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

Abstract

The invention provides a polypeptide with GLP-1 (glucagon-like peptide-1) R / GIPR / GCGR triple receptor agonistic activity or a pharmaceutically acceptable salt thereof, and the polypeptide or the pharmaceutically acceptable salt thereof has obvious triple receptor agonistic activity of glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and glucagon (GCG) and an anti-DPP-IV enzyme digestion effect. 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] Diabetes is a global health problem that urgently needs to be addressed. It is a metabolic disease characterized by persistently high blood sugar, which severely damages multiple organ systems and causes various complications. The prevalence of obesity is also expanding, increasing the risk of various chronic and debilitating diseases, including but not limited to diabetes, hypertension, and dyslipidemia. Therefore, developing innovative drugs and treatment strategies for obesity and 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 limitations such as insufficient agonistic efficacy, short half-life, low stability and bioavailability, and dose-dependent gastrointestinal side 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 / CGCR triple receptor agonists. Retatrutide is the most advanced GLP-1R / GIPR / CGCR triple receptor agonist, currently in phase III clinical trials. Its half-life has been extended to 6 days, showing good blood sugar lowering and weight loss effects, but mild to moderate gastrointestinal reactions are common.

[0005] Although GLP-1R / GIPR / GCGR triple receptor agonists with different amino acid sequences have been developed in existing technologies, they still have many shortcomings. Therefore, developing GLP-1R / GIPR / GCGR triple receptor agonists with novel structures, higher bioavailability, better therapeutic effects, higher stability, lower production costs, and the ability to significantly improve glycemic and weight control efficacy with fewer side effects, in order to provide safer and more effective treatments for metabolic diseases such as obesity and diabetes and their complications, is an important problem that urgently needs to be solved in this field. 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 or a pharmaceutically acceptable salt thereof is provided, said polypeptide having an amino acid sequence as shown in formula (I): Xaa1Xaa2Xaa3GTFTSDXaa 10 SXaa 12 YLXaa 15 Xaa 16 RAAXaa 20 DFVQWLLDGGPSXaa 33 GAPPPXaa 39 (I) in, Xaa1 is M, H, or Y. Xaa2 can be S, V, Aib, or A. Xaa3 is either Q or M. Xaa10 is K or Y, or a modified K or Y. Xaa12 is either Y or K. Xaa15 is either D or H. Xaa16 is either I or E. Xaa20 is either Y or Q. Xaa33 is either S or T. Xaa39 is either S or T. and, The amino acid sequence of formula (I) is not the amino acid sequence shown in SEQ ID NO:2. The polypeptide or a pharmaceutically acceptable salt thereof has the activity of binding to and activating glucagon-like peptide-1 (GLP-1) receptor, gastric inhibitory peptide (GIP) receptor, and glucagon (GCG) receptor.

[0008] In another preferred embodiment, the polypeptide has high GLP-1R selectivity.

[0009] In another preferred embodiment, "high GLP-1R selectivity" means that, under the same reaction conditions, the GLP-1R selectivity of the polypeptide is Y1≤2. in, YA represents the agonistic activity of the test peptide for GLP-1R. YB represents the agonistic activity of the test peptide for GCGR.

[0010] In another preferred embodiment, Y1 ≤ 1.5, more preferably ≤ 1, and even more preferably ≤ 0.5.

[0011] In another preferred embodiment, the reaction conditions are: a polypeptide concentration of 10... -5 ~10 -12 The concentration was mol / L, the reaction temperature was 37℃, and the reaction time was 4 hours.

[0012] In another preferred embodiment, Y1 is 0.0001~2, more preferably 0.001~1.5, and even more preferably 0.01~1.

[0013] In another preferred embodiment, the polypeptide is a modified polypeptide or an unmodified polypeptide.

[0014] 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 spacers, glycosylated side chains, cholesterol groups, fatty acid side chains, hydrophobic alkyl chains, polypeptide or protein fusion tags, or combinations thereof; preferably long-chain fatty acids.

[0015] 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.

[0016] In another preferred embodiment, the long-chain fatty acid additionally comprises a group selected from the group consisting of γ-glutamyl (γGlu), 8-amino-3,6-dioxanoic acid (AEEA), or oligoethylene glycol (OEG), or combinations thereof; preferably oligoethylene glycol (OEG) or γ-glutamyl (γGlu).

[0017] In another preferred embodiment, the polypeptide is composed of an amino acid sequence as shown in formula (I).

[0018] In another preferred embodiment, the structure of the polypeptide is shown in formula (I).

[0019] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutation relative to SEQ ID NO:2: S33T and S39T.

[0020] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:13.

[0021] In another preferred embodiment, the amino acid sequence of the polypeptide has a core amino acid mutation selected from the group consisting of SEQ ID NO:13: H1M, Aib2S or Aib2V Q3M, K12Y, Q20Y E16I, or combinations thereof.

[0022] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NO:5-27.

[0023] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutations relative to SEQ ID NO:2: H1M, Aib2S, Q3M, Q20Y, S33T, and S39T.

[0024] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:20.

[0025] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutations relative to SEQ ID NO:2: H1M, Aib2S, K12Y, S33T, and S39T.

[0026] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:18.

[0027] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutations relative to SEQ ID NO:2: Aib2S, Q3M, Q20Y, S33T, and S39T.

[0028] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:21.

[0029] In another preferred embodiment, the amino acid sequence of the polypeptide has the following core amino acid mutations relative to SEQ ID NO:2: Aib2V, Q3M, K12Y, Q20Y, S33T, and S39T.

[0030] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO:23.

[0031] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NO:18, 20, 21, 23.

[0032] In another preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NO:18, 20, 21, 23, and a γ-glutamyl long-chain fatty acid is covalently linked to the 10th position of the N-terminus.

[0033] In another preferred embodiment, the covalent connection is a γ-carboxylic acid linkage.

[0034] 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).

[0035] In another preferred embodiment, the amino acid sequence of the polypeptide is covalently linked to a γ-glutamyl n-hexadecanoyl (C16) chain or a γ-glutamyl octadecanoyl (C18) chain at the 10th position of the N-terminus.

[0036] In another preferred embodiment, the polypeptide has anti-DPP-IV enzyme cleavage activity.

[0037] 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.

[0038] 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.

[0039] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: injections and lyophilized preparations.

[0040] 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.

