Polypeptide compound and application thereof

Abstract

The invention discloses a polypeptide with GLP-1R / GCGR double receptor agonistic activity and a preparation method thereof. The polypeptide is a polypeptide with high stability and high activity, has good DPP IV enzyme stability, plasma stability and sugar tolerance improving effect, has longer in-vivo half-life period and lasting action time compared with natural glucagon GLP-1, and can be used for treating metabolism-related diseases and complications thereof.

Description

Technical Field

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

[0002] Diabetes is a chronic disease characterized by hyperglycemia, caused by an absolute or relative deficiency in insulin secretion and impaired utilization. Persistent hyperglycemia can lead to a variety of complications, such as diabetic nephropathy, diabetic eye disease, diabetic cardiovascular disease, diabetic cerebrovascular disease, diabetic neuropathy, and diabetic foot, which can even be life-threatening in severe cases. With the increasing prevalence of obesity, the incidence of diabetes is rising annually. According to the International Diabetes Federation (IDF), the number of people with diabetes worldwide reached 463 million in 2019. It is estimated that by 2030, the number of people with diabetes worldwide will reach 578 million. In recent years, with rapid economic development, increased life expectancy, and the arrival of an aging society, the prevalence of diabetes in China has skyrocketed. Diabetes has become a major public health issue in my country, imposing a severe social and economic burden and impacting the country's economic development.

[0003] Obesity, a disorder of energy metabolism, has become an increasingly serious health problem in many countries and is associated with a wide range of dangerous conditions, such as cardiovascular and cerebrovascular disease, kidney disease, dyslipidemia, liver disease, and osteoporosis. Obesity is the primary cause of insulin resistance, and obesity and insulin resistance are two major causative factors for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH, also known as metabolic dysfunction-associated steatohepatitis (MASH)). Therefore, there is an increasingly urgent need to find safe and effective treatments for these metabolic disorders.

[0004] Incretins are substances secreted from the intestine in response to food intake under normal physiological conditions. They stimulate insulin secretion from pancreatic β-cells in response to glucose levels, regulating glucose homeostasis and protecting β-cells. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-releasing polypeptide (GIP) are the two currently discovered incretins (Glucagon-like peptide-1 and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients. Digestion 1995;56:117-126).

[0005] Diabetic patients need to take hypoglycemic drugs for a long time. The hypoglycemic drugs currently on the market include insulin injection preparations and oral hypoglycemic drugs. GLP-1 receptor agonists (GLP-1RA) have been a hot topic in the research and development of hypoglycemic drugs in recent years because of their significant effects in improving key pathophysiological defects of type 2 diabetes, reducing the risk of cardiovascular death, improving atherosclerosis, reducing weight, lowering systolic blood pressure and improving blood lipid profiles. GLP-1 is a hormone mainly produced by intestinal L cells and is an incretin. GLP-1RA is a new type of hypoglycemic drug in recent years. By activating the GLP-1 receptor, it enhances insulin secretion in a glucose concentration-dependent manner and inhibits glucagon secretion, thereby achieving the effects of lowering blood sugar and losing weight.

[0006] However, natural GLP-1 has a short half-life and is easily degraded by dipeptidyl peptidase IV (DPP-4) once it enters the circulation, preventing it from fully exerting its effects. This is why GLP-1RAs have emerged. Currently available GLP-1RAs can be divided into short-acting and long-acting formulations, each with distinct molecular structures, pharmacokinetics, and dosing frequencies.

[0007] GLP-1 and glucagon (GCG) analogs have shown great potential in the treatment of diabetes and obesity by mimicking the effects of natural hormones to regulate blood sugar and metabolic processes. To ensure the effectiveness of these peptide drugs, they must remain stable in the body and avoid rapid degradation and clearance.

[0008] Currently, there is still a need to develop drugs that have both GLP-1R / GCGR dual receptor agonist activity and are sufficiently stable, highly active and have low immunogenicity risk. Summary of the Invention

[0009] To overcome the deficiencies in the prior art, the present invention provides polypeptide drugs that stimulate GCG receptors and GLP-1 receptor analogs for treating metabolic diseases (such as diabetes, obesity, etc.) and their applications. Specifically,

[0010] In a first aspect of the present invention, a polypeptide having GLP-1R / GCGR dual receptor agonist activity is provided, wherein the polypeptide is a variant of SEQ ID NO: 1, and the variant comprises an amino acid sequence having at least one amino acid mutation at positions 19-30 of SEQ ID NO: 1.

