GLP-1R / GIPR / GCGR triple receptor agonist and application thereof

Abstract

The invention provides a GLP-1R / GIPR / GCGR triple receptor agonist and an application of the GLP-1R / GIPR / GCGR triple receptor Specifically, the invention provides a polypeptide with GLP-1 (glucagon-like peptide-1) R / GIPR / GCGR triple receptor agonist activity or a pharmaceutically acceptable salt thereof, and the polypeptide or the pharmaceutically acceptable salt thereof has obvious triple agonist activity of a glucagon-like peptide-1 (GLP-1) receptor, a gastric inhibitory polypeptide (GIP) receptor and a glucagon (GCG) receptor. The compound can be used for preparing pharmaceutical compositions for preventing or treating metabolic diseases such as type I diabetes mellitus, type II diabetes mellitus, gestational diabetes mellitus, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, hyperlipidemia and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to GLP-1R / GIPR / GCGR triple receptor agonists and their applications. Background Technology

[0002] Obesity is a pressing health problem that needs to be addressed in the 21st century. Severe obesity can lead to insulin resistance, which in turn can cause diabetes. Diabetes is a metabolic disease characterized by persistently high blood sugar, which can cause serious damage to multiple organ systems and lead to various complications over a long period. As the prevalence of obesity continues to expand, the risk of various chronic and debilitating diseases is also increasing, including but not limited to diabetes, hypertension, and dyslipidemia. Therefore, developing innovative drugs and treatment strategies for obesity, diabetes, and their complications is of great significance for improving human health.

[0003] Currently, drugs with certain therapeutic effects on obesity and diabetes have been developed and applied in clinical treatment. These include single-molecule single-receptor agonists targeting GLP-1R, such as liraglutide and semaglutide; and single-molecule dual-receptor agonists targeting GLP-1R / GIPR (such as telpolide) and GLP-1R / GCGR (such as cotadutide). However, both types of drugs generally exhibit problems such as insufficient agonist efficacy and poor blood sugar control and weight loss effects.

[0004] Currently, research on next-generation metabolic drugs for diabetes and obesity mainly focuses on single-molecule pleiotropic receptor agonists, such as GLP-1R / GIPR / GCGR triple receptor agonists. Retaglutide is the most advanced GLP-1R / GIPR / GCGR triple receptor agonist, currently in phase III clinical trials. Its half-life has been extended to 6 days, demonstrating good blood sugar-lowering and weight-loss effects. Therefore, GLP-1R / GIPR / GCGR triple receptor agonists with different amino acid sequences have been developed using existing technologies, but many shortcomings remain.

[0005] Therefore, this invention urgently needs to develop a GLP-1R / GIPR / GCG triple receptor agonist with excellent GLP-1R / GIPR / GCG triple receptor agonist efficacy and excellent blood sugar control and weight loss effects. Summary of the Invention

[0006] This invention provides GLP-1R / GIPR / GCGR triple receptor agonists or pharmaceutically acceptable salts thereof and their applications.

[0007] In a first aspect of the invention, a polypeptide having GLP-1R / GIPR / GCGR triple receptor agonistic activity or a pharmaceutically acceptable salt thereof is provided, said polypeptide having the amino acid sequence shown in formula (I): Xaa1Xaa2MGTFTSDKSKYLDERAAYDFVQWLLDGPSTGAPPPT (I) in, Xaa1 is H or M. Xaa2 can be V, Aib, or S.

[0008] In another preferred embodiment, the polypeptide is a modified polypeptide or an unmodified polypeptide.

[0009] In another preferred embodiment, the modified polypeptide has a side chain covalently linked to the amino acid sequence of the group consisting of: long-chain fatty acids, polyethylene glycol chains, hydrophilic polymers, hydrophilic spacer arms, glycosylated side chains, or combinations thereof; preferably long-chain fatty acids.

[0010] In another preferred embodiment, the long-chain fatty acid is selected from the group consisting of hexadecanoyl (C16) chains, octadecanoyl (C18) chains, or combinations thereof, preferably hexadecanoyl (C16) chains.

[0011] In another preferred embodiment, the covalent connection is a γ-carboxylic acid linkage.

[0012] In another preferred embodiment, the polypeptide or a pharmaceutically acceptable salt thereof simultaneously has the activity of binding to and activating glucagon-like peptide-1 (GLP-1) receptor, gastric inhibitory peptide (GIP) receptor, and glucagon (GCG) receptor.

[0013] In another preferred embodiment, the polypeptide is composed of an amino acid sequence as shown in formula (I).

[0014] In another preferred embodiment, the structure of the polypeptide is shown in formula (I).

[0015] In another preferred embodiment, Xaa1 is H and Xaa2 is Aib.

[0016] In another preferred embodiment, Xaa1 is M, and Xaa2 is Aib or V.

[0017] In another preferred embodiment, the amino acid sequence of the polypeptide is further covalently linked at the 10th position of the N-terminus to a long-chain fatty acid selected from the group consisting of γ-glutamyl (γGlu), 8-amino-3,6-dioxanoic acid (AEEA), or oligoethylene glycol (OEG), or combinations thereof; preferably 8-amino-3,6-dioxanoic acid (AEEA) or γ-glutamyl (γGlu).

[0018] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:6.

[0019] In another preferred embodiment, the polypeptide has anti-DPP-IV enzyme cleavage activity.

[0020] In another preferred embodiment, the pharmaceutically acceptable salt is an alkali metal salt (such as sodium or potassium salt), or an alkaline earth metal salt (such as calcium or magnesium salt), or a combination thereof.

[0021] In another preferred embodiment, the amino acid sequence of the polypeptide, relative to the amino acid sequence of the polypeptide of SEQ ID NO:2, has an amino acid mutation at a core site selected from the group consisting of: The 1st, 2nd, 3rd, 20th, 33rd, 39th, or combinations thereof; The peptide exhibits significantly enhanced GLP-1R / GIPR / GCGR triple receptor agonist activity.

[0022] In another preferred embodiment, the polypeptide has a core amino acid mutation selected from the group consisting of: (a)H1M (b) Aib2S or Aib2V; (c)Q3M; (d)Q20Y; (e)S33T; (f)S39T; (g) Any combination of (a) to (f) above.

[0023] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutation relative to SEQ ID NO:2: Q3M, Q20Y, S33T and S39T.

[0024] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutation relative to SEQ ID NO:2: H1M, Aib2S, Q3M, Q20Y, S33T, and S39T.

[0025] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutation relative to SEQ ID NO:2: Aib2V, Q3M, Q20Y, S33T, and S39T.

[0026] In another preferred embodiment, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO:6, 8, or 9.

[0027] In another preferred embodiment, the polypeptide comprises an amino acid sequence as shown in any of SEQ ID NO:6, 8, 9, and the 10th position of the polypeptide is a modified K.

[0028] In another preferred embodiment, the polypeptide comprises an amino acid sequence as shown in any of SEQ ID NO:6, 8, 9, and the 10th position of the polypeptide is a K modified with a long-chain fatty acid.