[0041] In another preferred embodiment, the pharmaceutical composition is used for: (a) Improve the effectiveness of acute glycemic control; (b) Improve blood lipid and body fat levels; (c) Weight control; and / or (d) Prevention or treatment of diseases associated with abnormal glucose and lipid metabolism.

[0042] 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.

[0043] 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.

[0044] In another preferred embodiment, the disease is a disease related to abnormal glucose and lipid metabolism.

[0045] 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.

[0046] In another preferred embodiment, the route of administration includes injection (e.g., subcutaneous or intramuscular) or transdermal administration.

[0047] 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.

[0048] In another preferred embodiment, the object is a human or a non-human mammal.

[0049] 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.

[0050] In another preferred embodiment, the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is administered once or twice daily.

[0051] 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

[0052] Figure 1 The 50 ns molecular dynamics simulation (MD) key trajectory frame of the GLP-1R-Peptide 20 and H1M unit point mutant peptides is shown, showing the interaction of the peptide 1 position with the receptor binding pocket.

[0053] Figure 2 The RMSD (left panel) and RMSF (right panel) analyses of the GLP-1R-Peptide 20 and H1M unit point mutant peptides after 50 ns MD are shown.

[0054] Figure 3The RMSD (left panel) and RMSF (right panel) analyses of the GIPR-Peptide 20 and H1M unit point mutant peptides after 50 ns MD are shown.

[0055] Figure 4 The RMSD (left panel) and RMSF (right panel) analyses of the GCGR-Peptide 20 and H1M unit point mutant peptides after 50 ns MD are shown.

[0056] Figure 5 The RMSD (left) and RMSF (right) analyses after 20 1000 ns MD are shown.

[0057] Figure 6 The RMSD (left panel) and RMSF (right panel) analyses of the H1M unit point mutant peptide after 1000 ns MD are shown.

[0058] Figure 7 The RMSD (left panel) and RMSF (right panel) analyses of the Q3M unit point mutant peptide after 1000 ns MD are shown.

[0059] Figure 8 The RMSD (left panel) and RMSF (right panel) analyses of the Q20Y unit point mutant peptide after 1000 ns MD are shown.

[0060] Figure 9 The RMSD (left panel) and RMSF (right panel) analyses of the S33TS39T dual-site mutant peptide after 1000 ns MD are shown.

[0061] Figure 10 The RMSD (left) and RMSF (right) analyses of P25 after 1000 ns MD are shown.

[0062] Figure 11 The 1000 ns MD trajectory analysis of P25 is shown: P25 interacts with GLP-1R, GIPR, and GCGR residues.

[0063] Figure 12 The left panel shows the blood glucose levels and AUC of ICR mice after an oral glucose tolerance test (OGTT). 0～120 min Value (right figure).

[0064] Figure 13 The left panel shows the blood glucose levels and AUC of db / db mice after an oral glucose tolerance test (OGTT). 0～120 min Value (right figure).

[0065] Figure 14 The left panel shows the blood glucose levels and AUC of DIO mice after an oral glucose tolerance test (OGTT). 0～120 minValue (right figure).

[0066] Figure 15 The 15-day weight monitoring of db / db mice is shown.

[0067] Figure 16 The image shows the weight monitoring of DIO mice over 15 days.

[0068] Figure 17 The left panel shows the blood glucose levels and AUC of db / db mice after 14 days of treatment via OGTT. 0～120 min Value (right figure).

[0069] Figure 18 The left panel shows the blood glucose levels and their AUC in DIO mice after 14 days of OGTT treatment. 0～120 min Value (right figure).

[0070] Figure 19 The results show the serum lipid-related biochemical results of DIO mice after 14 days of treatment.

[0071] Figure 20 The fat and liver indices of DIO mice after 14 days of treatment are shown.

[0072] In each figure, Semaglutide represents semaglutide. Detailed Implementation

[0073] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time a specific GLP-1R / GIPR / GCGR triple receptor agonist and its applications. Specifically, this invention utilizes computer-aided design combined with in vitro activity testing to discover that modifying amino acids at specific positions in peptides with GLP-1R / GIPR / GCGR triple receptor activity and attaching fatty acid side chains at those positions significantly improves the affinity and agonistic efficacy of the peptides for the three receptors compared to existing structures, and effectively resists DPP-IV enzymatic cleavage, thus improving peptide stability. Furthermore, this invention unexpectedly discovered that the obtained peptides significantly reduce body weight and blood glucose levels in db / db mice (T2DM model) and DIO mice (obesity model), and significantly reduce blood lipid levels in DIO mice. The reduction is significantly greater than that of smegglutinin, a GLP-1R receptor agonist, demonstrating a significant hypoglycemic and weight-loss effect, and can be used to treat metabolic diseases such as type 2 diabetes, obesity, and hyperlipidemia. This invention was completed based on these findings.

[0074] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0075] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0076] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0077] 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”.

[0078] As used in this article, "Retatrutide" and "Retatrutide" are used interchangeably. 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-1 / GIP / Gcg triple receptor agonist action 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 agonism of 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.

[0085] 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.

[0086] 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.

[0087] 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”, and “polypeptide of the present invention having GLP-1R / GIPR / GCGR triple receptor agonist activity” are used interchangeably and all refer to the polypeptide described in the first aspect of the present invention.

[0088] Specifically, the present invention uses computer-aided design to initially screen out a polypeptide with triple agonistic activity of GLP-1 receptor, GIP receptor and GCG receptor.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Typically, the present invention first describes a polypeptide or a pharmaceutically acceptable salt thereof having GLP-1R / GIPR / GCGR triple receptor agonistic activity, said polypeptide consisting of an amino acid sequence as shown in the following general formula (I): Xaa1Xaa2Xaa3GTFTSDXaa 10 SXaa 12 YLXaa 15 Xaa 16 RAAXaa 20 DFVQWLLDGGPSXaa 33 GAPPPXaa 39 (I); among which, Xaa1 is M, H, or Y. Xaa2 can be S, V, Aib, or A. Xaa3 is either Q or M. Xaa10 is K or a modified K. Xaa12 is either Y or K. Xaa15 is either D or H. Xaa16 is either I or E. Xaa20 is either Y or Q. Xaa33 is either S or T. Xaa39 is either S or T.

[0093] Preferably, Xaa1 is H, Xaa2 is Aib, Xaa3 is Q, Xaa10 is K, Xaa12 is K, Xaa15 is D, Xaa16 is E, Xaa20 is Q, Xaa33 is T, and Xaa39 is T, and the polypeptide has the following amino acid sequence: Aib-QGTFTSDKSKYLDERAAQDFVQWLLDGPSTGAPPPT (SEQ ID NO: 13).