[0011] Preferably, the polypeptide variant comprises a mutation of one or a combination of two or more of A19, D21, W25, N28 or G30 of SEQ ID NO: 1; further preferably, it comprises a mutation of one or a combination of two or more of A19G, D21E, W25F, N28E or G30A.

[0012] Preferably, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-6.

[0013] Preferably, the polypeptide may further comprise at least one modification. The modified polypeptide may have better properties than the corresponding unmodified polypeptide, such as higher receptor agonist activity, stability, lower immunostimulatory activity, longer half-life, etc.

[0014] Preferably, the modification includes but is not limited to amidation, acetylation, glycosylation, phosphorylation or the inclusion of other functional components.

[0015] Preferably, the glycosylation includes glycosylation at any amino acid position, in the form of N-linked or O-linked glycosylation.

[0016] Preferably, the other functional components include but are not limited to water-soluble polymers, serum albumin, transferrin, Fc fragments, unstructured polypeptides or fragments of any of the foregoing.

[0017] Preferably, the water-soluble polymer includes but is not limited to polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups and branched or unbranched C8-C30 alkyl groups.

[0018] Preferably, the unstructured polypeptides include, but are not limited to, XTEN and PAS polypeptides (eg, conformationally disordered polypeptide sequences composed of amino acids Pro, Ala, and / or Ser).

[0019] The modification can occur at the N-terminus, C-terminus or any position in the middle of the sequence.

[0020] The modification may be one or more, and the multiple modifications may be of the same or different modification types.

[0021] Preferably, the modification comprises directly or indirectly linking a modification component (such as other functional components) to the polypeptide.

[0022] Further preferably, the indirect connection comprises the use of a linker or a non-naturally encoded amino acid linker. Exemplary linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths (such as polyethylene glycol or polydextran) or polypeptides of various lengths.

[0023] In one embodiment, the polypeptide comprises an amidation modification at the C-terminus.

[0024] The polypeptide can be prepared by any method in the prior art, such as chemical synthesis, biosynthesis, etc.

[0025] The second aspect of the present invention provides a nucleic acid encoding the above polypeptide.

[0026] The third aspect of the present invention provides a product comprising the above-mentioned polypeptide and / or the above-mentioned nucleic acid, wherein the product includes a vector, a cell, a kit, a delivery system or a pharmaceutical composition.

[0027] Preferably, the vector comprises the above-mentioned nucleic acid.

[0028] Preferably, the vector can be any vector suitable for delivering the nucleic acid to the target tissue or target cell for expression.

[0029] Preferably, the vector is a viral vector. Preferably, the viral vector includes but is not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector, and the like.

[0030] Preferably, the vector is a non-viral vector. Preferably, the non-viral vector includes but is not limited to any one or a combination of two or more of liposomes, lipid nanoparticles (LNP), polymers, proteins, aptamers, and N-acetylgalactosamine (GalNAc). Further preferably, the non-viral vector includes lipid nanoparticles (LNP).

[0031] Preferably, the delivery system comprises the above polypeptide or the above carrier.

[0032] Preferably, the delivery system comprises further modifications of the above polypeptides. Further preferably, the modifications include but are not limited to fusion of penetrating peptides, responsive peptides or self-assembling oligomeric proteins.

[0033] More preferably, the penetrating peptide (CPP) includes but is not limited to protamine, Tat peptide, transportan peptide, penetratin peptide or oligoarginine peptide;

[0034] The responsive peptides include but are not limited to pH responsive peptides, enzyme responsive peptides, temperature responsive peptides or redox responsive peptides;

[0035] The oligomeric protein includes but is not limited to Helicobacter pylori ferritin, dioxotetrahydropterin synthase, C4b binding protein or dihydrosulfonyl acetyltransferase.

[0036] Preferably, the pharmaceutical composition comprises the polypeptide or the delivery system, and pharmaceutically acceptable excipients.

[0037] The fourth aspect of the present invention provides a method for preparing the above-mentioned polypeptide. The polypeptide can be prepared by any method in the prior art, such as chemical synthesis and / or biosynthesis.

[0038] Preferably, the chemical synthesis method includes solid phase synthesis method, liquid phase synthesis method, etc., and the biosynthesis method includes fermentation method, gene recombination method, enzymatic hydrolysis method, etc.

