Application of polypeptide in blood sugar regulation

Abstract

The invention provides application of polypeptide in blood sugar regulation, and relates to the field of biological medicine. The polypeptide GSGGL provided by the invention can promote glucose consumption of HepG2 cells and reduce the blood sugar content of db / db diabetic model mice. Therefore, the polypeptide GSGGL provided by the invention can be applied to development of hypoglycemic drugs, and has the advantages of stable chemical properties, high biocompatibility, small toxic and side effects and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a polypeptide in regulating blood sugar. Background Art

[0002] Diabetes is a very harmful metabolic disease characterized by high blood sugar. In recent years, due to many factors such as the improvement of living standards, changes in dietary structure, increasingly tense pace of life, and sedentary lifestyle, the global incidence of diabetes has increased rapidly, and the incidence population is gradually getting younger. Diabetes has become the third largest disease that seriously threatens human health after tumors and cardiovascular diseases, accounting for 95% of patients.

[0003] Patients with type 2 diabetes (T2DM) account for 95% of the total diabetic population, and its prevention and treatment are the focus of diabetes research. Type 2 diabetes is a metabolic disease characterized by insulin resistance (IR), mainly characterized by hyperglycemia, which usually causes disorders in the body's sugar, fat and protein metabolism. Long-term hyperglycemia can easily cause chronic damage to the eyes, kidneys, liver, blood vessels and nerves, causing functional disorders, which can be life-threatening in severe cases. In the early treatment of type 2 diabetes, controlling postprandial blood sugar levels is the key to treatment. Hypoglycemic chemical drugs can be roughly divided into oral hypoglycemic drugs and injectable hypoglycemic drugs. Commonly used oral hypoglycemic drugs include insulin sensitizers, including biguanides (such as metformin) that reduce glucose production in the liver and thiazolidinediones (such as glitazones) that increase muscle sensitivity to insulin, α-glucosidase inhibitors (such as acarbose, miglitol, voglibose) that reduce glucose production in the intestine, insulin secretagogues such as sulfonylureas (such as glipizide and glimepiride) and meglitinides, glucose-dependent insulinotropic polypeptides (such as glucagon-like peptide-1) and dipeptidyl peptidase IV inhibitors (glinides), etc. Injectable hypoglycemic drugs include insulin and similar drugs, GLP-1 receptor agonists, etc.

[0004] Traditional glucose-lowering drugs have obvious defects, mainly manifested in various side effects such as gastrointestinal and liver diseases in some patients. There is also the problem of high costs due to long-term use of drugs, which cannot meet the needs of modern society for diabetes treatment. Finding efficient and safe drugs to prevent and treat diabetes remains a hot topic of global concern. Summary of the invention

[0005] The purpose of the present invention is to provide a drug which can reduce blood sugar and has less toxic and side effects.

[0006] In a first aspect, the present invention provides use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in preparing a biguanide replacement drug, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1.

[0007] In the above application, the polypeptide with the amino acid sequence of SEQ ID NO: 1 is composed of five amino acids, which are glycine (Gly)-serine (Ser)-glycine (Gly)-glycine (Gly)-leucine (Leu) from N-terminus to C-terminus, abbreviated as GSGGL.

[0008] In the above applications, the pharmaceutically acceptable salt can be formed by cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, bismuth, etc., and can also be formed by bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane and tetramethylammonium hydroxide, etc. In addition, the pharmaceutically acceptable salts of the polypeptides of the present invention may also include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, e), calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, hydrochloride, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate,edisylate, pamoate, embonate, estolate, palmitate, esylate, pantothenate, fumarate, phosphate / diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycosaminoglycan, llylarsanilate), sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethiodide, lactate, valerate, etc.