[0094] Preferably, Xaa1 is H, Xaa2 is Aib, Xaa3 is Q, Xaa10 is K, Xaa12 is Y, Xaa15 is D, Xaa16 is E, Xaa20 is Q, Xaa33 is S, and Xaa39 is S, and the polypeptide has the following amino acid sequence: H-Aib-QGTFTSDKSYYLDERAAQDFVQWLLDGGPSSGAPPPS (SEQ ID NO: 9).

[0095] Preferably, Xaa1 is M, Xaa2 is S, Xaa3 is Q, Xaa10 is K, Xaa12 is Y, Xaa15 is D, Xaa16 is E, Xaa20 is Q, Xaa33 is T, and Xaa39 is T, and the polypeptide has the following amino acid sequence: MSQGTFTSDKSYYLDERAAQDFVQWLLDGPSTGAPPPT(SEQ ID NO:18), In particular, a γ-glutamyl-n-hexadecanoyl (C16) chain is covalently linked to the 10th position of the N-terminus of the amino acid sequence.

[0096] Preferably, Xaa1 is M, Xaa2 is S, Xaa3 is M, Xaa10 is K, Xaa12 is K, Xaa15 is D, Xaa16 is E, Xaa20 is Y, Xaa33 is T, and Xaa39 is T, and the polypeptide has the following amino acid sequence: MSMGTFTSDKSKYLDERAAYDFVQWLLDGPSTGAPPPT(SEQ ID NO:20); In this amino acid sequence, the 10th position of the N-terminus is covalently linked to a γ-glutamyl-hexadecanoyl (C16) chain or a γ-glutamyl-octadecanoyl (C18) chain.

[0097] Preferably, Xaa1 is H, Xaa2 is V, Xaa3 is M, Xaa10 is K, Xaa12 is Y, Xaa15 is D, Xaa16 is E, Xaa20 is Y, Xaa33 is T, and Xaa39 is T, and the polypeptide has the following amino acid sequence: HVMGTFTSDKSYYLDERAAYDFVQWLLDGPSTGAPPPT(SEQ ID NO:23), In particular, a γ-glutamyl-n-hexadecanoyl (C16) chain is covalently linked to the 10th position of the N-terminus of the amino acid sequence.

[0098] 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).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] A preferred method is to use liquid-phase synthesis or solid-phase synthesis techniques, such as Boc solid-phase synthesis, Fmoc solid-phase synthesis, or a combination of both. Solid-phase synthesis provides rapid sample preparation, and the appropriate resin carrier and synthesis system can be selected based on the sequence characteristics of the target peptide. For example, in this study, the target peptide sequence was synthesized using the Fmoc / tBu solid-phase peptide synthesis strategy (SPPS), with Fmoc-Ser(tBu)-Wang Resin (0.3 mmol degree of substitution) as the solid carrier. The initial resin was swollen with 10 ml of N,N-dimethylformamide (DMF) for 30 minutes, followed by treatment with 10 ml of 20% piperidine / DMF solution for 30 minutes to remove the Fmoc protecting groups of serine residues. The deprotected resin was washed five times with 10 ml of DMF for 30 seconds each time. To ensure complete deprotection, ninhydrin (Kaiser) detection was performed: a small amount of resin sample was added to ninhydrin reagent and heated at 100°C for 3 minutes; a blue color indicated 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).

[0103] 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.

[0104] In a preferred embodiment, the polypeptide of the present invention is prepared by solid-phase synthesis according to its sequence, purified by high-performance liquid chromatography to obtain a high-purity lyophilized peptide powder, which is then stored at -20°C.

[0105] Another method is to generate the polypeptides of the present invention using recombinant technology. The polynucleotides of the present invention can be used to express or produce recombinant polypeptides of the present invention using conventional recombinant DNA technology. Generally, the following steps are involved: (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0106] Recombinant peptides can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0107] Since the polypeptides of this invention are relatively short, it is possible to chain multiple polypeptides together, recombinantly express them to obtain the expression product in the form of a multimer, and then form the desired small peptides through methods such as enzyme digestion.

[0108] 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.

[0109] 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) Improve the effectiveness of acute glycemic control; (b) Improve blood lipid and body fat levels; (c) Weight control; and / or (d) Prevention or treatment of diseases associated with abnormal glucose and lipid metabolism.

[0110] 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.

[0111] The pharmaceutical composition of the present invention can improve / control blood glucose and blood lipid levels, especially improve blood lipid and body fat levels, control weight, and has acute blood glucose control efficacy.

[0112] 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.

[0113] 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.

[0114] Generally, pharmaceutical formulations should be matched with the method of administration. The dosage forms of the pharmaceutical compositions of the present invention are injections and lyophilized preparations.

[0115] 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.

[0116] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition may be used depending on the application, preferably injections or lyophilized preparations.

[0117] 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.

[0118] 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.

[0119] The main advantages of this invention include: (a) The polypeptide of the present invention has GLP-1R, GCGR and GIPR triple receptor agonist activity. By changing the amino acid at a specific position in the polypeptide with GLP-1R / GIPR / GCGR triple receptor activity, it has significantly improved affinity and agonist efficacy for the three receptors.

[0120] (b) The peptides of the present invention, by better balancing the agonist activity of GLP-1R / GIPR / GCGR, especially with high selectivity for GLP-1R, reduce side effects (gastrointestinal reactions or tachycardia) while ensuring the hypoglycemic and weight loss effects, thereby improving safety and patient compliance.

[0121] (c) The polypeptides of the present invention can effectively resist DPP-IV enzyme cleavage and have improved polypeptide stability.

[0122] (d) The polypeptides with added fatty chain side chains at specific positions in this invention have excellent activity and higher pharmacokinetic stability, and have long-lasting properties.

[0123] (e) The polypeptide of the present invention has a variety of significant effects. It can not only significantly improve the efficacy of acute blood glucose control, improve blood lipid and body fat levels, and control weight significantly, but also reduce weight significantly more than the yangshen drug smegglutinin with GLP-1R receptor agonist. It has obvious hypoglycemic and weight loss effects and can be used to prevent or treat metabolic diseases such as diabetes, obesity, and hyperlipidemia.

[0124] 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.