[0039] Preferably, the preparation method comprises:

[0040] (1) Synthesizing crude polypeptides using chemical synthesis methods;

[0041] (2) separating and purifying the crude product to obtain the polypeptide.

[0042] Specifically, the polypeptides of the present invention can be prepared by standard peptide synthesis methods, for example, by standard solid phase or liquid phase methods, stepwise or by fragment assembly, and isolation and purification of the final peptide compound product, or by biosynthesis or any combination thereof. Preferably, the polypeptides of the present invention can be synthesized by solid phase or liquid phase peptide synthesis methods.

[0043] In one embodiment, the preparation method includes solid-phase peptide synthesis (SPPS). SPPS involves sequentially coupling the amino acids of the polypeptide to a resin to form a peptide chain. After sequence synthesis is complete, the N-terminal Fmoc protecting group is first deprotected (or after N-terminal modification is completed), followed by deprotection of the side chain protecting groups, and the peptide is cleaved from the resin.

[0044] The fifth aspect of the present invention provides the use of the above-mentioned polypeptide, nucleic acid or product in the preparation of a drug for treating metabolic-related diseases or complications.

[0045] Preferably, the metabolic-related diseases include but are not limited to obesity, hyperlipidemia, diabetes or fatty liver.

[0046] The complications include but are not limited to diabetic eye disease, diabetic neuropathy, diabetic foot, cardiovascular and cerebrovascular diseases, kidney disease, liver disease or osteoporosis.

[0047] The eighth aspect of the present invention relates to a method for preventing and / or treating metabolic-related diseases or complications, wherein the method comprises administering an effective amount of the above-mentioned polypeptide, the above-mentioned nucleic acid or the above-mentioned product to a diseased individual.

[0048] Preferably, the metabolic-related diseases include but are not limited to obesity, hyperlipidemia, diabetes (type 1 or type 2 diabetes), and fatty liver.

[0049] The complications include but are not limited to diabetic eye disease, diabetic neuropathy, diabetic foot, cardiovascular and cerebrovascular diseases, kidney disease, liver disease or osteoporosis.

[0050] Preferably, the patient comprises a human or a non-human animal, such as a non-human mammal.

[0051] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating or reversing the progression or severity of a sign, symptom, disorder, condition or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions or disorders.

[0052] The "effective amount" of the present invention refers to the amount or dosage of the drug of the present invention that provides the desired treatment or prevention after administration to an individual or organ in a single or multiple doses.

[0053] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them.

[0054] The "individual" described in the present invention can be a human or a non-human animal, and the non-human animal can be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0055] Beneficial effects of the present invention:

[0056] The present application further replaces and optimizes the amino acid sequence of the dual-target polypeptide c381. The obtained mutant polypeptide is a sufficiently stable and highly active polypeptide that does not require cross-linking with fatty acids, polyethylene glycol, albumin, or immunoglobulin Fc fragments, and does not require mutation of the second Ser to a non-natural amino acid. Therefore, it can minimize the potential risk of immunogenicity, and omit tedious chemical modification / cross-linking steps, simplify the preparation process, and improve product consistency. Compared with c381, the polypeptide of the present application has better DPPIV enzyme stability and plasma stability, and improves glucose tolerance; compared with natural glucagon, GLP-1, it has a longer in vivo half-life and duration of action, and the polypeptide of the present application can be used to treat metabolic-related diseases and their complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 :A is the rat plasma stability result, B is the area under the plasma drug remaining %-time curve in A;

[0058] Figure 2 :A is the human plasma stability result, B is the area under the plasma drug remaining %-time curve in A;

[0059] Figure 3 :A is the result of human DPPIV enzyme stability test, B is the area under the curve of drug remaining %-time in A;

[0060] Figure 4 : A is the result of intraperitoneal glucose tolerance test, B is the area under the blood glucose concentration-time curve in A;

[0061] Figure 5 : Results of tolerance test 120 minutes after intraperitoneal injection of glucose;

[0062] Figure 6 : A is the result of intraperitoneal glucose tolerance test, B is the area under the blood glucose concentration-time curve in A;

[0063] Figure 7 : A is the result of intraperitoneal glucose tolerance test 3.75 hours after administration, B is the result of intraperitoneal glucose tolerance test 3.5 hours after administration, and C is the result of intraperitoneal glucose tolerance test 3.25 hours after administration;