[0009] Furthermore, the pharmaceutical salt can be a pharmaceutical salt suitable for use in polypeptides, generally taking into account the sequence characteristics and properties of the polypeptide, the stability, solubility, biological toxicity and other physical and chemical factors of the polypeptide, and the intended use of the polypeptide. Commonly used pharmaceutical salts of polypeptides include, but are not limited to, acetate, pamoate, citrate, sodium salt, ammonium salt, hydrochloride, etc.

[0010] In the above application, the biguanide substitute drug refers to a drug that can be used instead of biguanide drugs, which has the same or similar efficacy as biguanide drugs, such as lowering blood sugar, but has less toxic side effects than biguanide drugs. Further, the biguanide drug is metformin.

[0011] In a second aspect, the present invention provides use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in the preparation of a drug for reducing blood sugar, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1.

[0012] In a third aspect, the present invention provides use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in the preparation of a medicament for preventing and / or treating hyperglycemia, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1.

[0013] In the above application, the hyperglycemia refers to the situation where the glucose level in the blood is higher than the normal range of 7.3 mmol / L. Hyperglycemia can be divided into two categories according to the cause: physiological and pathological; among them, physiological hyperglycemia is usually temporary and may be caused by factors such as strenuous exercise, high-sugar diet, emotional tension, etc., and blood sugar can return to normal after removing the inducement; pathological hyperglycemia is caused by disease. Further, the hyperglycemia is pathological hyperglycemia. Further, the hyperglycemia is diabetes. Further, the hyperglycemia is type 2 diabetes.

[0014] In the above application, the dosage form of the drug is selected from any one of tablets, capsules, granules, oral liquids, syrups, sprays, inhalants, ointments, powders, injections or freeze-dried powder injections.

[0015] In a fourth aspect, the present invention provides a polypeptide derivative, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1, and the polypeptide derivative is selected from at least one of B1) to B2): B1) a polypeptide derivative obtained by subjecting the polypeptide to at least one of PEG modification, fluorine element modification, biotin modification, and fluorescent group modification; B2) A polypeptide derivative formed by linking RGD to the N-terminus and / or C-terminus of a polypeptide.

[0016] In the above-mentioned polypeptide derivatives, the PEG modification refers to the modification of the amino group at the N-terminus of the polypeptide and / or the carboxyl group at the C-terminus of the polypeptide using polyethylene glycol (PEG) so that PEG is connected to the polypeptide through a covalent bond, which helps to increase the biocompatibility of the polypeptide and reduce toxicity; fluorine element modification refers to the introduction of fluorine atoms or fluorinated groups into the polypeptide molecule to increase the stability of the polypeptide and improve membrane permeability. The fluorination modification can be performed at the N-terminus or C-terminus of the polypeptide. The amino group at the N-terminus can be fluorinated to trifluoromethylamine (-CF3NH2), and the carboxyl group at the C-terminus can be fluorinated to trifluoroacetic acid (-CF3COOH). A fluorine atom or a trifluoromethyl (-CF3) group can also be introduced at the hydrogen atom position on the leucine side chain; biotin modification refers to the connection of biotin to the N-terminus of the polypeptide through a covalent bond, thereby improving the stability of the polypeptide in vivo and in vitro; fluorescent group modification refers to the connection of the fluorescent group to the serine hydroxyl side chain of the polypeptide through a covalent bond, which facilitates the detection and analysis of the polypeptide and intracellular tracking.

[0017] Among the above-mentioned polypeptide derivatives, RGD refers to a tripeptide containing three amino acids, and the specific amino acid connection method is arginine-glycine-aspartic acid. Connecting RGD to the N-terminus and / or C-terminus of the polypeptide helps to improve the targeting of the drug, thereby increasing the concentration of the drug in the lesion site, reducing the impact on normal cells, improving the efficacy and reducing side effects.

[0018] In a fifth aspect, the present invention provides the use of the above polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the use is selected from at least one of A1) to A3): A1) Application in the preparation of biguanide substitute drugs; A2) Use in the preparation of drugs for lowering blood sugar; A3) Use in the preparation of a medicament for preventing and / or treating hyperglycemia.