[0125] Materials and Methods: Animal models: male ICR mice (6-8 weeks old), male db / db mice (C57BLKS, leptin receptor deficient, 6-8 weeks old), and male artificially induced obese (DIO) mice (C57BL / 6J modeled for 8 weeks, each weighing approximately 35g). 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.

[0126] Detection kit: Bright-Lite™ Fluorescent Reporter Gene Assay Kit.

[0127] The reagents, cells, and instruments used in the embodiments of this invention are commercially available or prepared using conventional methods.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 3. Animal experiments 3.1 Oral Glucose Tolerance Test (OGTT) Mice were fasted for 12 hours and deprived of water for 4 hours the day before the OGTT. Blood glucose levels were measured using a glucometer via tail clipping 30 minutes before the OGTT. Mice were also subcutaneously injected with PBS (100 µL / mouse), Peptide 20-C16 (30 nmol / kg), smegglutinin (30 nmol / kg), P26 (30 nmol / kg), P27 (30 nmol / kg), and P32 (30 nmol / kg), respectively. Blood glucose was measured at 0 minutes, and mice were also administered glucose solution (2 g / kg) via gavage. Blood glucose levels were subsequently measured at 15, 30, 60, and 120 minutes. The area under the blood glucose curve (AUC) from 0 to 120 minutes was calculated.

[0134] 3.2 14-day weight monitoring The γ-glutamyl hexadecanoyl (C16) side chain of liraglutide increases its affinity for albumin, thereby extending its half-life and enabling once-daily dosing. Following the liraglutide dosing frequency, mice were subcutaneously injected with the drug at 8:00 AM daily for 14 consecutive days (physiological saline (100 µl / mouse), semaglutide (30 nmol / kg), P26 (30 nmol / kg), P27 (30 nmol / kg), P32 (30 nmol / kg)). Daily body weight was measured using an electronic balance (for a total of 15 days). Mice were fasted for 12 hours and deprived of water for 4 hours before body weight measurements on days 0 and 14 to eliminate the influence of food and water intake on the initial and final body weight data.

[0135] 3.3 OGTT after 14 days of continuous administration Starting from day 0, mice were subcutaneously injected with the following drugs at 8:00 AM daily for 14 consecutive days: saline (100 µl / mouse), semaglutide (30 nmol / kg), P26 (30 nmol / kg), P27 (30 nmol / kg), and P32 (30 nmol / kg). On day 14, mice that had been fasted for 12 hours and deprived of water for 4 hours underwent an oral glucose tolerance test (OGTT). Glucose was administered via gavage (2 g / kg), and blood glucose levels were measured at 15 min, 30 min, 60 min, and 120 min. The area under the blood glucose curve (AUC) from 0 to 120 min was calculated. 0-120min ).

[0136] 3.4 Detection of serum biochemical indicators in mice The following lipid-related biochemical indicators were detected in the serum of mice that were subcutaneously injected with physiological saline (100 µl / mouse), semaglutide (30 nmol / kg), P26 (30 nmol / kg), P27 (30 nmol / kg), and P32 (30 nmol / kg) using a Hitachi 3500 fully automated biochemical analysis system: (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.

[0137] 3.5 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.

[0138] 4. In vitro cell experiments on the anti-DPP-IV enzyme activity of peptides The test peptide was prepared into a high-concentration stock solution (4.5 μM), and recombinant DPP-IV enzyme was added to bring the final concentration to 10 nM. Enzymatic digestion was performed by incubation at 37 °C for 24 hours in reaction buffer (50 mM Tris-HCl, pH 7.5). After the reaction, the mixture was serially diluted using RPMI 1640 (containing 0.05 mg / ml BSA). The diluted peptides were then used to assess GLP-1R agonist activity at the cellular level using a luciferase reporter gene assay, ensuring the final peptide concentration ranged from 0.05 pM to 500 nM, with a concentration measurement point every 10-fold. The experiment was repeated three times, and concentration-signal response curves were plotted for eight concentration points. EC50 was predicted based on curve fitting. 50 Numerical value.

[0139] ECG of the test peptide for GLP-1 receptor agonist activity 50 The formula for calculating the change factor Y1 is as follows: YA / YB. in, YA represents the EC50 of the polypeptide in the presence of DPP-IV, which exhibits agonistic activity against the GLP-1 receptor. 50 value; YB represents the EC50 of the polypeptide for GLP-1 receptor agonistic activity in the absence of DPP-IV. 50value.

[0140] Furthermore, the formula for calculating the peptide degradation rate estimate D based on receptor agonist activity is: D = (1 - 1 / Y1) × 100%.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Example 1: Evaluation of the in vitro activity of H1M unit point mutant peptides Computer-based predictive analysis suggests that the His-to-Met mutation of the first amino acid in Peptide 20 may not disrupt the agonistic effects of the peptide molecule on GLP-1R, GIPR, and GCGR. Furthermore, the codon resulting from the single-point mutation of H1M is also the initiation codon for DNA transcription and translation. Therefore, the single-point mutation of H1M allows us to obtain peptide molecules through biological expression methods, such as expression of peptides in E. coli.

[0145] 1) Experimental methods In Schrodinger Bioluminate, the first position of Peptide 20 in the prepared complex structure of GLP-1R, GIPR, GCGR and Peptide 20 was mutated to Met. The local energy of this amino acid was minimized using the Minimize component, and six complexes were constructed: GLP-1R-Peptide 20, GLP-1R-H1M, GIPR-Peptide 20, GIPR-H1M, GCGR-Peptide 20 and GCGR-H1M.

[0146] Molecular dynamics simulations of the six complexes using Max Flow for 50 ns revealed that after the mutation of Peptide 20 from His to Met at position 1, the hydrogen bond interaction between this site and the GCGR binding pocket weakened. However, in both Peptide 20 and the H1M point-mutated peptide, the first amino acid at position 1 is located within the receptor binding pocket, and all first amino acids are oriented towards the hydrophobic pocket (red indicates nonpolar amino acids). Met, as a nonpolar amino acid, may have formed a hydrophobic interaction with the pocket, the principle of which is illustrated in [the diagram]. Figure 1 .

[0147] Based on computer predictions, the in vitro activity of the H1M unit point mutant peptide against three receptors was detected.

[0148] 2) Experimental Results After 50 ns simulation using MM-GBSA, the binding free energy of the Peptide 20 or H1M unit point mutant peptide with the three receptors was less than 0 kcal / mol. The results are shown in Table 1.

[0149] Table 1. Binding free energy results of Peptide 20 & H1M after 50 ns MD The results showed that the H1M unit point mutant peptides still had strong affinity for all three receptors.