[0064] Figure 8 : Results of intraperitoneal glucose tolerance test 0.25h to 4.75h after administration;

[0065] Figure 9 : A is the hypoglycemic effect of db / db mice after drug administration in each group; B is the area under the glucose concentration-time curve in A. DETAILED DESCRIPTION

[0066] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0067] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0068] Example 1: Design and synthesis of polypeptides

[0069] 1. Peptide sequence design

[0070] The amino acid sequence of the target peptide (c381) obtained by retrieval is:

[0071] HSQGTFTSDY SKYLDSQAAQ DFVQWLMNGGPSSGAPPPS (SEQ ID NO: 1)-NH2 and based on this, some amino acids were replaced and optimized to obtain the following sequence:

[0072] Table 1 Polypeptide sequences

[0073]

[0074] 2. Synthesis and purification of peptides

[0075] 2.1 Synthesis of peptides

[0076] Peptides are produced using solid-phase peptide synthesis (SPPS). SPPS involves sequentially coupling amino acids to a resin to form a peptide chain. After sequence synthesis, the N-terminal Fmoc protecting group is first deprotected (or after N-terminal modification), followed by deprotection of the side chain protecting groups, and the peptide is cleaved from the resin. The preparation process is as follows:

[0077] 1) Coupling the first amino acid: Take an appropriate amount of modified resin, add the prepared amino acid solution and coupling reagent to the resin, and react for a period of time;

[0078] 2) Removal of Fmoc: After adding Pip / DMF solution for a period of time, vacuum filtration was performed to remove the solvent;

[0079] 3) Washing: Add DMF to the resin (washing step) and remove the solvent by vacuum filtration;

[0080] 4) Resin test: Place ninhydrin, N,N-dimethylformamide, and a small amount of resin in a test tube. Place the test tube in a metal bath for a few seconds and check to see if the resin changes color. If the resin changes color, the Fmoc group has been successfully removed.

[0081] 5) Amino acid condensation: Add the prepared amino acid solution to the resin. Then add the coupling reagent, shake well for a while, and vacuum filter to remove the solvent.

[0082] 6) Repeat steps 2-5 until the last amino acid is synthesized.

[0083] 2.2 Peptide purification

[0084] The purification process is as follows:

[0085] 1) Dissolve the crude polypeptide: Based on the nature of the sequence, select an appropriate reagent to completely dissolve the crude polypeptide.

[0086] 2) Set the purification gradient: Design the purification gradient based on the sequence properties.

[0087] 3) The crude polypeptide is filtered through a membrane and injected into a high performance liquid chromatography machine, and the absorption peaks in the chromatogram are collected separately.

[0088] 4) Test the molecular weight and purity of the fraction to determine the target fraction.

[0089] The purified peptides were analyzed by HPLC and MS, and the results are shown in Table 2.

[0090] Table 2 Parameters after peptide purification

[0091]

[0092]

[0093] Example 2 Plasma stability test of polypeptide

[0094] 1. Rat plasma stability

[0095] Add 14 μl of target peptide solution to 686 μl of rat plasma and vortex to mix. Place this solution in a 37°C water bath. Remove 100 μl of the solution at 0 h, 2 h, 4 h, 6 h, and 24 h. Add 200 μl of 0.1% TFA in acetonitrile (pre-chilled at -20°C). Vortex for 1 min. Centrifuge at 6000 rpm for 5 min at 15°C. Remove 140 μl of the supernatant and add 10 μl of the internal standard solution. Vortex to mix thoroughly before performing Q-TOF analysis. Prepare two replicates. This is equivalent to spiking 100 μl of plasma with 2 μl of target peptide.

[0096] The chromatographic parameters are as follows:

[0097] Table 3 Chromatographic parameters

[0098]

[0099] The mass spectrometry parameters are as follows:

[0100] Table 4 Mass spectrometry parameters

[0101]

[0102]

[0103] The final test results are as follows Figure 1 As shown, the stability of rat plasma from strong to weak is 012>015≈c381≈014≈013.

[0104] 2. Human plasma stability

[0105] Take 180 μl of the target peptide solution, add 1620 μl of human plasma, and vortex to mix. Place this solution in a 37°C waterbath. Remove 100 μl of the solution at 0, 1, 2, 4, 6, 8, and 22 hours, add 200 μl of 0.1% TFA in methanol (pre-chilled at -20°C), vortex for 1 minute, and centrifuge at 15,000 rpm for 10 minutes at 10°C. Take 145 μl of the supernatant, add 5 μl of the internal standard solution, vortex to mix, and perform Q-TOF analysis. Prepare duplicate samples.