[0019] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising a first blood sugar lowering active ingredient and a pharmaceutically acceptable carrier, wherein the first blood sugar lowering active ingredient is selected from at least one of the above-mentioned polypeptides, pharmaceutically acceptable salts of polypeptides, derivatives of polypeptides, and pharmaceutically acceptable salts of derivatives of polypeptides.

[0020] The pharmaceutical composition provided by the present invention has polypeptide GSGGL as the main active ingredient, and its content is not less than the therapeutically effective amount. It is understandable that the pharmaceutical composition provided by the present invention may also include other second hypoglycemic active ingredients with hypoglycemic effects, such as insulin or insulin analogs.

[0021] The carrier in the pharmaceutical composition provided by the present invention includes excipients, lubricants, pH regulators, stabilizers, wetting agents, emulsifiers, suspending agents, preservatives, sweeteners, colorants, flavoring agents, osmotic pressure regulators, etc. Specifically, excipients can be, for example, lactose, galactose, mannitol, low molecular weight dextran, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose and polyacrylic acid, such as Carbopol, etc. Lubricants can be, for example, talc, magnesium stearate and mineral oil. pH regulators can be, for example, organic or inorganic acids such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, and one or more of the physiologically acceptable inorganic bases or salts such as potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium bicarbonate salts. The stabilizer can be selected from one or more of EDTA-2Na, sodium thiosulfate, sodium pyrosulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, lysine, glutamic acid, aspartic acid, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyl / hydroxycellulose or its derivatives such as HPC, HPC-SL, HPC-L or HPMC, cyclodextrin, sodium dodecyl sulfate or tris(hydroxymethyl)aminomethane, etc. The osmotic pressure regulator is a salt (e.g., sodium chloride, potassium chloride), a sugar or sugar alcohol, an amino acid, an alditol (e.g., glycerol, propylene glycol, 1,3-propylene glycol, 1,3-butylene glycol), a polyethylene glycol (e.g., PEG400) or a mixture thereof.

[0022] The pharmaceutical composition may further comprise biodegradable polymer beads, dextran and cyclodextrin inclusion complexes.

[0023] The dosage form of the above-mentioned pharmaceutical composition can be an existing dosage form, for example, for oral administration, the composition can be in the form of tablets, capsules, granules, oral liquids, syrups, sprays, inhalants, pastes, powders and sustained release preparations. Suitable excipients for oral administration include pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0024] The above-mentioned pharmaceutical composition can also be in the form of pills, tablets or capsules, and the composition contains any one of the following substances: a diluent, such as lactose, sucrose, dicalcium phosphate, etc.; a disintegrant, such as starch or its derivatives; a lubricant, such as magnesium stearate, etc.; and a binder, such as starch, gum arabic, polyvinyl pyrrolidone, gelatin, cellulose and its derivatives.

[0025] The above-mentioned pharmaceutical composition can also be a liquid composition, which can be prepared by dissolving or dispersing the polypeptide of the present invention and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as saline solution (e.g., 0.9% w / v sodium chloride), dextrose aqueous solution, glycerol, ethanol, etc. to form a solution or suspension for oral, topical or intravenous administration.

[0026] The above-mentioned pharmaceutical compositions can also be provided in lyophilized form. Such compositions can include a buffer (e.g., bicarbonate) for rehydration before administration, or a buffer can be included in the lyophilized composition for rehydration, for example, with water. The lyophilized composition can additionally contain a suitable medicament, such as other drugs for treating diabetes. The lyophilized composition can optionally be packaged in combination with a rehydration buffer to be provided in a syringe so that the rehydrated composition can be immediately administered to the patient.

[0027] In some embodiments, the polypeptides of the present invention can be prepared into controlled or sustained release preparations, such as injectable microspheres, bioerodible particles, polymer compounds, beads, or liposomes or other biocompatible matrices, and the preparations can then be delivered by injection. For example, the polypeptides of the present invention can be encapsulated in liposomes, or formulated into microparticles or microcapsules, or can be incorporated into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins, poly (lactic acid-co-glycolic acid) (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or delivered via protein carriers or using conjugates.