[0029] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (1) The polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention; and (2) Pharmaceutically acceptable carrier.

[0030] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: injections and lyophilized preparations.

[0031] In a third aspect of the invention, the use of the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is provided for the preparation of pharmaceutical compositions.

[0032] In another preferred embodiment, the pharmaceutical composition is used for: (a) Lowering fasting blood glucose levels; (b) Improve the effectiveness of acute glycemic control; (c) Improve blood lipid and body fat levels; (d) Controlling weight; and / or (e) Prevention or treatment of diseases associated with abnormal glucose and lipid metabolism.

[0033] In another preferred embodiment, the use of the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention in the preparation of a pharmaceutical composition for the prevention or treatment of metabolic diseases selected from the group consisting of: type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

[0034] In a fourth aspect of the invention, a method for preventing or treating a disease is provided, the method comprising the step of: applying a therapeutically effective amount of a polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention to a subject in need.

[0035] In another preferred embodiment, the disease is a disease related to abnormal glucose and lipid metabolism.

[0036] In another preferred embodiment, the diseases associated with abnormal glucose and lipid metabolism are selected from the group consisting of: type 1 diabetes, type 2 diabetes, gestational diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), obesity, and hyperlipidemia.

[0037] In another preferred embodiment, the route of administration includes injection (e.g., subcutaneous or intramuscular) or transdermal administration.

[0038] In another preferred embodiment, the therapeutically effective amount of the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is 0.5-5 nmol / kg, preferably 1-2.5 nmol / kg, more preferably 1-1.5 nmol / kg, such as 1.22 μg / ml, based on human body weight.

[0039] In another preferred embodiment, the concentration of the therapeutically effective amount of the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is 0.1-100 mg / week, more preferably 0.25-50 mg / week, and even more preferably 0.5-30 mg / week.

[0040] In another preferred embodiment, the object is a human or a non-human mammal.

[0041] In another preferred embodiment, the subject is a subject diagnosed with a disease associated with abnormal glucose and lipid metabolism, or a subject with a susceptibility (or risk) to a disease associated with abnormal glucose and lipid metabolism.

[0042] In another preferred embodiment, the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is administered once daily, twice daily, or three times daily.

[0043] In a fifth aspect of the present invention, a method for modifying a polypeptide having GLP-1R / GIPR / GCGR receptor agonist activity is provided, the method comprising the steps of: performing an amino acid mutation at a core site relative to a control peptide having GLP-1R / GIPR / GCGR receptor agonist activity, thereby obtaining a polypeptide having GLP-1R / GIPR / GCGR receptor agonist activity. The core site has a core amino acid mutation selected from the following group: (c)Q3M, Q20Y, S33T and S39T.

[0044] In another preferred embodiment, the core site also has a core amino acid mutation selected from the group consisting of (a) H1M; or (b) Aib2S or Aib2V.

[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0046] Figure 1The EC50 results of the candidate drug’s agonistic activity against three receptors are shown: (A) EC50 results of the candidate drug’s agonistic activity against GLP-1R; (B) EC50 results of the candidate drug’s agonistic activity against GIR; (C) EC50 results of the candidate drug’s agonistic activity against GCGR.

[0047] Figure 2 The oral glucose tolerance curves of DIO mice after drug injection on day 0 are shown.

[0048] Figure 3 The area under the glucose tolerance curve (AUC) of DIO mice after oral glucose tolerance test on day 0 is shown.

[0049] Figure 4 The results of three weeks of weight monitoring in DIO mice are shown.

[0050] Figure 5 The results show the changes in body weight of DIO mice after three weeks of treatment.

[0051] Figure 6 The oral glucose tolerance test (OGTT) curves of DIO mice after three weeks of treatment are shown.

[0052] Figure 7 The fasting blood glucose levels of DIO mice after three weeks of treatment are shown.

[0053] Figure 8 The area under the glucose tolerance curve (AUC) of DIO mice after three weeks of treatment is shown.

[0054] Figure 9 The blood biochemical parameters of DIO mice after three weeks of treatment are shown.

[0055] Figure 10 The fat index and liver index of DIO mice after three weeks of treatment are shown.

[0056] Figure 11 The oral glucose tolerance curves of dbdb mice after drug injection on day 0 are shown.

[0057] Figure 12 The area under the glucose tolerance curve for dbdb mice after drug injection on day 0 is shown.

[0058] Figure 13 The results of three weeks of weight monitoring in dbdb mice are shown.

[0059] Figure 14 The data show the weight changes of dbdb mice after three weeks of treatment.

[0060] Figure 15 The survival curves of dbdb mice are shown.

[0061] Figure 16 The oral glucose tolerance test (A) and fasting blood glucose levels (B) of dbdb mice after three weeks of treatment are shown.

[0062] Figure 17 Area under the oral glucose tolerance test curve after three weeks of treatment Figure 18 The blood biochemical parameters of dbdb mice after three weeks of treatment are shown.

[0063] Figure 19 The fat and liver indices of dbdb mice after three weeks of treatment are shown.

[0064] Figure 20 The diagram shows the structural schematics of the initiating peptide and candidate polypeptides of the present invention.

[0065] In each figure, Retatrutide represents Retatrutide. Detailed Implementation

[0066] Through extensive and in-depth research, and after numerous screenings and experiments, the inventors have unexpectedly developed for the first time a polypeptide or a pharmaceutically acceptable salt thereof with triple receptor agonistic activity of GLP-1R / GIPR / GCGR. This polypeptide can maintain affinity and agonistic efficacy for the three GLP-1R / GIPR / GCGR receptors. Surprisingly, compared with compounds with existing structures, the polypeptide of this invention can have a significantly improved hypoglycemic and weight-loss effect.

[0067] Specifically, experiments show that the peptides obtained in this invention can significantly improve the fasting blood glucose reduction effect and short-term or long-term blood glucose control efficacy in db / db mice (T2DM model) and DIO mice (obesity model), significantly improve weight loss efficacy, significantly reduce blood lipid levels in DIO mice, and significantly reduce blood lipids or body fat. The reduction is significantly greater than that of the commercially available GLP-1R / GIPR / GCGR triple receptor agonist retaglutide, and has obvious hypoglycemic and weight loss effects. It can be used to treat metabolic diseases such as type 2 diabetes, obesity, and hyperlipidemia.

[0068] Based on this, the present invention was completed.

[0069] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0070] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0071] As used in this article, "Ritaglutide" and "Ritaglutide" are interchangeable. They refer to a compound released by Eli Lilly in June 2023 that has GLP-1R / GIPR / CGCR triple receptor agonist activity. It is currently the most advanced and has entered Phase III clinical trials. The drug achieves GLP-1R / GIPR / CGCR triple receptor agonist activity by using a hybrid glucagon-related structure based on the GLP / GIP dual receptor agonist Tirzepatide. It is used to treat obesity and type II diabetes, and also helps control blood sugar and manage weight loss.