[0106] The chromatographic parameters are as follows:

[0107] Table 5 Chromatographic parameters

[0108]

[0109] The mass spectrometry parameters are as follows:

[0110] Table 6 Mass spectrometry parameters

[0111]

[0112]

[0113] The final test results are as follows Figure 2 As shown, the order of human plasma stability is as follows: 015>014>012≈c381. The polypeptide of the present application is a GLP-1 / GCG analogue, a new type of peptide drug that combines the biological functions of GLP-1 and GCG, and is intended to simultaneously regulate blood sugar levels and affect energy balance, thereby combating diabetes and obesity. The design of this type of peptide drug needs to consider its stability in the human body, especially plasma stability, because they are subject to degradation by multiple enzymes in the body. In the drug development process, plasma stability is one of the key factors determining the bioavailability and efficacy of peptide drugs. Drugs with good plasma stability have a longer half-life in the body and can more effectively exert their therapeutic effects. The human plasma stability of GLP-1 / GCG analogues is crucial to the drugability of peptide drugs, because higher stability means that the degradation rate of the drug in the body is slower, and it can maintain activity for a longer time, thereby enhancing efficacy and reducing the frequency of administration, which helps to improve the therapeutic effect of the drug, reduce the cost and side effects of long-term treatment, and reduce the medication burden on patients.

[0114] Judging from the results, the human plasma stability (area under the curve AUC) of 014 and 015 was significantly better than that of c381 (increased by 15% to 36%), which indicates that 014 and 015 have a longer half-life and better stability in the human body. This improvement in stability is achieved through structural optimization in drug design.

[0115] Example 3: Human DPP IV enzyme stability test

[0116] Dissolve each peptide sample in Milli-Q water and dilute to a concentration of 1 mg / ml. Add 40 μL of the target peptide solution to 39.2 μL of 40 mM ammonium bicarbonate (pH 7.9) and 0.8 μL of DPP IV enzyme (Abcam, PN AB79138) and vortex to mix thoroughly. Place the solution in a 37°C metal bath. Remove 10 ml of the solution at 0, 2, 4, 6, and 24 hours. Terminate the reaction with 90 μL of 1% TFA solution before performing Q-TOF analysis.

[0117] like Figure 3As shown, the stability of DPP IV is as follows: 014>015>012≈c381≈011.

[0118] Among them, the DPP IV enzyme stability of 014 was much better than that of 015, 012 and c381 (AUC was higher by 19%, 25% and 27% respectively).

[0119] The DPPIV enzyme stability of GLP-1 / GCG analogs is crucial for the drug development of peptides. DPPIV (Dipeptidyl Peptidase IV) is a ubiquitous enzyme in the body that rapidly degrades peptide hormones like GLP-1, affecting their effective concentration and duration of action. Improving the DPPIV enzyme stability of analogs can significantly prolong the drug's in vivo half-life, enhancing its pharmacological activity and therapeutic efficacy. For GLP-1 / GCG analogs, enhanced DPPIV enzyme stability means these molecules can function longer in the body, more effectively regulating blood glucose levels and promoting insulin secretion. Furthermore, improved stability can reduce drug dosing frequency, improve patient compliance, and enhance therapeutic efficacy. Specific amino acid sequence modifications and structural optimization can significantly enhance the DPPIV enzyme stability of peptides. For example, the optimized design of 014 significantly reduces its degradation rate in the body, resulting in a stronger glucose-lowering effect in the treatment of conditions such as type 2 diabetes and obesity.

[0120] Example 4: Cell activity test

[0121] 1. Methods: Functional activity was determined by cAMP formation in HEK-293 clonal cell lines expressing hGIPR, hGLP-1R, or hGCGR.

[0122] 1. Preparation of test culture medium and diluent

[0123] Prepare plating medium, which is shelf-stable for 6 months at 2-8°C. Mix 90% DMEM and 5% FBS (e.g., 450ml DMEM and 50ml FBS).