[0028] In a seventh aspect, the present invention provides a method for preventing and / or treating hyperglycemia, comprising administering to a subject a therapeutically effective amount of the above polypeptide or a pharmaceutically acceptable salt of the polypeptide or the above pharmaceutical composition.

[0029] In the present invention, the subject may be a subject with elevated blood sugar or hyperglycemia, or a patient with a second disease or functional disorder other than hyperglycemia who also has hyperglycemia. The above-mentioned method for preventing or treating hyperglycemia can reduce or eliminate the clinical symptoms related to hyperglycemia in the subject, or reduce blood sugar.

[0030] In the present invention, the subject can be a mammal, which can be selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents and primates, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, gorillas) and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys and humans.

[0031] The dosage of the pharmaceutical composition for treating hyperglycemia is variable, depending on the mode of administration, route of administration, age and / or weight of the individual, and the condition of the individual being treated, and is ultimately determined by the attending physician. The dosage administered to an individual, in the context of the present invention, should be sufficient to induce a beneficial response in the individual over a period of time. The dosage is a "therapeutically effective amount". In one embodiment of the invention, for mice, the therapeutically effective amount may be 50-200 mg polypeptide / kg, preferably 200 mg polypeptide / kg.

[0032] The polypeptide of the present invention can be administered with suitable pharmaceutical excipients as required. It will be appreciated by those skilled in the art that the composition will vary depending on the mode of administration and dosage unit.

[0033] It is understood that the polypeptides and pharmaceutical compositions provided by the invention can be co-administered with other therapeutic agents for the treatment or prevention of hyperglycemia. Co-administration can be simultaneous, for example, in the form of a single pharmaceutical composition or an independent composition. The compositions of the invention can also be administered separately from another therapeutic agent, for example, with an independent administration schedule.

[0034] The present invention provides an application of a polypeptide in regulating blood sugar, and relates to the field of biomedicine. The polypeptide GSGGL provided by the present invention can promote the consumption of glucose by HepG2 cells, and can reduce the blood sugar content of db / db diabetic model mice; therefore, the polypeptide GSGGL provided by the present invention can be applied to the development of hypoglycemic drugs, and has the advantages of stable chemical properties, high biocompatibility, and low toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result diagram of MS identification of peptide molecular weight; Figure 2 This is the result diagram of HPLC testing of peptide purity; Figure 3 The effect of peptides on glucose consumption in HepG2 cells; ns means no significant difference compared with the control group, *** means P < 0.001, **** means P < 0.0001; Figure 4 The effect of peptides on the activity of HepG2 cells; **** indicates P < 0.0001; Figure 5 The effect of peptides on the water intake of mice; Figure 6 The effect of peptides on fasting blood glucose levels in mice; Figure 7 Results of oral glucose tolerance test (OGTT) in mice; Figure 8 The results of the mouse insulin tolerance test (ITT); Fig. 9 The results of the determination of serum insulin content in mice; **** indicates P < 0.0001; Fig.10 The graphs are the results of mouse serum ELISA experiments; A is the results of the BUN level experiment in mouse serum; B is the results of the CK level experiment in mouse serum; C is the results of the ALT level experiment in mouse serum; D is the results of the ALS level experiment in mouse serum; ns means that there is no significant difference between the treatment group and the model group. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] Example 1 Preparation of polypeptide In this example, a polypeptide having an amino acid sequence of GSGGL was synthesized by solid phase synthesis.

[0039] The molecular weight and HPLC tests of the pure peptides were performed, and the test results were as follows: Figure 1-2 As shown, it can be seen that the molecular weight of the polypeptide is 389.41 Da and the purity is 99.6%.