[0072] As used in this article, "db / db mice" refers to a type 2 diabetes animal model caused by spontaneous mutations. The gene mutations prevent the normal encoding of the leptin receptor, which in turn leads to typical type 2 diabetes characteristics such as obesity, hyperglycemia, and insulin resistance. Common symptoms include obesity, hyperglycemia and insulin resistance, metabolic abnormalities, reproductive disorders, and other complications.

[0073] As used in this article, "DIO mice" refers to a mouse model of obesity induced by a high-fat diet, used to study obesity, diabetes and related metabolic diseases. Typically, mouse strains sensitive to high-fat diets (such as C57BL / 6J) are selected, and after consuming a high-fat diet, they gradually develop obesity-related symptoms such as weight gain, elevated blood sugar, and insulin resistance.

[0074] In this invention, the term "Peptide 20" refers to a peptide having the amino acid sequence shown in SEQ ID NO:2, and wherein the 10th position of the polypeptide is covalently linked to a γ-glutamyl hexadecanoyl (C16) chain.

[0075] Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 (GLP-1) is a 37-amino acid incretin that stimulates insulin secretion, protects pancreatic β-cells, and inhibits glucagon secretion, gastric emptying, and food intake, leading to weight loss. GLP-1R, primarily expressed in the pancreatic islets, is a G protein-coupled receptor (GPCR) that plays a crucial role in mediating various physiological effects related to glucose homeostasis. Activation of this receptor by endogenous GLP-1 secreted by intestinal L cells leads to the accumulation of intracellular cyclic adenosine monophosphate (cAMP), promoting insulin secretion in a glucose-dependent manner (hyperglycemic environment) to lower blood glucose and inhibiting glucagon (GCG) release. However, it does not promote insulin secretion in a hypoglycemic environment. Furthermore, GLP-1R activation also suppresses appetite and delays gastrointestinal transit.

[0076] Liraglutide and semaglutide are GLP-1R agonists approved for the treatment of type 2 diabetes and obesity. However, single-molecule, single-receptor GLP-1R agonists face challenges such as insufficient agonistic efficacy and side effects. Many patients with type 2 diabetes and obesity still do not have adequately controlled conditions. Currently available single-molecule, single-receptor agonists or single-molecule, dual-receptor agonists for blood glucose and weight control still have limitations such as side effects, desensitization due to single-target activation, and difficulties in production and yield.

[0077] Glucagon (GCG) Glucagon (GCG) is also derived from proglucagon. It binds to and activates the glucagon receptor, initiating the corresponding signaling pathway to regulate gluconeogenesis and glycogenolysis, thereby raising blood glucose levels and maintaining blood glucose levels. Previous studies have shown that glucagon can suppress appetite, reduce food intake, and also has effects such as fat degradation and weight reduction. Pocai et al. (Obesity 2012; 20:1566–1571; Diabetes 2009, 58, 2258) and Day et al. (Nat Chem Biol 2009; 5:749) described a dual agonist of the GLP-1 receptor and glucagon receptor, which combines the effects of GLP-1 and glucagon within a single molecule to produce a therapeutic mechanism with anti-diabetic effects and significant weight loss.

[0078] Glucose-dependent insulinotropic peptide (GIP) Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β-cells in the presence of glucose and protecting pancreatic β-cells. GIP is secreted by intestinal K cells, and GIPR is a member of the GPCR family. GIP activating GIPR promotes insulin secretion from pancreatic β-cells and also has a dual role in regulating GCG secretion: promoting GCG release in hypoglycemic states and inhibiting GCG release in hyperglycemic states. GIPR activation also plays an important role in regulating appetite and weight; central activation of GIPR is associated with inhibiting food intake and promoting weight loss. GIP can also regulate lipid metabolism by targeting adipose tissue. Given the multifaceted roles of GIP and GIPR in metabolic regulation, the design of GLP-1R / GIPR dual-receptor agonists holds promise for broad therapeutic applications.

[0079] The published patent CN201680005007.X describes a GLP-1 / GIP receptor dual agonist compound, LY3298176, based on a natural GIP polypeptide sequence. It has good effects in reducing blood sugar and weight. Currently, this product has been approved by the FDA for the treatment of type 2 diabetes, with the generic name Tirzepatide.

[0080] Both natural GIP and GLP-1 can be rapidly inactivated by the ubiquitous protease DPP-IV, thus limiting their use to short-term metabolic control. Current technologies generally employ peptide linkage to fatty acid chains and the addition of unconventional amino acids to increase the peptide's half-life in vivo. For example, Tirzepatide modifies the lysine residue at position 20 using GLP-1 drug modification techniques, and mutates the residues at positions 2 and 13 to the unconventional amino acid Aib.

[0081] The active ingredient of the present invention As used herein, the terms “active ingredient of the present invention”, “GLP-1R / GIPR / GCGR triple receptor agonist of the present invention”, “polypeptide of the present invention having GLP-1R / GIPR / GCGR triple receptor agonist activity”, and “polypeptide of the present invention” are used interchangeably and all refer to the polypeptide described in the first aspect of the present invention.

[0082] As used herein, “triple agonist activity” refers to an incretin analogue that is active at each of the GLP-1, GIP, and glucagon receptors. This balanced activity at each receptor allows the administered dose to provide sufficient activity at each receptor to deliver the agonist benefit, while avoiding the undesirable side effects associated with excessively high activity. Furthermore, peptides with triple agonist activity at the GLP-1, GIP, and GCG receptors have prolonged durations of action at each of the GIP, GLP-1, and GCG receptors, which advantageously allows for low-frequency dosing such as once daily, three times weekly, twice weekly, or once weekly.

[0083] The structural features of the GLP-1R / GIPR / GCGR triple receptor agonist described herein result in analogs with sufficient activity at each of the GIP, GLP-1, and GCG receptors to achieve the beneficial effect of activity at each receptor (i.e., triple agonist activity), but the activity at any one receptor is not high enough to overwhelm the activity at the other two receptors or to cause undesirable side effects when administered at a dose sufficient to produce activity at all three receptors.

[0084] The structural features of the GLP-1R / GIPR / GCGR triple receptor agonist described in this article include modifying amino acids at specific positions and covalently linking fatty acid side chains at specific positions to promote optimal binding and efficacy at each receptor, improve its stability, and reduce its immunogenicity.

[0085] Typically, the present invention first describes polypeptides or pharmaceutically acceptable salts thereof having GLP-1R / GIPR / GCGR triple receptor agonistic activity, as described in the first aspect of the invention.

[0086] The long-acting GLP-1R / GIPR / GCGR triple receptor agonist described in this invention includes naturally occurring amino acids and non-natural amino acids, such as α-aminoisobutyric acid (Aib).

[0087] As used in this article, "modified K" or "K modified with fatty acid chain" refers to a polypeptide whose N-terminal 10 position K is covalently linked to a γ-glutamyl fatty acid chain.

[0088] In addition to the modifications described herein, the GLP-1R / GIPR / GCGR triple receptor agonists described herein may also include one or more additional amino acid modifications, provided that the analogue can still bind to and activate each of the GIP, GLP-1 and GCG receptors.

