polypeptide from Example 1 (0.001-300 nM), the conjugate from Example 2 (0.001-300 nM), natural glucagon (SEQ ID NO: 1: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT, 0.013-300 nM), and GLP-1 (SEQ ID NO: 6: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, 0.001-300 nM). The cells were then incubated in a CO2 incubator at 37°C for 4 hours. Subsequently, the amount of cAMP (luciferase reporter) produced was measured using the One-Glo® luciferase assay system (Promega) to determine the EC of the GLP-1 receptor. 50 The values ​​were calculated. The results are shown in Table 4 below.

[0162] Next, to determine the activity against the glucagon receptor (GCGR), we used DiscoverX's cAMP Hunter® eXpress GCGR CHO-K1 GPCR assay kit. Using CHO-K1 cells expressing human glucagon receptor, we plated 3 × 10⁶ cells in a 96-well plate. 4 Cells were seeded at a cell / well density. Each well was then treated with the polypeptide from Example 1 (0.013–300 nM), the conjugate from Example 2 (0.013–300 nM), natural glucagon (0.015–33.33 nM), and GLP-1 (0.001–30.00 nM), and the cells were then incubated in a CO2 incubator at 37°C for 30 minutes. The amount of cAMP produced was then measured to determine the EC of the glucagon receptor (GCGR). 50 The values ​​were calculated. The results are shown in Table 4 below.

[0163] [Table 4]

[0164] As shown in Table 4, GLP-1 showed high activity towards the GLP-1 receptor, but its activity towards the glucagon receptor was very low and could not be measured. On the other hand, natural glucagon showed very high activity towards the glucagon receptor, but low activity towards the GLP-1 receptor. Based on these results, the selectivity of this experimental method was confirmed.

[0165] On the other hand, the polypeptide of Example 1 has an EC of 0.13 relative to the GLP-1 receptor. 50 The polypeptide exhibited a value that was found to be nearly identical to that of GLP-1. This indicates that the polypeptide of Example 1 showed very high activity against the GLP-1 receptor, and that the polypeptide of Example 1 also had activity against the glucagon receptor. Furthermore, animal experiments confirmed that the polypeptide of Example 1 has an anti-obesity effect.

[0166] Furthermore, the conjugate of Example 2 was confirmed to retain similar activity to the polypeptide of Example 1 for both the GLP-1 receptor and the glucagon receptor. Generally, when a non-peptide polymer (e.g., PEG) is bound to a polypeptide, the activity against the receptor is significantly reduced compared to before binding. On the other hand, the conjugate of Example 2 showed little reduction in receptor activity even when a non-peptide polymer was bound to the polypeptide. This indicates that the conjugate of Example 2 maintained high activity while simultaneously exhibiting an extended in vivo half-life.

[0167] Therefore, the pharmaceutical composition according to the present invention has excellent activity against glucagon receptors and GLP-1 receptors, and has been confirmed to have anti-diabetic and anti-obesity effects, as well as triglyceride-reducing effects by suppressing appetite, promoting insulin secretion, and promoting lipolysis of adipocytes.

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

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

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

[0171] In this case, body weight and blood glucose were expressed as percentages (%) relative to 100% of the pre-administration (day 0) value.

[0172] [Table 5]

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

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

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

[0176] Furthermore, a time-series analysis of the blood glucose change pattern revealed that blood glucose levels decreased by approximately 80% compared to the blood glucose levels measured before administration. This indicates that the conjugate in Example 2 has a blood glucose-lowering effect, while the positive control did not have an insufficient blood glucose-lowering effect.

[0177] Based on these results, it can be seen that the effect of the conjugate in Example 2 on weight loss was brought about by an increase in energy metabolism in the body and a simple decrease in food intake. The pharmaceutical composition according to the present invention had the effect of reducing food intake, suppressing gastric emptying, and promoting lipolysis.