[0040] Example 2 Effects of polypeptides on glucose consumption and cell activity of HepG2 cells In order to observe the effect of the polypeptide synthesized in Example 1 on the glucose consumption of hepatocytes and its toxic effects, human hepatoma cells (HepG2) were selected as experimental materials in this example, the glucose consumption of cells was measured using a glucose detection kit, and the survival rate of cells after treatment with different concentrations of polypeptides was measured using the MTT method.

[0041] GraphPad Prism software was used for statistical analysis, and each data was expressed as mean ± standard deviation. The Student's t test was used to compare two means, and p < 0.05 was considered statistically significant.

[0042] 1. HepG2 cell culture Take out the cryopreserved tube of HepG2 cells stored in liquid nitrogen, immediately put it in a 37℃ water bath, and shake it slightly. After the liquid melts, aspirate the cell suspension into a culture bottle containing DMEM high-glucose culture medium (Sigma, D6429) including 10% fetal bovine serum and 2% penicillin-streptomycin. Place the cells in a 37℃, 5% CO2 incubator for constant temperature culture, change the medium overnight, and when the cells cover 80%-90% of the bottom of the culture bottle, add 0.25% trypsin EDTA solution for digestion, transfer to other containers at a ratio of 1:2 or 1:3 for expansion culture, and select cells in the logarithmic growth phase with good growth status for experiments.

[0043] 2. Determination of Glucose Consumption by HepG2 Cells HepG2 cells in the logarithmic growth phase were taken, washed twice with PBS, and digested from the culture flask by trypsin digestion to prepare a cell suspension, which was inoculated in a 96-well plate, and 100 μL of cell suspension was added to each well. The plate was placed in a 37°C, 5% CO2 incubator for constant temperature culture for 24 h. After the cells adhered to the wall, the culture medium was poured off. The cells were divided into 7 groups, each with 5 wells. Groups 1-5 were experimental groups, and 100 μL of the above-mentioned DMEM high-glucose culture medium containing different concentrations (10, 50, 100, 200, 400 μg / mL) of polypeptides were added respectively; Group 6 was a blank control group, and DMEM high-glucose culture medium without polypeptides was added; Group 7 was a positive control group, and DMEM high-glucose culture medium containing 2 mM metformin was added, and the cells were cultured in an incubator for 12 h. At the same time, a blank well was set, that is, no cells were added, only culture medium was added.

[0044] After the cell culture was completed, the culture medium was collected and the glucose content in the culture medium was determined using a glucose kit (Nanjing Jiancheng Bioengineering Institute, A154-1-1). The glucose consumption (Mm) was calculated as follows: (glucose content in the culture medium in the blank well) - (glucose content in the culture medium in the control / experimental group).

[0045] The results are as follows Figure 3 As shown, it can be seen that the peptide concentration of 100-200 µg / mL can promote the consumption of glucose by cells, and the effect is close to that of the positive control metformin.

[0046] 3. Determination of HepG2 Cell Activity According to the culture method of HepG2 cells in 2, the culture medium was discarded after the culture was completed, and MTT solution with a final concentration of 5 mg / mL was added to each well. The culture was terminated after incubation for 4 h. The same volume of dimethyl sulfoxide solution was added to each well and shaken for 10 min to fully dissolve the crystalline material. The absorbance value of the solution was measured at a wavelength of 570 nm using an ELISA reader, and the calculation was performed according to cell activity (%) = (absorbance value of the solution in the control / experimental group) / (absorbance value of the solution in the blank well) × 100% to analyze the cell activity and number.

[0047] The results are as follows Figure 4 As shown in the figure, the peptides within 200 μg / mL had no significant effect on the activity of HepG2 cells.