[0089] Preparation of peptides The polypeptides of this invention can be recombinant polypeptides or synthetic polypeptides. The polypeptides of this invention can be chemically synthesized or recombinant. Accordingly, the polypeptides of this invention can be artificially synthesized using conventional methods or produced using recombinant methods.

[0090] A preferred method employs the Fmoc / tBu solid-phase peptide synthesis strategy (SPPS), using Fmoc-Ser(tBu)-Wang Resin (degree of substitution 0.3 mmol) as the solid support. The initial resin is swollen with 10 ml of N,N-dimethylformamide (DMF) for 30 min, followed by treatment with 10 ml of 20% piperidine / DMF solution for 30 min to deprotect the Fmoc protecting groups of serine residues. The deprotected resin is washed five times with 10 ml of DMF for 30 seconds each time. To ensure complete deprotection, ninhydrin (Kaiser) assay is performed: a small amount of resin sample is added to ninhydrin reagent and heated at 100 °C for 3 min; a blue color indicates the presence of free amino groups. Amino acid coupling was then performed: 0.9 mmol Fmoc-Pro-OH, 0.9 mmol 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), and 1.5 mmol N,N-diisopropylethylamine (DIEA) were added and dissolved in 10 ml DMF. The reaction was carried out under nitrogen protection for 1 hour. After coupling, the resin was washed three times with 10 ml DMF for 30 seconds each time. Ninhydrin detection showed a colorless result, indicating complete coupling. The Fmoc group of the N-terminal histidine was removed by treatment with 20% piperidine / DMF. To ensure the presence of the free N-terminal amino group, ninhydrin detection showed a blue result. Subsequently, the N-terminus was protected with a tert-butyloxycarbonyl (Boc): a mixed solution of 1 ml ditert-butyl dicarbonate (Boc2O), 1 ml DMF, and 1 ml DIEA was reacted for 1 hour. The protected ninhydrin test was colorless, indicating that the N-terminal amino group was protected. Next, the lysine side chain in the sequence was specifically modified: the Dde protecting group was selectively removed by reacting with 10 ml of 3% hydrazine hydrate / DMF solution for 30 minutes. The Dde-removed resin was washed five times with 10 ml of DMF for 30 seconds each time; the ninhydrin test showed a blue color, confirming the exposure of the free amino group on the lysine side chain. Subsequently, γ-carboxyl-protected glutamic acid was coupled to the exposed lysine side chain amino group: 0.9 mmol Fmoc-Glu(OtBu)-OH, 0.9 mmol HBTU, and 1.5 mmol DIEA were added and dissolved in 10 ml of DMF, reacting for 1 hour. After coupling, washing and ninhydrin testing showed a colorless result. The Fmoc protecting group of this glutamic acid residue was removed with 20% piperidine / DMF, and subsequent ninhydrin testing showed a blue color, indicating the exposure of the γ-carboxyl-terminal amino group. Finally, under the same coupling conditions (0.9 mmol stearic acid, 0.9 mmol HBTU, and 1.5 mmol DIEA reacted in 10 ml DMF for 1 hour), stearic acid (C16) was linked to the γ-carboxyl group of glutamic acid to form an esterification modification (γGlu-C16). The final ninhydrin test was colorless (negative).

[0091] After full-sequence assembly, the peptide-resin complex was thoroughly washed with DMF and methanol and then vacuum dried. The peptide was cleaved from the resin and all acid-labile side-chain protecting groups (tBu, Trt, Boc, Pbf) were simultaneously removed using a cleavage mixture (15 ml, v / v ratio 95% trifluoroacetic acid (TFA) / 2.5% triisopropylsilane (TIS) / 2.5% H2O) with shaking at room temperature for 3 hours. The reaction mixture was filtered through a sintered glass funnel to remove the resin, and the resulting TFA filtrate containing the peptide was transferred to a centrifuge tube containing approximately 45 ml of pre-chilled anhydrous diethyl ether to precipitate. The precipitate was collected by centrifugation, and the resulting white solid particles were washed three times thoroughly with ice-cold anhydrous diethyl ether, followed by centrifugation and decantation after each wash. The final crude peptide was dried under vacuum and used as the crude material for subsequent analysis.

[0092] treat As used herein, the term “treatment” generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a patient’s disease, including: (a) prevention of the disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the onset of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0093] The inventors unexpectedly discovered that the active ingredient of this invention can effectively prevent or treat diseases related to abnormal glucose and lipid metabolism. Experiments have shown that when the active ingredient of this invention or a formulation or drug containing said active ingredient is administered to subjects with diabetes or obesity, one or more efficacies selected from the group consisting of: (a) Lowering fasting blood glucose levels; (b) Improve the effectiveness of acute glycemic control; (c) Improve blood lipid and body fat levels; (d) Controlling weight; and / or (e) Prevention or treatment of diseases associated with abnormal glucose and lipid metabolism.

[0094] Pharmaceutical Composition The present invention also provides pharmaceutical compositions for the prevention or treatment of diseases related to abnormal glucose and lipid metabolism. Typically, the pharmaceutical compositions of the present invention comprise: (a) an active ingredient: a therapeutically effective amount of a polypeptide having GLP-1R / GIPR / GCGR triple receptor agonist activity or a pharmaceutically acceptable salt thereof, or a combination thereof; and (b) any one or more combinations of a pharmaceutically acceptable carrier, excipient, or diluent.

[0095] The pharmaceutical composition of the present invention can improve / control blood glucose and blood lipid levels, especially reduce fasting blood glucose levels, improve blood lipid and body fat levels, control weight, and has acute blood glucose control efficacy.

[0096] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0097] As used herein, the term "therapeuticly effective amount" refers to any amount of a drug as described below, which, when used alone or in combination with another therapeutic agent, promotes disease remission. Disease remission may manifest as a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability caused by the disease. The "therapeuticly effective amount" of the drug of this invention also includes a "preventive effective amount," which is any amount of a drug as described below, which, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or experiencing a recurrence of the disease, is sufficient to treat the disease and / or delay the onset or progression of the disease and / or alleviate one or more symptoms of the disease. As used herein, a "pharmaceutically acceptable carrier" is one or more compatible solid or liquid fillers or gel substances suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and must have sufficient purity and sufficiently low toxicity, i.e., a reasonable benefit / risk ratio. "Compatibility" here refers to the ability of the components in the composition to interact with and be mixed with the compounds of the present invention without significantly reducing the efficacy of the compounds. In this invention, a "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents; generally, satisfactory effects are obtained when the active ingredient of the present invention is administered daily at a dose of about 30 nmol / kg animal body weight (for rodents such as mice). The choice of carrier should be matched with the manner of administration of the pharmaceutical composition, as is well known to those skilled in the art.

[0098] Generally, pharmaceutical formulations should be matched with the method of administration. The dosage forms of the pharmaceutical compositions of the present invention are lyophilized formulations and injections.

[0099] There are no particular limitations on the administration method of the pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): intravenous injection, subcutaneous or intradermal administration, etc. The subjects to be prevented or treated can be animals; especially humans.