[0150] RMSD & RMSF analyses were performed on the H1M single-point mutant peptide, Peptide 20 naked peptide, and three receptors (GLP-1R, GIPR, and GCGR) after 50 ns MD. The results are shown in [Figure / Table / Incomplete]. Figure 2-4 .

[0151] The results showed that the RMSD of the single point mutant peptides Peptide 20 and H1M remained stable within 1-3 Å, indicating that the system was in equilibrium during the simulation and that neither Peptide 20 nor H1M underwent significant conformational changes. Trajectory analysis of the first position showed that after the mutation to Met, the first position did not undergo significant perturbation within the binding pocket.

[0152] To further verify that the H1M unit point mutant peptide can effectively bind to three receptors, long-term molecular dynamics simulations (1000 ns) were performed on the above six complexes. After long-term molecular dynamics (MD) simulations, RMSD and RMSF analyses were performed on the trajectories. The results are shown in... Figure 5 , Figure 6 And Table 2.

[0153] Table 2. Average binding free energy of each peptide to the three receptors over 1000 ns MD The results showed that Peptide 20 and the H1M single point mutant peptide did not undergo significant conformational changes within the binding pockets of the three receptors and remained in a steady state. Furthermore, the binding free energy calculation results (Table 2) indicated that the H1M single point mutant peptide had good binding ability with GLP-1R, GIPR, and GCGR.

[0154] Example 2: Evaluation of the in vitro activity of GLP-1R / GIPR / GCGR triple receptor agonist peptides with different site mutations First, in vitro cell experiments were conducted using chemically synthesized high-purity peptides. Based on the MD post-binding free energy results at 1000 ns and the RMSD & RMSF analysis results, the advantageous point mutations that can be used to design GLP-1R / GIPR / GCGR triple receptor agonist sequences were further evaluated.

[0155] The results of 1000 ns MD-bound free energy and RMSD & RMSF analysis of dominant point mutations are shown in the figure. Figure 7 , 8 And Table 3.

[0156] Table 3. Average binding free energy of each polypeptide to the three receptors over 1000 ns MD The results showed that the Q3M and Q20Y point mutant peptides exhibited good affinity (ΔG) for all three receptors. bind <0), and it binds stably within the receptor-binding pocket.

[0157] Furthermore, the tryptophan cage (Trp cage) is a key structure derived from Exendin-4, located at Leu21 to Ser39 of Exendin-4. This structure enhances the helicity of the peptide's C-terminus, surrounding Trp25. Introducing Exendin-4 at the C-terminus of similar peptides prevents their side chains from being exposed to solvents, resisting cleavage by Neutral Endopeptidase 24.11 (NEP 24.11), thus improving the solubility of similar peptides while enhancing their chemical stability. Choosing Thr, which is more hydrophobic than Ser and does not affect its agonistic efficacy, may enhance local hydrophobicity and thus improve the efficacy of the tryptophan cage.

[0158] The results of the 1000 ns MD combined with the free energy results and the RMSD & RMSF analyses are shown below. Figure 9 and Table 3; The results showed that simultaneous S33T and S39T mutations (S33TS39T) in Peptide20 resulted in good affinity for all three receptors.

[0159] Further prediction of peptide mutation sites was conducted, and computer-aided design was used to predict peptides that could simultaneously meet the requirements of effective resistance to DPP-IV cleavage and possess GLP-1R, GIPR, and GCGR triple receptor agonist activities. Finally, the top-ranked peptide sequence P25(M) was selected. 1 S 2 M 3 Y 20 T 33 T 39 ).

[0160] The 1000 ns MD post-combination free energy results of P25 with RMSD & RMSF analysis results are shown below. Figure 10 And Table 3.

[0161] The results showed that P25 had good binding ability to all three receptors. Based on the 1000 ns MD trajectory of P25, the interactions between P25 and GLP-1R, GIPR, and GCGR residues were analyzed, and the results are shown in... Figure 11 .

[0162] The results showed that P25 formed several interactions with amino acids in the binding pockets of the three receptors, including salt bridges, hydrogen bonds, Pi-Pi stacking, and van der Waals interactions (Vdw).

[0163] Example 3: Determination of the agonistic activity of peptides carrying triple mutations (MSM, HVM, HSM, HAibM) against GLP-1R, GIPR, and GCGR. 1) Experimental methods Based on this, the in vitro activity of the Aib2V, Q3M, and Q20Y unit point mutant peptides against the three receptors GLP-1R, GIPR, and GCGR was detected.

[0164] To determine the agonistic activities of these peptides carrying triplet mutations on GLP-1R, GIPR, and GCGR, HEK293 cell lines stably expressing CRE / luc2P-GLP-1R, CRE / luc2P-GIPR, and CRE / luc2P-GCGR were first constructed. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase, then passaged and seeded into 96-well white-walled, black-bottomed plates, approximately 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours until they reached 80% confluence, then synchronized for 6 hours in serum-free RPMI-1640 medium containing 0.1% BSA. Subsequently, different concentrations of mutant peptide solutions were added from 10 µmol / L... -1 To begin, a tenfold decrease method was used to prepare 10 to 12 concentration gradients, with a final concentration range of 10. -5 Up to 10 -12 mol L -1 100 µL of solution was added to each well; the control group was added with an equal volume of serum-free culture medium. After co-incubating the peptide with the cells for 4 hours, an equal volume of Bright-Lite luciferase substrate was added, and the reaction was carried out in the dark for 2 minutes. Finally, the relative luminescence units (RLU) after the reaction were measured using a multi-functional microplate reader.

[0165] The experiment included a positive control (Peptide 20) and a blank control (without ligand). The RLU values ​​at each concentration were normalized to the percentage response to the positive control by subtracting the blank RLU values. All data were fitted using four-parameter logistic regression with GraphPad Prism software to plot dose-response curves and calculate the half-maximal effective concentration (EC50) and its 95% confidence interval. Triple replicates were set up for each concentration, and the entire experiment was repeated twice to ensure data accuracy and reliability.

[0166] 2) Experimental Results The in vitro activity assays of the H1M unit point mutant peptide and the naked Peptide 20 peptide with the three receptors GLP-1R, GIPR and GCGR were performed, and the results are shown in Tables 4a and 4b.