[0048] Example 3 Verification of the hypoglycemic activity of the peptide on type 2 diabetic mice 1. Drug treatment of mice Six 6-week-old C57BLKS / J male mice (The Jackson Laboratory, 000662) were selected as the control group (normal group), and 6-week-old C57BLKS / J db / db male mice (The Jackson Laboratory, 008340) were selected as type 2 diabetes model mice. The C57BLKS / J db / db male mice were randomly divided into 5 groups, 6 mice in each group, namely low-dose group, medium-dose group, high-dose group, model group and metformin group. The mice were adapted to feeding for two weeks, 3-4 mice per cage, temperature of 25±2℃, humidity of 55±5%, free food and water during the period. After the mice were adapted, they were housed individually in one cage and intraperitoneally injected once a day for 5 consecutive weeks. The low-dose group was intraperitoneally injected with 50 mg / kg BW of polypeptide, the medium-dose group was intraperitoneally injected with 100 mg / kg BW of polypeptide, the high-dose group was intraperitoneally injected with 200 mg / kg BW of polypeptide, and the metformin group was intraperitoneally injected with 200 mg / kg BW of metformin. The model group and the normal group were intraperitoneally injected with normal saline. During the entire experiment, the amount of water consumed by each mouse was recorded every 7 days.

[0049] The amount of water consumed by mice in each group was Figure 5 As shown in the figure, db / db mice showed polydipsia due to increased blood sugar. The water intake of mice in the polypeptide group and metformin group was not much different from that of the model group in the first 2 weeks, and the water intake began to decrease in the 3rd week.

[0050] 2. Determination of fasting blood glucose in mice Before the start of treatment (day 0) and throughout the experiment, the fasting blood glucose level of mice was measured every 7 days using a blood glucose meter. The mice were fasted for 12 hours before measurement, and blood samples were collected from the tail vein. The mice were taken out of the cage and gently placed on the iron grid. The tail of the mouse was cut off about 1-2 mm from the end with scissors. The tail of the mouse was gently squeezed to let the blood flow out, the first drop was discarded, and the second drop of blood was used to measure the fasting blood glucose level (mmol / L) with a blood glucose meter.

[0051] The results are as follows Figure 6 As shown, during the 5 weeks of continuous intraperitoneal injection, the blood glucose levels of mice in the model group were significantly higher than those in the normal group, and the blood glucose levels of mice in the drug-treated groups decreased rapidly. The effect of the high-dose polypeptide group was better than that of the metformin group, indicating that the polypeptide has a better blood glucose-lowering effect.

[0052] 3. Oral Glucose Tolerance Test (OGTT) in Mice In the fifth week of treatment, the mice were fasted for 12 h and then subjected to an oral glucose tolerance test by gavage with 2 g / kg BW of glucose solution. Blood was then collected through the tail vein at 0 min, 30 min, 60 min, 90 min, and 120 min to measure the blood glucose level of each mouse at each time point.

[0053] The results are as follows Figure 7 As shown, compared with the model group mice, the plasma glucose level of the drug-treated group mice was significantly reduced, and the hypoglycemic effect of the peptide at a concentration of 200 mg / kg was comparable to that of metformin.

[0054] 4. Insulin tolerance test (ITT) in mice In the fifth week of treatment, the mice were fasted for 12 h, insulin (0.2 U / kg BW) was injected intraperitoneally, and then blood was collected through the tail vein at 0 min, 30 min, 60 min, 90 min, and 120 min to measure the blood glucose level of each mouse at each time point.

[0055] The results are as follows Figure 8 As shown in the figure, 30 minutes after insulin injection, the plasma glucose levels of mice in the model group, metformin group and high-dose group decreased by 26.9%, 52.7% and 48.9%, respectively. 60 minutes after insulin injection, the plasma glucose levels of mice in the model group, metformin group and high-dose group decreased by 43.2%, 67.6% and 73.8%, respectively. This indicates that the 200 mg / kg dose of peptide treatment significantly enhanced the insulin-induced decrease in plasma glucose levels.

[0056] 5. Determination of Insulin Levels in Mice Blood samples were collected from the tail vein of mice and placed in a 4°C refrigerator for 2 h. The samples were centrifuged at 3000 r / min for 10 min to separate plasma and serum. The serum was placed in a clean centrifuge tube and the insulin content (mU / L) in mouse serum was determined using an insulin Elisa detection kit (BBI Life Sciences Co., Ltd., D721159).