[0100] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally by adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in the conventional manner depending on the dosage form.

[0101] When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the polypeptide of the present invention or its pharmaceutically acceptable salt as the active ingredient can be reasonably determined based on the weight, age, sex, and symptom severity of each patient to be treated.

[0102] The main advantages of this invention include: The polypeptide of this invention possesses triple receptor agonist activity of GLP-1R, GCGR, and GIPR. By altering amino acids at specific positions in the polypeptide with GLP-1R / GIPR / GCGR triple receptor activity, not only can the affinity and agonist efficacy for all three receptors be maintained, but it also exhibits a variety of significant effects, namely: (a) Significantly reduced fasting blood glucose levels; (b) Significantly improved acute glycemic control efficacy; (c) Improved blood lipid and body fat levels; (d) Demonstrated significant weight control effects. Furthermore, its reduction is significantly greater than that of Yangshen drugs Retaglutide and Peptide20, which contain GLP-1R receptor agonists, and it has a significant hypoglycemic and weight-loss effect. It can be used to prevent or treat metabolic diseases such as diabetes, obesity, and hyperlipidemia.

[0103] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0104] Materials and Methods: Animal models: male ICR mice (6-8 weeks old), male db / db mice (C57BLKS, leptin receptor defective, 6-8 weeks old), and male artificially induced obese (DIO) mice (C57BL / 6J modeled for 8 weeks, each weighing approximately 35g).

[0105] Cells: HEK293 cells overexpressing CRE / luc2P / GLP-1R, HEK293 cells overexpressing CRE / luc2P / GIPR, and HEK293 cells overexpressing CRE / luc2P / GCGR were purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.

[0106] Test kit: Bright-Lite™ Fluorescent Reporter Gene Assay Kit.

[0107] The reagents, cells, and instruments used in the embodiments of this invention are commercially available or prepared using conventional methods.

[0108] 1. Cell Culture 1.1 Cell resuscitation (1) Transfer the cells to be revived in liquid nitrogen to a 37°C water bath and shake and heat until the cryopreservation solution in the cryovial is completely thawed; (2) Dilute the cell cryopreservation liquid to 10 mL using the corresponding culture medium in a biosafety cabinet, centrifuge at 800 rpm for 5 min, and discard the supernatant; (3) Resuspend the cells in 10 mL of culture medium and repeatedly pipette them evenly. Transfer them to T75 cell culture flasks and place them in a carbon dioxide incubator at 37 °C for static culture. Observe the growth status daily.

[0109] 1.2 Cell passage (1) Cells in a cell culture flask can be passaged once the cell confluence reaches about 80%. (2) Remove the culture medium, add 2 mL of 0.25% trypsin containing EDTA, shake the culture flask to distribute it evenly, and place it in a 37 ℃ carbon dioxide incubator to digest the cells. Observe the cell status every 30 s, and stop digestion when the cells detach in large areas. (3) Add 10 mL of culture medium to stop digestion, repeatedly pipette the cell clusters to make them into single cells, take 1 / 3 of the cell suspension and add it to a new cell culture flask, add an appropriate amount of culture medium and place the new cell culture flask in a carbon dioxide incubator at 37 ℃ for static culture.

[0110] 1.3 Cell cryopreservation (1) Take T225 cells with a confluence of about 80% to be frozen and digest them, then centrifuge at 800 rpm for 5 min; (2) Resuspend the cells in 5 mL of NBCS containing 10% DMSO as the cell cryopreservation solution and pipette them evenly. (3) Each cryovial was filled with 1 mL of cell suspension, placed in a programmed cooling box and placed in a -80 ℃ freezer for gradient cooling overnight. The cryovial was then transferred to liquid nitrogen for storage the next day.

[0111] 2. Luciferase reporter gene assay for chemically synthesized peptides 2.1 Cell Plating 10 saturates were inoculated per well using an automated pipetting station on a 96-well transparent black plate. 3 50 µL of cell suspension per well was incubated at 37 °C in a CO2 incubator for 24 h. 2.2 Co-incubation of peptides with effector cells The peptides were serially diluted using an automated pipetting workstation, starting at 10,000 nM and then serially diluted 10-fold using complete culture medium. 50 µL of peptide solution (v:v=1:1) was added to each well of cell culture medium and incubated at 37 °C in a CO2 incubator for 4 h. 2.3 Bright-Lite™ Detection Reagent Reaction Place the 96 plate at room temperature to equilibrate to room temperature, add 50 µL of Bright-Lite™ assay reagent to each well, and react at room temperature in the dark for at least 2 min. 2.4 Reading Chemiluminescence Signals The chemiluminescence signal was detected at all wavelengths using a multi-functional microplate reader within 30 minutes.

[0112] 2.5 Data Analysis Using Prism (10.2.1), the curve was fitted using a nonlinear (four-parameter dose-response curve model) and the EC50 was calculated.

[0113] 4. Animal experiments 4.1 Oral Glucose Tolerance Test (OGTT) To study the short-term glycemic control effect of drugs on DIO mice, mice weighing over 35g and with similar weights were selected and grouped into groups of 6 (n=6). Mice were fasted for 12 hours the day before the oral glucose tolerance test. For the oral glucose tolerance test, fasting blood glucose was measured using a glucometer via tail clipping 30 minutes before the test (referred to as -30 min). Based on an average mouse weight of 38g, mice were subcutaneously injected with a solution of retaglutide diluted in physiological saline, PBS, P37, P38, P27, and Peptide 20 at a dose of 15 nmol / kg. Blood glucose was measured at 0 min, and the mice were administered glucose solution via gavage (2g / kg). Blood glucose was then measured at 15 min, 30 min, 60 min, and 120 min. The area under the blood glucose curve (AUC) from 30 min before the test to 120 min after the test was calculated to represent the drug's glycemic control ability.

[0114] To study the short-term glycemic control effect of drugs on dbdb mice, 6-week-old dbdb mice of similar weight were selected and divided into groups of 6 mice each, with each group receiving one drug. An oral glucose tolerance test was conducted after drug administration. Mice were fasted for 12 hours the day before the experiment. On the day of the experiment, fasting blood glucose was measured using a glucometer via tail clipping at -30 minutes. Based on an average mouse weight of 40g, mice were subcutaneously injected with a solution of retaglutide diluted in physiological saline, PBS, P38, P27, and Peptide 20 at a dose of 15 nmol / kg. Blood glucose was measured at 0 minutes, and glucose solution (2g / kg) was administered via gavage. Blood glucose was then measured at 15, 30, 60, and 120 minutes. The area under the blood glucose curve from -30 to 120 minutes was calculated to represent the drug's glycemic control ability in mice.

[0115] 4.2 Weight monitoring experiment lasting three weeks To investigate the effect of the drug on weight control in DIO mice over a period of time, a strategy of administering the drug twice a week (with two administrations 2 days and 3 days apart) was adopted during a three-week (21-day) experimental period. Based on an average mouse weight of 38g, DIO mice were subcutaneously injected with a dose of 15 nmol / kg of retaglutide diluted with physiological saline, PBS, P37, P38, P27, and Peptide 20. The injection dose was adjusted weekly based on the latest average mouse weight. Mouse weight was monitored at fixed times daily. Mice were fasted for 12 hours before weight measurements on days 0 and 21 to eliminate the influence of diet on the starting and ending weight data.