[0167] Table 4a. The various peptides (naked peptides) that stimulate GLP-1R, GIPR, and GCGR EC, respectively. 50 The results showed that, compared with Peptide 20 (naked peptide), P16 significantly enhanced the agonistic activity of GLP-1R and GCGR, and was able to maintain the agonistic activity of GIPR (Table 4a).

[0168] In addition, the Q3M unit point mutant peptide showed improved in vitro agonistic effect on GIPR compared to the naked peptide Peptide 20; it also showed effective agonistic ability against GLP-1R and GCGR, but with a slight decrease.

[0169] Similarly, the Q20Y single-point mutant peptide can effectively activate three receptors, with improved in vitro activation of GLP-1R and GCGR; it has effective activation of GIPR, but with a slight decrease.

[0170] The in vitro activity assays of the Q3M and Q20Y mutant peptides and the naked Peptide 20 peptide for the agonistic efficacy against the three receptors GLP-1R, GIPR, and GCGR are shown in Table 4a.

[0171] The above results demonstrate that the Q3M or Q20Y mutation sites and their mutant peptides can be used to design triple receptor agonists.

[0172] To balance the agonistic effects of the three receptors and to enhance the agonistic effect of the peptide on GCGR under the Met condition at position 1, the amino acid at position 2 of the peptide was mutated to Ser (derived from the second amino acid of GCG), and in vitro activity was tested. The results are shown in Table 4a.

[0173] The results showed that simultaneous H1M and Aib2S mutations in Peptide20 (compared to P20 in P19) significantly enhanced the agonistic effect of the peptide on GCGR.

[0174] The in vitro activity of the two-site mutant peptides of S33T and S39T was detected, and the results are shown in Table 4a.

[0175] The results showed that simultaneous S33T and S39T mutations in Peptide20 (compared to P19) did not affect the agonistic effect on the three receptors. P18 and P19 showed comparable agonistic activity for GIPR and GLP-1R, but significantly enhanced agonistic activity for GCGR.

[0176] The in vitro activity of P25 was subsequently tested, and the results are shown in Table 4a.

[0177] However, unexpectedly, the agonistic efficacy of the sequence-modified P25 with the three receptors was reduced (compared to Peptide20).

[0178] Previous studies have found that the 10th amino acid is crucial for altering the lipophilicity or hydrophobicity of a polypeptide. Therefore, further, based on simultaneous H1M and Aib2A mutations, the 10th amino acid was mutated by K10Y, and its agonistic efficacy was tested in vitro. The results are shown in Table 4a.

[0179] The results showed that the K10Y mutation at the 10th amino acid significantly enhanced the agonistic effect of the peptide on the three receptors (compared to P17 and P18).

[0180] Table 4b. The activation of GLP-1R, GIPR, and GCGR EC by each polypeptide (after side chain modification). 50 Where <0.01 represents *****; 0.01-0.1 represents ****; 0.1-1 represents ***; 1-10 represents **; 10-100 represents *; >100 represents -.

[0181] The formula for calculating the selectivity (Y1) of GLP-1R is as follows: Y1 = YA / YB. in, YA represents the agonistic activity of the test peptide for GLP-1R. YB represents the agonistic activity of the assay peptide for GCGR. When Y1≤2, the target peptide exhibits high selectivity for GLP-1R.

[0182] The results showed that, compared with Peptide 20-C16, P23, P26, P27, P29, and P30 significantly enhanced the agonistic activity of GLP-1R and were able to maintain the agonistic activity of GIPR and GCGR (Table 4b).

[0183] Furthermore, compared to peptide 20-C16, the peptides P23, P26, and P27 of the present invention exhibit slightly decreased GCGR agonistic activity, with Y1 ≤ 2 (preferably Y1 ≤ 1.5). Therefore, P23, P26, and P27 possess GLP-1R / GIPR / GCGR triple receptor agonistic activity while also exhibiting high GLP-1R selectivity, thereby achieving a balanced triple receptor agonistic activity of GLP-1R / GIPR / GCGR. This results in both hypoglycemic and weight-loss effects while reducing side effects such as nausea, vomiting, elevated liver enzymes, and tachycardia caused by high GCGR activation.

[0184] Furthermore, based on simultaneous mutations of H1M, Aib2S, Q3M, Q20Y, S33T, and S39T, a γ-glutamyl fatty chain was attached to the 10th amino acid K position, i.e., a γ-glutamyl n-hexadecanoyl (C16) chain (P26) or a γ-glutamyl octadecanoyl (C18) chain (P27) was attached to the 10th amino acid K position of P25; or based on simultaneous mutations of H1M, Aib2S, K12Y, S33T, and S39T, a γ-glutamyl fatty chain was attached to the 10th amino acid K position, i.e., a γ-glutamyl octadecanoyl (C18) chain (P23) was attached to the 10th amino acid K position of P21, and in vitro activity was detected. The results are shown in Tables 4a and 4b.

[0185] This invention unexpectedly discovered that, compared to other naked peptide sequences, modifications to the K at the N-terminal 10 position of P21 and P25 significantly enhance the triple receptor agonist activity of GLP-1R / GIPR / GCGR. When P21 and P25 are covalently linked to an OEG-OEG-γ-glutamyl-octadecanoyl (C18) chain or a γ-glutamyl-n-hexadecanoyl (C16) chain at the N-terminal 10 position of K in the peptide sequence (i.e., P23, P26, P27), they exhibit significantly enhanced triple receptor agonist activity of GLP-1R / GIPR / GCGR. Specifically, compared to naked peptide P21, P23 shows an approximately 223.8-fold increase in GLP-1R agonist activity (3.805 / 0.017) and an approximately [missing value] increase in GIPR agonist activity. The activity of P26 was 766.5 times greater than that of P25, and its GCGR agonist activity was increased by approximately 86,677 times. Compared with naked peptide P25, P26 showed an increase of approximately 158.5 times in GLP-1R agonist activity, approximately 25,621 times in GIPR agonist activity, and approximately 39,844 times in GCGR agonist activity. Compared with naked peptide P25, P27 showed an increase of approximately 190.2 times in GLP-1R agonist activity, approximately 2,233 times in GIPR agonist activity, and approximately 1,074 times in GCGR agonist activity (Tables 4a and 4b).

[0186] The above results indicate that selecting K-linking of the γ-glutamyl fatty chain at position 10 in structural modification can significantly enhance the hydrophobicity near the 10th amino acid, which helps to improve the cell membrane localization of the peptide and its binding to the transmembrane domains of GLP-1R, GIPR, and GCGR (P21 vs. P22 & 23). Furthermore, comparing P22 and P23, it was found that the hexadecanoyl (C16) side chain has a slightly better agonistic effect than the octadecanoyl (C18) side chain.