[0178] Experimental Example 3: Measurement of In Vivo Activity of Example 2 - 2

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

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

[0181] [Table 6]

[0182] As shown in Table 6, from the results obtained after administering the drug for 2 weeks, in the non - treated group, the blood glucose level increased rapidly after glucose administration and then decreased, but in the mouse group administered with the conjugate of Example 2, it was confirmed that the increase in blood glucose was significantly reduced. Therefore, it was confirmed that the glucose tolerance of the conjugate of Example 2 increased compared to the non - treated group. Also, in the mouse group administered with the conjugate of Example 2, it was revealed that the increase in blood glucose was smaller compared to the positive control.

[0183] Experimental Example 4: Measurement of In Vivo Activity of Example 2 - 3

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

[0185] First, the conjugate from 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. During the 12 days of drug administration, changes in blood glucose and body weight were measured every other day after drug administration. The results are shown in Table 7 below.

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

[0187] [Table 7]

[0188] [Table 8]

[0189] As shown in Table 7, weight loss was observed in the mouse group administered the conjugate of Example 2 compared to the untreated group. Furthermore, while high blood glucose levels were maintained for two weeks in the untreated group, blood glucose levels decreased in the mouse group administered the conjugate of Example 2.

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

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

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

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

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

[0195] [Table 9]

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

[0197] Experimental Example 6: Measurement of In vivo activity of Examples 2 and 6 5

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

[0199] As shown in Figure 5, it was revealed that in mice administered the conjugates of Example 2 or 6, blood glucose levels decreased compared to the control, while in the control administered PBS, blood glucose levels increased.

[0200] As shown in Figure 6, in the control group, blood glucose levels rose sharply due to administered glucose and then decreased, but in mice administered the conjugates of Example 2 or 6 for two weeks, the increase in blood glucose was significantly reduced 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 increased compared to the glucose tolerance of the control group.

[0201] Therefore, as shown in Figures 5 and 6, the pharmaceutical composition containing the polypeptide according to the present invention was confirmed to have preventive or therapeutic effects against obesity and diabetes.

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

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

[0204] Specifically, a non-alcoholic fatty liver disease (NAF) experimental animal model was created by first feeding normal C57BL / 6 mice (approximately 6 weeks old) a 60% high-fat diet for about 24 weeks, increasing the mice's body weight to an average of approximately 50g. Subsequently, the conjugate from Example 2 was administered by subcutaneous injection at a dose of 20 nmol / kg once every other day for 2 weeks. As a control, the GLP-1 agonist "liraglutide" was also administered by subcutaneous injection at a dose of 100 nmol / kg once daily for 2 weeks. After 2 weeks of drug administration, blood was collected from the mice, serum cholesterol concentration was measured, the liver was excised, weighed, embedded in paraffin, and then sectioned with a microtome. Liver biopsy was then performed using hematoxylin and eosin (H&E).

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

[0206] Therefore, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention has been shown to be effective in preventing and treating non-alcoholic fatty liver disease by reducing liver weight, serum cholesterol levels, and hepatic steatohepatia in animal models of non-alcoholic fatty liver disease.

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

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

[0209] First, an experimental animal model of non-alcoholic fatty liver disease was created by feeding normal C57BL / 6 mice (approximately 6 weeks old) a high-trans fatty acid diet containing 40% high fat, 20% fructose, and 2% cholesterol for approximately 16 weeks. Subsequently, the conjugate from Example 2 was administered by subcutaneous injection at a dose of 20 nmol / kg once every 3 days for 4 weeks. As a positive control, the GLP-1 agonist "liraglutide" was also administered by subcutaneous injection at a dose of 53 nmol / kg once daily for 4 weeks. After the 4-week experiment was completed, serum cholesterol levels, liver weight, and liver triglycerides (liver TG) were measured.