[0116] For dbdb mice: To study the effect of the drug on weight control in mice over a period of time, a strategy of administering the drug twice a week (with two administrations 2 days and 3 days apart) was adopted during a three-week (21-day) experimental period. Based on an average mouse weight of 38g, DIO mice were subcutaneously injected with 15 nmol / kg of retaglutide, PBS, P37, P38, P27, and Peptide 20 diluted with physiological saline. The injection dosage was adjusted weekly based on the latest average mouse weight. Mouse weight was monitored at fixed times daily. Mice were fasted for 12 hours before weight measurements on days 0 and 21 to eliminate the influence of diet on the starting and ending weight data. After the 21-day period, the ratio of daily weight to initial weight was calculated, and a weight change curve was plotted.

[0117] 4.3 Oral glucose tolerance test after 3 consecutive weeks of treatment To investigate the effect of drug treatment on glycemic control in DIO mice after a period of treatment, mice treated for three weeks were fasted for 12 hours on day 21 and then subjected to an oral glucose tolerance test. Immediately after blood glucose measurement, mice were administered glucose solution (2 g / kg) via gavage. Blood glucose levels were then measured at 15, 30, 60, and 120 minutes via tail clipping using a glucometer. The area under the blood glucose curve (AUC) from 0 to 120 minutes was calculated to represent the mice's glycemic control ability after drug treatment.

[0118] To study the improvement of glycemic control in dbdb mice after a period of drug treatment, mice treated for three weeks were fasted for 12 hours on day 21 and then subjected to an oral glucose tolerance test. Immediately after 0 min of blood glucose measurement, the mice were administered glucose solution by gavage (2 g / kg). Blood glucose levels were then measured using a glucometer via tail clipping at 15 min, 30 min, 60 min, and 120 min. The area under the blood glucose curve from 0 min to 120 min was calculated to represent the mice's glycemic control ability after drug treatment.

[0119] 4.4 Detection of serum biochemical indicators and liver and fat in mice The treated mice were dissected. Blood was collected and serum was separated. Five indicators in the serum were measured: total cholesterol (T-CHO), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), triglycerides (TG), and free fatty acids (NEFA). (1) Total cholesterol (T-CHO): Detected using cholesterol oxidase and HMMPS method; (2) Triglycerides (TG): Detected using GPO, HMMPS method, and free glycerol removal method; (3) High-density lipoprotein (HDL-C): Detected using direct assay and antibody blocking assay; (4) Low-density lipoprotein (LDL-C): Detected using the direct assay method and the selective protection method; (5) Free fatty acids (NEFA): Detected using ACS and ACOD methods.

[0120] Furthermore, the weight of the visceral fat and liver of the mice was weighed and recorded.

[0121] 4.5 Fasting blood glucose measurement test after treatment This study investigated the effect of drug treatment on the control of fasting blood glucose in mice after a period of time. Mice treated for three weeks were fasted for 12 hours on day 21, and their fasting blood glucose levels were measured. The measured blood glucose values ​​were also used as the initial blood glucose values ​​before gavage in the oral glucose tolerance test. Blood glucose levels in mice were measured using a glucometer after tail clipping.

[0122] 4.6 Data Analysis Using Prism (10.2.1), one-way ANOVA was performed on the data from different experimental groups to calculate the p-value and assess whether there were significant differences between the groups.

[0123] 5. Peptide synthesis This study employed a solid-phase peptide synthesis strategy (SPPS) using Fmoc / tBu, with Fmoc-Ser(tBu)-Wang Resin (0.3 mmol degree of substitution) as the starting material. After swelling the resin in DMF, the serine Fmoc protecting group was removed using 20% ​​piperidine / DMF, and the exposure of the free amino group was confirmed by a blue ninhydrin test. Subsequently, the peptide chain was progressively extended using an HBTU / DIEA activation coupling system (0.9 mmol Fmoc-Pro-OH, 0.9 mmol HBTU, 1.5 mmol DIEA, DMF solvent, reaction under nitrogen protection for 1 hour). Colorless ninhydrin detection after each coupling step indicated complete coupling. After the main chain sequence was assembled, the N-terminal histidine was de-Fmoc-protected and Boc-protected (Boc₂O / DIEA / DMF mixture reaction for 1 hour), and colorless ninhydrin detection confirmed successful protection.

[0124] To target the lysine side chain in the sequence, the Dde protecting group was selectively removed using 3% hydrazine hydrate / DMF. The exposure of the amino group in the ninhydrin blue color confirmed the formation of the side chain amino group. Subsequently, Fmoc-Glu(OtBu)-OH was coupled to form a γ-carboxyl-protected structure. After removing the glutamic acid Fmoc, its γ-carboxyl-terminal amino group was linked to stearic acid (C16) under the same coupling conditions, forming an esterification modification (γGlu-C16). A negative final ninhydrin test indicated the completion of the modification.

[0125] Finally, the peptide-resin complex was washed with DMF / methanol and dried. The peptide was then cleaved simultaneously with a TFA / TIS / H2O (95:2.5:2.5) mixture and shaken at room temperature for 3 hours to remove acid-labile protecting groups (tBu, Trt, Boc, Pbf). The TFA filtrate containing the peptide was precipitated with cold diethyl ether, collected by centrifugation, and washed three times with ice-cold diethyl ether. After vacuum drying, the crude peptide was obtained for subsequent analysis.

[0126] Example 1: In vitro evaluation of the GLP-1R / GIPR / GCGR triple receptor agonist activity of candidate drugs using tool cell lines 1) Experimental methods The receptor agonistic activity of three candidate drugs was measured using HEK293 cells overexpressing GLP-1R, GIPR, and GCGR receptors. Three HEK293 cell lines overexpressed GLP-1R, GIPR, and GCGR receptors, respectively, and carried coupled luciferase reporter genes. Cells were seeded in 96-well plates 24 hours in advance, and the test drugs were added. Fluorescence intensity was detected 4 hours later by adding luciferase. Fluorescence intensity was positively correlated with receptor agonistic activity.

[0127] We selected retaliutide, the most advanced tri-agonist currently under investigation, and compared it with nine candidate drugs to compare their agonistic activities on three different receptor cell types. The agonistic capacity of each drug was determined by calculating EC50.

[0128] 2) Experimental Results The results of the candidate drugs and Yangshen drugs are shown in Table 1 and Figure 1 As shown.

[0129] Table 1. Agonistaltic activity (EC50) of candidate drugs against three receptors The results showed that five candidate drugs, P27, P37, P38, P47, and P48, were superior to retaglutide in activating GLP1R. Among them, P38 and P47 were superior to retaglutide in activating GCGR, and the above candidate drugs were close to the control drug retaglutide in activating GIPR.