[0187] Furthermore, linking a γ-glutamyl n-hexadecanoyl (C16) chain (P26) or a γ-glutamyl octadecanoyl (C18) chain (P27) at the 10th amino acid K position helps restore the agonistic efficacy of P25 for all three receptors (P25 vs. P26 / P27); it was also found that P26 has a stronger agonistic efficacy against GLP-1R than smegglutinin.

[0188] Furthermore, fine-tuning of the agonistic efficacy of the three receptors can be achieved by mutating other sites in the amino acid sequence (e.g., K12Y mutation).

[0189] To this end, Peptide20 was subjected to multiple site mutations (P32) at Aib2V, Q3M, K12Y, Q20Y, S33T and S39T, and in vitro activity tests were performed. The results are shown in Table 4b.

[0190] The results showed that P32 has potent triadic receptor agonist efficacy.

[0191] The above results indicate that P26, P27, and P32 all possess potent triadic receptor agonistic efficacy.

[0192] Example 4: Evaluation of the effect of the first three amino acid mutation combinations of the peptide on its resistance to DPP-IV restriction enzyme cleavage. To assess the tolerance of these peptides to DPP-IV degradation, in vitro enzymatic digestion experiments were first performed using DPP-IV. The test peptides were prepared into high-concentration stock solutions. The reaction system consisted of recombinant DPP-IV enzyme (final concentration 10 nM), the test peptide (4.5 μM), and reaction buffer (50 mM Tris-HCl, pH 7.5), and was incubated at 37°C for 24 hours. After the reaction, the mixture was serially diluted and added to the reporter gene cells (final concentration from 0.05 pM to 500 nM, 10-fold dilution). Following the reaction, the GLP-1R activating activity before and after DPP-IV digestion was assessed using the methods described in Examples 2 and 3 (luciferase reporter gene assay). The reporter gene cell line was the HEK293 cell line stably expressing CRE / luc2P-GLP-1R. The experiment was performed three times, and the mean and standard deviation were reported.

[0193] 2) Experimental Results EC before and after DPP-IV reaction 50 The change factors are shown in Table 5.

[0194] Table 5. Agonistaltic activity of the test peptides against GLP-1R and EC5 before and after DPP-IV reaction. 50 Change factor The results showed that after DPP-IV digestion, the fold change Y1 of P26, P27, P30, P31, P32 and P35 was significantly lower than that of the control peptide (Peptide20-C16, Y1=3.15) (all less than 3.15, corresponding to a degradation rate of less than 68.25%), indicating fewer degradation products. This suggests that these mutants can significantly improve the anti-DPP-IV digestion activity of the peptides.

[0195] Furthermore, the agonistic activity of these peptides on GLP-1R remains at a low EC50. 50 Within the specified range, it exhibits significant agonistic activity.

[0196] The above results indicate that P26, P27, and P32 possess significant triple receptor agonist activity and resistance to DPP-IV cleavage.

[0197] Example 5: Evaluation of the potent acute glycemic control efficacy of P26, P27, and P32 In this embodiment, OGTT was performed on three mouse models: ICR mice (healthy model), db / db mice (C57BLKS, T2DM model), and DIO mice (C57BL / 6J, obese model) to evaluate the efficacy of P26, P27, and P32 in acute glycemic control of these mice. Mice given PBS served as the blank control group, while mice given smegglutinin and Peptide 20-C16 served as the corresponding positive control groups.

[0198] ICR mice, db / db mice, and DIO mice were administered PBS, smegglutinin, Peptide 20-C16, P26, P27, and P32, respectively, and the AUC was measured. 0-120min The results are shown in Figure 12-14 See Tables 6-8.

[0199] Table 6. Area under the curve (AUC) of blood glucose levels after OGTT in ICR mice 0～120 min ) Table 7. Area under the curve (AUC) of blood glucose levels after OGTT in db / db mice 0～120 min ) Table 8. Area under the curve (AUC) of blood glucose levels after OGTT in DIO mice 0～120 min ) The results showed that, compared with the blank control group, model mice administered semaglutide, Peptide 20-C16, P26, P27, and P32 had a higher AUC.0-120min All were significantly reduced (****, p<0.0001), and the AUC of the P26, P27, and P32 treatment groups in db / db mice was significantly lower. 0-120min The levels were significantly lower than those in the Semaglutide treatment group (****, p<0.0001), indicating that P26, P27, and P32 all had significantly better acute glycemic control efficacy than P07.

[0200] The above results indicate that P26, P27, and P32 have extremely potent acute glycemic control efficacy in ICR mice, db / db mice, and DIO mice.

[0201] Example 6: Evaluation of the weight control efficacy of P26, P27, and P32 mice after 14 days of treatment using db / db and DIO model mice. Body weight was monitored for 15 days in db / db mice and DIO mice in the PBS blank control group, smegglutide treatment group, and P26, P27, and P32 treatment groups, respectively.

[0202] The weight monitoring results of db / db mice are shown in... Figure 15 .

[0203] Weight monitoring results in db / db mice showed that the weight gain rate on day 14 in the P26, P27, and P32 treatment groups was significantly lower than that in the PBS control group (****, p<0.0001). The weight gain rate on day 14 in the semaglutide treatment group was lower than that in the PBS group (***, p=0.0012). There was no significant difference in weight gain rate on day 14 between the P26 and semaglutide treatment groups, while the weight gain rates on day 14 in the P27 and P32 treatment groups were significantly lower than those in the semaglutide treatment group (****, p<0.0001). In other words, treatment with P26, P27, and P32 significantly inhibited weight gain in db / db mice compared to PBS administration.

[0204] The 15-day weight monitoring results of DIO mice are shown in... Figure 16 .

[0205] The results showed that the weight loss rate on day 14 in DIO mice treated with P26, P27, and P32, as well as in the semaglutide treatment group, was significantly higher than that in the PBS control group (****, p<0.0001). There was no significant difference between the P26 and semaglutide treatment groups, while the weight loss rate on day 14 in the P27 treatment group was higher than that in the semaglutide treatment group (***, p=0.0012). Treatment with P26, P27, and P32 for 14 days significantly improved weight loss efficacy in DIO mice compared to 14 days of PBS administration.