[0124] 2. Preparation of subculture medium

[0125] DMEM,10% FBS,300μg / ml G418,50μg / ml HygromycinB

[0126] 3. Preparation of detection solution: Using the Bright-Lite Luciferase Assay System, add 100 ml of staining solution to the substrate and mix thoroughly to obtain the detection solution.

[0127] 4. Preparation of sample gradient solution

[0128] Weigh a certain amount of sample and reference standard and dissolve them in sterile water to prepare a 1 mM stock solution.

[0129] The stock solution was diluted to 120 nM (working concentration 60 nM) with stimulation buffer to obtain solution SD1, and 4-fold serial dilutions were performed starting from SD1 for a total of 10 concentration gradients.

[0130] 5. Cell seeding

[0131] The cells were passaged and expanded in advance. When the cell density reached 80%, the cells were washed 1-2 times with PBS and digested with 0.25% trypsin. The digestion was terminated when the cells became round under microscope. The cells were stained with trypan blue to determine if the cell viability was greater than 95%. The cells were prepared into a density of 0.3×10 6 cells / ml, and 20 μl of the cell suspension was inoculated into each well of a 384-well plate (6000 cells / well).

[0132] 6. Drug treatment

[0133] The next day, 20 μl of serially diluted samples were added to each well and the cells were transferred to a 37°C cell culture incubator and incubated for 3 h.

[0134] 7. Detection fluid treatment

[0135] After incubation, remove the cell plate and allow it to equilibrate to room temperature for 15 minutes. Then, add 40 μl of the test solution, which has been equilibrated to room temperature, to each well. Place the culture plate on a microplate shaker at 500 rpm for 3 minutes, and finally, collect data using a microplate reader.

[0136] 8. Detection and data processing

[0137] Use a SpectraMax Paradigm microplate reader with excitation at 340 nm, collect signal values ​​under emission light conditions of 616 nm and 665 nm, calculate the ratio of acceptor and donor emission signals for each individual well, plot the relationship between HTRF ratio and compound concentration, determine the concentration of cAMP by standard curve analysis, plot the relationship between compound concentration and cAMP concentration, and give Emax and EC50 values.

[0138] 2. Results

[0139] Table 7 Functional hGIPR, hGLP-1R and hGCGR assays

[0140]

[0141] The experimental results are shown in Table 7. The specific analysis is as follows:

[0142] (1) GLP-1R activity: c381, 011 to 015 all have GLP-1R activity, and 012 and 015 have better GLP-1R activity.

[0143] (2) GCGR activity: c381, 011-015 all have GCGR activity, and 014 has better GLP-1R activity.

[0144] (3) GIPR activity: c381 and 011-015 had no GIPR activation activity.

[0145] Example 5: Glucose tolerance test in mice

[0146] 1. Glucose tolerance 0.25 to 2.25 hours after taking the drug

[0147] Mice were acclimated for 3 days, then randomly divided into groups based on body weight and placed in metabolic cages for 12 hours of fasting. Blood glucose levels were measured by tail bleeding 1 hour before glucose administration. Forty-two animals were randomly divided into groups based on body weight and blood glucose levels, with six animals per group.

[0148] Mice were placed in metabolic cages and fasted for 12 h. Blood was collected from the tail 1 h before glucose administration (recorded as 0-minute blood glucose), the corresponding drugs were injected subcutaneously, and glucose (2 g / kg) was injected intraperitoneally 15 minutes later. Blood glucose levels were measured 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 120 minutes after glucose. The animals remained fasting during the experiment to prevent interference with food intake.

[0149] The experimental design is as follows:

[0150] Table 8 Design of glucose tolerance experiment 0.25 to 2.25 hours after drug administration

[0151]

[0152]

[0153] The results of the intraperitoneal glucose tolerance test and the area under the blood glucose concentration-time curve are as follows Figure 4 As shown, the blood glucose level 120 minutes after glucose injection is Figure 5 shown.

[0154] The blood glucose of wild-type mice in the vehicle group rose rapidly 15 minutes after intraperitoneal injection of glucose and then dropped to near the baseline level at 120 minutes.

[0155] Compared with the vehicle group, JK-0246R-011 and JK-0246R-014 significantly improved glucose tolerance within 2.25 hours after a single subcutaneous injection of 30 nmol / kg into wild-type mice (p<0.05). c381, JK-0246R-012, JK-0246R-013, and JK-0246R-015 also significantly improved glucose tolerance within 2.25 hours after a single subcutaneous injection of 30 nmol / kg into wild-type mice.