[0210] Referring to Figures 10-12, which show measurements of serum cholesterol levels, liver weight, and liver triglycerides, it can be seen that mice administered the conjugate of Example 2 showed a significant decrease in serum cholesterol levels, liver weight, and liver triglycerides compared to the untreated group administered physiological saline, and also decreased compared to the positive control (i.e., the liraglutide-treated group). Therefore, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention is effective in preventing and treating non-alcoholic fatty liver disease by reducing liver weight, serum cholesterol, and liver triglycerides in an animal model of non-alcoholic fatty liver disease.

[0211] Experimental Example 9: Measurement of In vivo activity of Example 2 (8)

[0212] After performing the experiment using the same method as in Experimental Example 8, liver biopsies were performed and the NAFLD activity score (NAS) was measured to investigate the preventive or therapeutic effect on non-alcoholic fatty liver disease. Following the same experiment as in Experimental Example 8, the conjugate from Example 2 was administered for 4 weeks, after which the mouse livers were excised, embedded in paraffin, and sectioned with a microtome. Subsequently, hematoxylin-eosin (H&E) staining and oil red O staining were performed.

[0213] As a result, as shown in Figures 13 and 14, the liver tissue images and NAS results after 4 weeks of administration showed that mice administered with the conjugate of Example 2 had significantly reduced hepatic steatosis and NAS compared to the untreated group administered with physiological saline and the positive control (i.e., the liraglutide-treated group). Therefore, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention is effective in preventing or treating non-alcoholic fatty liver disease by reducing hepatic steatosis and decreasing NAS in an animal model of non-alcoholic fatty liver disease.