[0130] Compared to peptide P38, where the fatty acid chain is chemically coupled at position 10, peptide P40, where the fatty acid chain is chemically coupled at position 20, showed a significant decrease in the agonistic activity of the three receptors. This suggests that chemical coupling of the fatty acid chain at different positions of the peptide (such as coupling at position 20) can lead to a substantial decrease in the agonistic activity of the peptide for the three receptors.

[0131] Furthermore, P38 and P47, and P27 and P48 exhibit similar agonistic activities for the three receptors, likely due to their identical sequence and fatty acid chain coupling positions. This suggests that altering the fatty acid chain configuration does not affect the peptide's agonistic activity.

[0132] Example 2: Evaluation of the potent acute glycemic control efficacy of candidate drugs 1) Experimental methods In this embodiment, following the experimental method of the oral glucose tolerance test after drug administration described above, three mouse models, namely db / db mice (C57BLKS, T2DM model) and DIO mice (C57BL / 6J, obesity model), were subjected to OGTT to evaluate the efficacy of P38 in controlling acute blood glucose in these mice. Mice given PBS served as the blank control group, while mice given retaliutide and Peptide20 served as the corresponding positive control groups.

[0133] The AUC of blood glucose in an oral glucose tolerance test was measured by administering PBS, retaglutide, P38, P27, P37, and Peptide 20 to db / db mice, and to DIO mice, respectively. 0-120min The results are shown in Figures 2-3 , Figures 11-12 And Tables 2-3.

[0134] 2) Experimental Results Results of the candidate drug's potent acute glycemic control efficacy in DIO mice are as follows: Figure 2-3 As shown in Table 2.

[0135] Table 2. Area under the blood glucose curve (AUC) in DIO mice after oral glucose tolerance test 0～120 min ) The results showed that, compared with the PBS-only injection group and the Peptide20 injection group, the area under the blood glucose curve from 0 to 120 min was significantly reduced in DIO mice after injection of retaglutide, P37, P38, and P27.

[0136] However, unexpectedly, compared with the PBS-only injection group and the Peptide20-only treatment group, DIO mice showed a significantly reduced area under the blood glucose curve from 30 min before to 120 min after the formal experiment following injection of P38 or P27 (****, p<0.0001). Furthermore, candidate drug P38 demonstrated the best short-term glycemic control effect in mice, outperforming the positive control drugs Peptide20 and P27; this indicates that both P38 and P27 have significant short-term glycemic control effects in mice, with candidate drug P38 being more effective than the positive control drugs Peptide20 and P27.

[0137] Furthermore, the results of the candidate drug's potent acute glycemic control efficacy in db / db mice are as follows: Figures 11-12 As shown in Table 3.

[0138] Table 3. Area under the blood glucose curve (AUC) in db / db mice after oral glucose tolerance test 0～120 min ) The results showed that, compared with the PBS-only injection group and the treatment groups injected with retaglutide or Peptide20, dbdb mice exhibited significantly reduced areas under the blood glucose curve from 30 min before to 120 min after the formal experiment following injection of P38 or P27, respectively. Furthermore, the areas under the blood glucose curve from 0 to 120 min were comparable to those in the PBS-only injection group and the Peptide20 injection group, indicating that peptide20 has no short-term effect on blood glucose in dbdb mice and does not provide short-term glycemic control.

[0139] The above results all indicate that P27 or P38 has extremely potent acute glycemic control efficacy in db / db mice and DIO mice.

[0140] Example 3: Evaluation of the efficacy of candidate drugs in controlling body weight after three weeks of continuous treatment using db / db and DIO model mice. 1) Experimental methods Following the experimental method described above, db / db mice were administered PBS, retaglutide, P38, P27, P37, and Peptide 20, and DIO mice were administered PBS, retaglutide, P38, P27, P37, and Peptide 20, with body weight monitored during 21 days of treatment.

[0141] 2) Experimental Results The results of the candidate drug's efficacy in controlling body weight in DIO mice are as follows: Figure 4 , Figure 5 As shown in Table 4.

[0142] Table 4. Percentage of body weight relative to initial body weight in DIO mice on day 21 The results showed that after 21 days of treatment, the percentage of weight loss at the endpoint in the P38 treatment group was significantly lower than that in the control group PBS (****, p<0.0001) and the Peptide20 treatment group (**, p=0.0020), and also lower than that in the retaglutide treatment group (*, p=0.0308).

[0143] In addition, the growth rate of body weight in mice in the P37 treatment group, P27 treatment group and positive control group (retaglutide treatment group, Peptide20 treatment group) was significantly lower than that in the PBS group (****, p<0.0001).

[0144] This indicates that 21 days of continuous P38 treatment has a significant effect on weight loss in DIO mice, and the effect is better than that of Peptide20 and Retaglutide.

[0145] Results of the candidate drug's efficacy in controlling body weight in db / db mice are as follows: Figure 13 , Figure 14 As shown in Table 5.

[0146] Table 5. Percentage of body weight relative to initial body weight on day 21 in db / db mice The results showed that after three weeks of treatment, the weight gain rate of mice in the P38 experimental group was significantly lower than that in the PBS group, and the weight loss in the P38 group was comparable to that in the retaglutide group.

[0147] Furthermore, in db / db mice, due to adverse reactions to Peptide20, all six mice in the Peptide20 group died starting on day 8 and all died by day 16; while one mouse in the reinaglutide group died on day 16, and no mice died in the P38 and P27 groups. This indicates that P38 is superior to reinaglutide in terms of safety. Figure 15 ).

[0148] In summary, P38 has better therapeutic efficacy and safety than retaglutide.

[0149] Example 4: Evaluation of the glycemic control efficacy of candidate drugs after 21 days of treatment in db / db and DIO model mice. 1) Experimental methods Following the experimental method described above, db / db mice were administered PBS, retaliutide, P38, P27, P37, and Peptide20, respectively, and DIO mice were administered PBS, retaliutide, P38, P27, P37, and Peptide20, respectively, and their glycemic control ability after 21 days of treatment was assessed.

[0150] 2) Experimental Results The results of the candidate drug's efficacy in controlling blood glucose in DIO mice after 21 days of treatment are as follows: Figure 7 As shown in Table 6.

[0151] Table 6. Area under the blood glucose curve in DIO mice after 21 days of oral glucose tolerance test The results showed that, for DIO mice, after 21 days of treatment, fasting blood glucose levels in the retaliatory, P38, and Peptide20 groups were significantly lower than those in the PBS group, and fasting blood glucose levels in the P38 group were significantly lower than those in the retaliatory and Peptide20 groups.

[0152] Furthermore, the results of the candidate drug's efficacy in controlling fasting blood glucose in DIO mice after 21 days of treatment are as follows: Figure 6 , Figure 8 As shown in Table 7.

[0153] Table 7. Fasting blood glucose levels in DIO mice after 21 days The results showed that, for DIO mice, after 21 days of treatment, the oral glucose tolerance of mice in the retaglutide group, P38 group, and Peptide20 group was significantly improved compared with the PBS group after three weeks of treatment. Furthermore, the oral glucose tolerance of mice in the P38 group was significantly improved compared with the retaglutide group and the Peptide20 group after three weeks of treatment.