[0206] The above results indicate that 14 days of treatment with P26, P27, and P32 has significant efficacy in weight control in both db / db mice and DIO mice, and is comparable to that of smegglutinin.

[0207] Example 7: Evaluation of the acute glycemic control efficacy of P26 after 14 days of treatment in db / db and DIO model mice After db / db mice and DIO mice were treated with smegglutinin and P26 for 14 days, respectively, the drugs were stopped for 1 day without other injections, and the OGTT values ​​were measured.

[0208] OGTT values ​​of db / db mice, results are shown in Figure 17 and Table 9 Table 9. Area under the curve (AUC) of blood glucose levels after OGTT in db / db mice 14 days after treatment 0～120 min ) The results showed that, compared with the PBS blank control group, the AUC of the semaglutide and P26, P27, and P32 treatment groups was significantly higher. 0-120min There were no significant differences, indicating that smegglutide, P26, P27, and P32 did not improve the acute glycemic control efficacy of db / db mice after 14 days of treatment.

[0209] OGTT values ​​of DIO mice are shown in Figure 18 See Table 10.

[0210] Table 10. Area under the curve (AUC) of blood glucose levels after OGTT in DIO mice 14 days after treatment 0～120 min ) The results showed that, compared with the PBS blank control group, the AUC of the P26, P27, and P32 treatment groups was significantly higher. 0-120min All were significantly reduced, with the AUC in the semaglutide treatment group being significantly lower. 0-120min It also decreased significantly.

[0211] However, the AUC in the P26 treatment group was lower than that in the semaglutide treatment group. 0-120min In comparison, the P26 treatment group was lower than the semaglutide treatment group (*, p=0.0172), while the P27 and P32 treatment groups were significantly lower than the semaglutide treatment group (***, p=0.0001), indicating that P26, P27, and P32 were all superior to semaglutide in controlling acute blood glucose in DIO mice after 14 days of treatment.

[0212] Example 8: Evaluation of the efficacy of DIO model mice in improving blood lipid and body fat levels after 14 days of treatment at P26, P27, and P32. DIO mice were treated with smegglutinin, P26, P27, and P32 for 14 days, respectively. Serum lipid-related biochemical indicators were then measured. The results are shown in... Figure 19 See Table 11.

[0213] Table 11. Serum lipid-related biochemical results (mmol / L) in DIO mice after 14 days of treatment (Mean±SEM) The results show that: Compared with the PBS blank control group, in terms of total cholesterol (T-CHO), the Semaglutide group showed a significant decrease (**, p=0.0044), the P26 group showed a significant decrease (*, p=0.034), and the P27 group showed a significant decrease (****, p<0.0001); in terms of high-density lipoprotein (HDL-C), compared with the PBS blank control group, the Semaglutide group showed a significant decrease (**, p=0.0059), and the P27 group showed a significant decrease (****, p<0.0001), and the P27 group... The ability to lower HDL-C was significantly better in the Semaglutide group than in the PBS control group (*, p=0.0342); regarding low-density lipoprotein (LDL-C), compared with the PBS blank control group, the Semaglutide group showed a significant decrease (**, p=0.0018), the P26 group showed a significant decrease (*, p=0.016), and the P27 group showed a significant decrease (****, p<0.0001); regarding triglycerides (TG) and free fatty acids (NEFA), there were no significant differences between each drug treatment group and the PBS blank control group.

[0214] The above results indicate that serum total cholesterol and low-density lipoprotein (LDL) concentrations decreased in DIO mice after 14 days of treatment with P26 and P27; serum total cholesterol, LDL, and HDL concentrations decreased after 14 days of treatment with semaglutide in DIO mice. This suggests that 14 days of treatment with P26 and P27 in DIO mice can improve blood lipid levels by reducing LDL and decreasing fat transport from the liver to the bloodstream.

[0215] After 14 days of treatment with smegglutinin and P26, P27, and P32, the adipose tissue index of DIO mice was calculated, and the results are shown in... Figure 20 And Table 12.

[0216] Table 12. Fat and liver index (%) in DIO mice after 14 days of treatment (Mean±SEM) The results showed that, compared with the PBS control group, mice treated with semaglutide had significantly less adipose tissue (*, p=0.0113), mice treated with P26 had significantly less adipose tissue (*, p=0.0296), and mice treated with P27 had significantly less adipose tissue (*, p=0.0416). There were no significant differences in liver index among the groups. Analysis of the adipose and liver indices indicates that DIO mice treated with P26 or P27 for 14 consecutive days have a certain ability to improve fat-related body fat levels and achieve weight loss, an ability comparable to that of semaglutide.

[0217] 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.

[0218] The sequence information of this invention is shown in Table A.

[0219] Table A

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that, The polypeptide has the amino acid sequence shown in formula (I): Xaa1Xaa2Xaa3GTFTSDXaa 10 SXaa 12 YLXaa 15 Yes 16 COMFORTABLE 20 DFVQWLLDGGPSXaa 33 GAPPPXaa 39 (I) in, Xaa1 is M, H, or Y. Xaa2 can be S, V, Aib, or A. Xaa3 is either Q or M. Xaa10 is K or Y, or a modified K or Y. Xaa12 is either Y or K. Xaa15 is either D or H. Xaa16 is either I or E. Xaa20 is either Y or Q. Xaa33 is either S or T. Xaa39 is either S or T. and, The amino acid sequence of formula (I) is not the amino acid sequence shown in SEQ ID NO:

2. The polypeptide or a pharmaceutically acceptable salt thereof has the activity of binding to and activating glucagon-like peptide-1 (GLP-1) receptor, gastric inhibitory peptide (GIP) receptor, and glucagon (GCG) receptor.

2. The polypeptide according to claim 1, characterized in that, The polypeptide exhibits high GLP-1R selectivity, meaning that under the same reaction conditions, the GLP-1R selectivity of the polypeptide is Y1≤2. in, YA represents the agonistic activity of the test peptide for GLP-1R. YB represents the agonistic activity of the test peptide for GCGR.

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

4. The polypeptide according to claim 3, 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, cholesterol groups, fatty acid side chains, hydrophobic alkyl chains, polypeptide or protein fusion tags, or combinations thereof; preferably long-chain fatty acids.

5. The polypeptide according to claim 4, 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.

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: 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 or 7, characterized in that, The polypeptide has an amino acid sequence as shown in SEQ ID NO:

20.

9. 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.

10. The use of the polypeptide or a pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that, Used in the preparation of pharmaceutical compositions.

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