[0057] The results are as follows Fig. 9 As shown in the figure, compared with normal mice, the serum insulin level of mice in the model group was significantly increased. Compared with the model group, the serum insulin content of mice was reduced after 5 weeks of treatment with low, medium and high doses of peptides and metformin.

[0058] Example 4 Biosafety Evaluation of Peptides In order to evaluate the safety of the peptide in vivo, whole blood was drawn from mice 5 weeks after intraperitoneal injection, placed in a 4℃ refrigerator for 2 h, centrifuged at 3000 r / min for 10 min, and plasma and serum were separated. The serum was collected in a clean centrifuge tube, and the ALT (alanine aminotransferase) and ALS (aldosterone) levels in the serum after treatment were detected by ELISA to evaluate liver function; the CK (creatine kinase) level in the serum was detected to evaluate myocardial damage; the BUN (blood urea nitrogen) level was detected to evaluate renal function. The mouse urea nitrogen (BUN) enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Ke Ai Bo Biotechnology, catalog number CB10533-Mu; the mouse creatine kinase (CK) ELISA kit was purchased from Jiang Lai Biotechnology, catalog number JL18284; the mouse alanine aminotransferase (ALT) ELISA kit was purchased from Jiang Lai Biotechnology, catalog number JL12668; the aldosterone (ALS) ELISA kit was purchased from BBI Life Sciences Co., Ltd., catalog number D751031. Perform the test according to the kit instructions.

[0059] Test results such as Fig.10 As shown, there was no significant difference in the levels of ALT, ALS, CK and BUN in the serum before and after the peptide drug treatment, indicating that the liver function, heart function and kidney function of the peptide mice were not significantly affected, indicating that the peptide has good biosafety.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in the preparation of a biguanide substitute drug, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. Use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in the preparation of a drug for lowering blood sugar, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

3. Use of a polypeptide or a pharmaceutically acceptable salt of a polypeptide in the preparation of a medicament for preventing and / or treating hyperglycemia, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

4. The use according to claim 3, characterized in that: The hyperglycemia is diabetes.

5. The use according to any one of claims 1 to 4, characterized in that: The pharmaceutically acceptable salt of the polypeptide is at least one of acetate, pamoate, citrate, sodium salt, ammonium salt and hydrochloride of the polypeptide.

6. The use according to any one of claims 1 to 5, characterized in that: The dosage form of the drug is selected from any one of tablets, capsules, granules, oral liquids, syrups, sprays, inhalants, pastes, powders, injections or freeze-dried powder injections.

7. A polypeptide derivative, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO: 1, and the derivative of the polypeptide is selected from at least one of B1) to B2): B1) a polypeptide derivative obtained by subjecting the polypeptide to at least one of PEG modification, fluorine element modification, biotin modification, and fluorescent group modification; B2) A polypeptide derivative formed by linking RGD to the N-terminus and / or C-terminus of a polypeptide.

8. Use of the polypeptide derivative or pharmaceutically acceptable salt thereof according to claim 7, characterized in that: The application is selected from at least one of A1) to A3): A1) Application in the preparation of biguanide substitute drugs; A2) Use in the preparation of drugs for lowering blood sugar; A3) Use in the preparation of a medicament for preventing and / or treating hyperglycemia.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a first hypoglycemic active ingredient and a pharmaceutically acceptable carrier, wherein the first hypoglycemic active ingredient is selected from at least one of the polypeptide in the application according to any one of claims 1 to 6, the pharmaceutically acceptable salt of the polypeptide in the application according to any one of claims 1 to 6, the polypeptide derivative according to claim 7, and the pharmaceutically acceptable salt of the polypeptide derivative according to claim 7.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further comprises a second hypoglycemic active ingredient.

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