[0156] Compared with the c381 group, there was no difference in the glucose tolerance improving activity of JK-0246R-011, JK-0246R-012, JK-0246R-013, JK-0246R-014 and JK-0246R-015 (p>0.05).

[0157] There was no difference in the glucose tolerance improving activity among JK-0246R-011, JK-0246R-012, JK-0246R-013, JK-0246R-014 and JK-0246R-015 (p>0.05).

[0158] Compared with the vehicle group, JK-0246R-011, JK-0246R-012, JK-0246R-013, JK-0246R-014 and JK-0246R-015 had significant activity in improving glucose tolerance 2.25 hours after administration.

[0159] Conclusion: c381, JK-0246R-011, JK-0246R-012, JK-0246R-013, JK-0246R-014 and JK-0246R-015 all have the effect of improving glucose tolerance in mice 2.25 hours after administration.

[0160] 2. Glucose tolerance 2.5 to 4.5 hours after taking the drug

[0161] Mice were weighed and randomly divided into groups based on body weight. They were placed in metabolic cages and fasted for 12 hours. Blood glucose levels were measured by tail bleeding 1 hour before glucose administration. Forty-two animals were randomly divided into groups based on body weight and blood glucose levels, with six animals per group.

[0162] Mice were placed in metabolic cages and fasted for 12 h. Blood was collected from the tail before drug administration (recorded as 0-minute blood glucose), and the corresponding drugs were injected subcutaneously. Glucose (2 g / kg) was injected intraperitoneally 150 minutes later, and blood glucose levels were measured 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 120 minutes after glucose administration. The animals remained fasting during the experiment to prevent interference with food intake.

[0163] The experimental design is as follows:

[0164] Table 9 Design of glucose tolerance experiment 2.5 to 4.5 hours after drug administration

[0165]

[0166] The results of the intraperitoneal glucose tolerance test and the area under the blood glucose concentration-time curve are as follows Figure 6 shown.

[0167] The blood glucose of wild-type mice in the vehicle group rose rapidly 15 minutes after intraperitoneal injection of glucose and then dropped to near the baseline level at 120 minutes.

[0168] Compared with the vehicle group, c381, JK-0246R-011 to JK-0246R-015 30 nmol / kg single subcutaneous injection into wild-type mice had significant activity in improving glucose tolerance within 3 to 4.75 hours (p<0.05).

[0169] Compared with the c381 group, JK-0246R-012 had a significant activity in improving glucose tolerance (p<0.05), and the activities of JK-0246R-013 to JK-0246R-015 were equivalent to those of c381 (p>0.05).

[0170] Compared with the JK-0246R-012 group, the activity of JK-0246R-013 in improving glucose tolerance within 3 to 4.75 hours after a single subcutaneous injection into wild-type mice was significantly weaker than that of JK-0246R-012 (p<0.05), and the activity of JK-0246R-014~015 was equivalent to that of JK-0246R-012 (p>0.05).

[0171] Compared with the JK-0246R-013 group, JK-0246R-015 had a significantly stronger activity in improving glucose tolerance within 3 to 4.75 hours after a single subcutaneous injection into wild-type mice (p<0.05), and the activity of JK-0246R-014 was equivalent to that of JK-0246R-013 (p>0.05).

[0172] Compared with the JK-0246R-014 group, JK-0246R-015 had the same activity of improving glucose tolerance as JK-0246R-014 within 3 to 4.75 hours after a single subcutaneous injection into wild-type mice (p>0.05).

[0173] 3. Analysis of improved glucose tolerance 3.25h-3.75h after taking the drug:

[0174] The results of glucose tolerance improvement at 3.75h, 3.5h and 3.25h after taking the drug are as follows Figure 7 shown.

[0175] (1) Glucose tolerance 3.75 hours after medication:

[0176] Compared with the vehicle group, c381, JK-0246R-012, JK-0246R-013, JK-0246R-014, and JK-0246R-015 were active 3.75 hours after a single subcutaneous injection into wild-type mice (p<0.05). This suggests that the activity of c381, JK-0246R-012, JK-0246R-013, JK-0246R-014, and JK-0246R-015 can persist up to 3.75 hours after administration.

[0177] Compared with c381, there was no difference in the activity of JK-0246R-012~015 (p>0.05).

[0178] There was no difference in the activity of JK-0246R-012~015 among wild-type mice 3.75 hours after a single subcutaneous injection (p>0.05).