[0154] This indicates that the P38 candidate drug provides the best glycemic control in mice, and all three candidate drugs effectively improve glycemic control.

[0155] Furthermore, the results of the candidate drug's efficacy in controlling blood glucose in dbdb mice after 21 days of treatment are as follows: Figure 16-17 As shown in Table 8-9.

[0156] Table 8. Area under the blood glucose curve in dbdb mice after 21 days of oral glucose tolerance test Table 9. Fasting blood glucose levels in dbdb mice after 21 days The results showed that, in dbdb mice, after 21 days of treatment, the fasting blood glucose level in the P38 experimental group was significantly lower than that in the PBS group and the retaglutide group. Figure 16 -B and Table 9) After three weeks of treatment, the oral glucose tolerance test (ORT) level in the P38 experimental group mice was significantly lower than that in the PBS group, and comparable to that in the retaglutide group. Figure 16 -A、 Figure 17 (and Table 8).

[0157] Furthermore, after 21 days of treatment, the fasting blood glucose levels of mice in the P38 group were significantly lower than those in the PBS-only group (*, p=0.0395), while there was no significant difference in fasting blood glucose levels between the retaglutide and P27 groups and the PBS-only group after 21 days of treatment. Mice in the Peptide20 group died on day 16, so fasting blood glucose results for day 21 were unavailable. Figure 16 -B and Table 9).

[0158] This indicates that only the P38 drug group showed some improvement in blood glucose levels in dbdb mice after 21 days of treatment.

[0159] The above results indicate that P38 significantly improves both blood glucose levels and blood glucose control in mice.

[0160] Example 5: Evaluation of the efficacy of candidate drugs in improving blood lipid and body fat levels in DIO and dbdb mice after 21 days of treatment. 1) Experimental methods Following the experimental method described above, the treated mice were dissected. Blood was collected and serum was separated. Five indicators in the serum—total cholesterol (T-CHO), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), triglycerides (TG), and free fatty acids (NEFA)—were measured. The visceral fat and liver of the mice were weighed and recorded.

[0161] The ability of db / db mice and DIO mice to improve blood lipid and body fat levels after 21 days of treatment in PBS blank control group, retaglutide treatment group, P38 treatment group, P37 treatment group, P27 treatment group, Peptide20 treatment group, and other groups.

[0162] 2) Experimental Results The results of 21 days of treatment with the candidate drug on blood lipid levels in DIO mice are as follows: Figure 9 As shown in Table 10.

[0163] Table 10. Blood biochemical indices of DIO mice The results showed that, through analysis of blood biochemical indicators in DIO mice, the total cholesterol (T-CHO), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels in the P38 group were significantly lower than those in the retaglutide, Peptide20, and PBS groups. Furthermore, compared with the Peptide20 group, the total cholesterol, LDL-C, and HDL-C levels in the P37 and P27 groups were significantly lower.

[0164] There were no significant differences between the five experimental groups and the control group in terms of triglycerides (TG) and free fatty acids (NEFA) in DIO mice.

[0165] In addition, the results of 21 days of treatment with the candidate drug on the blood lipid levels of dbdb mice are as follows: Figure 18 As shown in Table 11.

[0166] Table 11. Blood biochemical indices of dbdb mice The results showed that analysis of blood biochemical parameters in dbdb mice revealed no significant differences between the experimental group and the PBS group in terms of total cholesterol (T-CHO), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C). However, triglycerides (TG) were significantly higher in the P38 group compared to the PBS group (*, p=0.0298). Furthermore, the level of free fatty acids (NEFA) was significantly higher in the P38 group compared to the PBS group (*, p=0.0019).

[0167] In addition, the visceral fat and liver mass of DIO mice and dbdb mice were compared with body weight to calculate the fat index and liver index.

[0168] In DIO mice, the adipose tissue index of P38 mice was significantly lower compared with the PBS control group, the retaglutide treatment group, and the Peptide20 treatment group, while there was no significant difference in the liver index between the experimental group and the PBS group. Figure 10 (Tables 12 and 13).

[0169] Table 12. Liver index and fat index of DIO mice Table 13. Average liver weight of DIO mice In dbdb mice, the adipose tissue index was significantly lower in the P38 group compared to the PBS control group, and the adipose tissue index level in the P38 group was comparable to that in the retaglutide treatment group; the liver index was significantly higher in the P38 group compared to the PBS control group, and also relatively higher in the P38 group compared to the retaglutide treatment group. Figure 19 (and Tables 14-15).

[0170] The calculation formula is as follows: Liver index = liver weight / body weight; Fat index = fat weight / body weight.

[0171] Table 14. Liver fat index of dbdb mice Table 15. Average liver weight of dbdb mice The above results indicate that the average liver weight of DIO and dbdb mice was within a reasonable range and there was no significant difference. Therefore, the changes in the liver index were more due to the fluctuations in body weight than to the changes in the liver weight caused by the drug.

[0172] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0173] The sequence information of this invention is shown in Table A.

[0174] Table A

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof having GLP-1R / GIPR / GCGR triple receptor agonistic activity, characterized in that, The polypeptide has the amino acid sequence shown in formula (I): Xaa1Xaa2MGTFTSDKSKYLDERAAYDFVQWLLDGPSTGAPPPT(I) in, Xaa1 is H or M. Xaa2 can be V, Aib, or S.

2. The polypeptide according to claim 1, characterized in that, The polypeptide can be a modified polypeptide or an unmodified polypeptide.

3. The polypeptide according to claim 2, characterized in that, The modified polypeptide has a side chain covalently linked to the amino acid sequence of the group consisting of: long-chain fatty acids, polyethylene glycol chains, hydrophilic polymers, hydrophilic spacers, glycosylated side chains, or combinations thereof; preferably, long-chain fatty acids.

4. The polypeptide according to claim 3, characterized in that, The long-chain fatty acid is selected from the group consisting of hexadecanoyl (C16) chain, octadecanoyl (C18) chain, or combinations thereof, preferably hexadecanoyl (C16) chain.

5. The polypeptide according to claim 4, characterized in that, The covalent connection is a γ-carboxylic acid linkage.

6. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide, relative to SEQ ID NO:2, has the following core amino acid mutations: Q3M, Q20Y, S33T and S39T.

7. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide, relative to SEQ ID NO:2, has the following core amino acid mutations: H1M, Aib2S, Q3M, Q20Y, S33T, and S39T.

8. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide, relative to SEQ ID NO:2, has the following core amino acid mutations: Aib2V, Q3M, Q20Y, S33T, and S39T.

9. The polypeptide according to claim 1, characterized in that, The polypeptide contains an amino acid sequence as shown in any of SEQ ID NO:6, 8, or 9, and the 10th position of the polypeptide is a modified K.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (1) the polypeptide or a pharmaceutically acceptable salt thereof as described in claim 1; and (2) Pharmaceutically acceptable carrier.

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