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

[0215] The pharmaceutical composition containing the polypeptide according to the present invention is useful for the safe prevention or treatment of obesity, diabetes, or non-alcoholic fatty liver disease because it does not cause side effects such as vomiting or nausea, reduces food intake, improves insulin secretion, suppresses gastric emptying, promotes lipolysis, and lowers triglyceride levels. In certain embodiments, for example, the following items are provided: (Item 1) The following general formula 1: [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (Sequence IDs 7-9), (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is either a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (sequence number 10), EQAAK (sequence number 11), or EEAVK (sequence number 12); and, R3 is a deletion, cysteine, lysine, or methionine. A polypeptide having the amino acid sequence represented by the symbol. (Item 2) The following general formula 1: [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (Sequence IDs 7-9), (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is either a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (sequence number 10), EQAAK (sequence number 11), or EEAVK (sequence number 12); and, R3 is a deletion, cysteine, lysine, or methionine. A pharmaceutical composition comprising a polypeptide having the amino acid sequence represented by [the symbol shown]. (Item 3) The pharmaceutical composition according to item 2, wherein the polypeptide is covalently bonded to or forms microspheres with one or more selected from the group consisting of non-peptide polymers, fatty acids, cholesterol, antibodies, antibody fragments, albumin and its fragments, nucleotides, fibronectin, transferrin, FcRn conjugates, sugars, elastin, heparin, and derivatives thereof. (Item 4) The pharmaceutical composition according to item 2, wherein R2 comprises glutamic acid (E) and lysine (K), and the glutamic acid and lysine together form a ring via an amide bond. (Item 5) The pharmaceutical composition according to item 2, wherein the non-peptidic polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic acid-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. (Item 6) The pharmaceutical composition according to item 3, wherein the non-peptidic polymer is polyethylene glycol or a derivative thereof. (Item 7) The pharmaceutical composition according to item 6, wherein the molecular weight of the non-peptidic polymer is 3,000 to 100,000 Da. (Item 8) The polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidylpropionate (PEG succinimidylpropionate), methoxypolyethylene glycol succinimidylpropionate (methoxyPEG succinimidylpropionate), acrylate polyethylene glycol succinimidylpropionate (acrylatePEG succinimidylpropionate), thiol polyethylene glycol succinimidylpropionate (thiol PEG succinimidylpropionate), and hydroxysuccini. The pharmaceutical composition according to item 6, comprising at least one selected from the group consisting of imidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxy polyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), derivatives thereof, and multi-branched forms of those derivatives. (Item 9) The pharmaceutical composition according to item 6, wherein the polyethylene glycol or its derivative is linear or branched. (Item 10) The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used to prevent or treat obesity. (Item 11) The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used to prevent or treat diabetes. (Item 12) The pharmaceutical composition according to item 2, wherein the pharmaceutical composition is used to prevent or treat non-alcoholic fatty liver disease. (Item 13) The pharmaceutical composition according to item 12, wherein the non-alcoholic fatty liver disease comprises one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer. (Item 14) A method for preparing a pharmaceutical composition, wherein the pharmaceutical composition is Polypeptides having an amino acid sequence represented by the following general formula 1; and Conjugate containing non-peptide polymers The method includes, A method comprising the step of mixing the non-peptide polymer and the polypeptide and reacting them with each other: [General formula 1] R1-X1-QGTFTSDYSKYLD-R2-EFVQWLMNT-R3 (Sequence IDs 7-9), (wherein R1 is histidine, desaminohistidyl, N-dimethylhistidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, or β-carboxyimidazopropionyl; X1 is either a deletion, glycine, or aminoisobutyric acid (Aib); R2 is EKRAK (sequence number 10), EQAAK (sequence number 11), or EEAVK (sequence number 12); and, R3 is a deletion, cysteine, lysine, or methionine. (Item 15) The method according to item 14, wherein R2 comprises glutamic acid (E) and lysine (K), and the glutamic acid and lysine together form a ring via an amide bond. (Item 16) The method according to item 14, wherein the non-peptidic polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol (PVA), polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylactic acid-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof. (Item 17) The method according to item 14, wherein the non-peptidic polymer is polyethylene glycol or a derivative thereof. (Item 18) The polyethylene glycol derivatives include methoxypolyethylene glycol, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, polyethylene glycol succinimidylpropionate (PEG succinimidylpropionate), methoxypolyethylene glycol succinimidylpropionate (methoxyPEG succinimidylpropionate), acrylate polyethylene glycol succinimidylpropionate (acrylatePEG succinimidylpropionate), thiol polyethylene glycol succinimidylpropionate (thiol PEG succinimidylpropionate), and hydroxysucci The method according to item 17, wherein the present material is at least one selected from the group consisting of cinimidyl polyethylene glycol (hydroxysuccinimidyl PEG), methoxypolyethylene glycol succinimidyl carboxymethyl ester (mPEG succinimidyl carboxymethyl ester), acrylate polyethylene glycol succinimidyl carboxymethyl ester (acrylate PEG succinimidyl carboxymethyl ester), polyethylene glycol succinimidyl carbonate (PEG succinimidyl carbonate), polyethylene glycol propionaldehyde (PEG propionaldehyde), polyethylene glycol butyraldehyde (PEG butyraldehyde), its derivatives, and multi-branched forms of its derivatives. (Item 19) The method according to item 14, wherein the mixing for reacting the non-peptidic polymer and the polypeptide with each other comprises reacting the polypeptide and the non-peptidic polymer in a molar ratio of 1:1 to 1:5. (Item 20) The method according to item 14, wherein the mixing for reacting the non-peptide polymer and the polypeptide is carried out at a pH of 4.0 to 9.0. (Item 21) The method according to item 14, wherein the reaction time in the mixing for reacting the non-peptide polymer and the polypeptide is in the range of 0.5 to 24 hours. (Item 22) The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat obesity. (Item 23) The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat diabetes. (Item 24) The method according to item 14, wherein the pharmaceutical composition is used to prevent or treat non-alcoholic fatty liver disease. (Item 25) The method according to item 24, wherein the non-alcoholic fatty liver disease comprises one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer. (Item 26) A method for preventing or treating one or more diseases selected from the group consisting of obesity, diabetes mellitus, and non-alcoholic fatty liver disease, A method comprising the step of administering a pharmaceutical composition as defined in item 2 to a subject.

Claims

[Claim 1] The invention as shown in the drawings.

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