[0179] (2) Glucose tolerance 3.5 hours after medication:

[0180] Compared with the vehicle group, c381, JK-0246R-012, JK-0246R-013, JK-0246R-014, and JK-0246R-015 were active 3.5 h after a single subcutaneous injection into wild-type mice (p<0.05).

[0181] (3) Glucose tolerance 3.25 h after drug administration: Compared with the vehicle group, c381, JK-0246R-012, JK-0246R-013, JK-0246R-014, and JK-0246R-015 all showed activity in improving glucose tolerance (p<0.05).

[0182] The results of improving glucose tolerance in mice from 0.25h to 4.75h after administration are shown in Figure 8 .

[0183] Example 6: Application of polypeptides in lowering blood sugar

[0184] 1. Lowering blood sugar in db / db mice

[0185] Mice were acclimated for 4 days and then randomly divided into three groups of 8 mice each based on body weight and pre-drug blood glucose. Blood glucose levels were measured at the corresponding time points according to the grouping table. The animals had free access to food and drink during the experiment. The grouping design was as follows:

[0186] Table 10 Experimental groups

[0187]

[0188] The final test results are as follows Figure 9 As shown, the non-fasting blood glucose levels of db / db mice in the vehicle group had no statistical difference at all time points within 6 hours after drug administration compared with those before drug administration, and were relatively stable.

[0189] Compared with the vehicle group at the same time points after administration, the non-fasting blood glucose and corresponding AUC of db / db mice in the JK-0246R-014 group were reduced 1, 3, and 6 hours after a single subcutaneous injection of 30 nmol / kg (p<0.05).

[0190] Conclusion: The hypoglycemic activity of JK-0246R-014 at 30 nmol / kg can persist for 3 hours after a single subcutaneous injection into db / db mice.

[0191] Based on the above test results on plasma stability, DPPIV enzyme stability, GLP-1R / GCGR activity, glucose tolerance improvement activity, and blood sugar lowering activity, all modified peptides have good effects, among which JK-0246R-014 has the best effect.

[0192] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A polypeptide having GLP-1R / GCGR dual receptor agonist activity, characterized in that: The polypeptide is a variant of SEQ ID NO: 1, and the variant comprises an amino acid sequence having at least one amino acid mutation at positions 19-30 of SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The variant includes a mutation in one or a combination of two or more of A19, D21, W25, N28 or G30 of SEQ ID NO: 1; preferably includes a mutation in one or a combination of two or more of A19G, D21E, W25F, N28E or G30A.

3. The polypeptide according to any one of claims 1-2, characterized in that The polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-6.

4. The polypeptide according to any one of claims 1 to 3, characterized in that The polypeptide may further comprise at least one modification, including amidation, acetylation, glycosylation, phosphorylation or the inclusion of other functional components.

5. The polypeptide according to claim 4, characterized in that The other functional components include water-soluble polymers, serum albumin, transferrin, Fc fragments, unstructured polypeptides or fragments of any of the foregoing.

6. A nucleic acid encoding the polypeptide according to any one of claims 1 to 5.

7. A product comprising the polypeptide according to any one of claims 1 to 5 and / or the nucleic acid according to claim 6, characterized in that: The products include vectors, cells, kits, delivery systems or pharmaceutical compositions.

8. The product according to claim 7, characterized in that The vector comprises the nucleic acid; The delivery system comprises the polypeptide or the carrier. Preferably, the delivery system comprises further modification of the polypeptide. Further preferably, the modification comprises fusion of a penetrating peptide, a responsive peptide or a self-assembling oligomeric protein. The pharmaceutical composition comprises the polypeptide or the delivery system, and pharmaceutically acceptable excipients.

9. Use of the polypeptide according to any one of claims 1 to 5, the nucleic acid according to claim 6, or the product according to any one of claims 7 to 8 in the preparation of a medicament for treating metabolic-related diseases or complications, preferably, the metabolic-related diseases include obesity, hyperlipidemia, diabetes, or fatty liver; More preferably, the complications include diabetic eye disease, diabetic neuropathy, diabetic foot, cardiovascular and cerebrovascular diseases, kidney disease, liver disease or osteoporosis.

10. The method for preparing the polypeptide according to any one of claims 1 to 5, characterized in that: The preparation method includes chemical synthesis and / or biological synthesis.

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