Multi-target polypeptide compound for long-acting blood glucose reduction and weight loss as well as preparation method and application of multi-target polypeptide compound

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a multi-target polypeptide compound for long-acting blood glucose reduction and weight loss as well as a preparation method and application of the multi-target polypeptide compound. Specifically, the present disclosure provides a polypeptide compound having agonistic activity on GLP-1R, GIPR and GCGR, or a pharmaceutically acceptable salt thereof, or a solvate thereof, the polypeptide compound has a polypeptide part and a modification part, the sequence of the polypeptide part comprises Y-Aib-EGT-X6-TSD-X10-SI-Iva-LDKKAQKAF-X23-X24-WLLEGGPSSGAPPPS (SEQ ID NO.1), and the sequence of the modification part comprises Y-Aib-EGT-X6-TSD-X10-SI-Iva-LDKKAQKAF-X23-X24-WLLEGGPSSGAPPPS (SEQ ID NO.2). The modification part comprises a bridge ring connection formed between the 17 lysine and the 20 lysine in SEQ ID NO. 1.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a long-acting multi-target polypeptide compound for lowering blood sugar and reducing weight, its preparation method, and its application. Background Technology

[0002] With changes in lifestyle, the prevalence of diabetes continues to rise. Diabetes has become a major chronic disease seriously affecting people's health and is one of the leading causes of death and disability. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, accounting for approximately 90% of all diabetes cases, characterized by hyperglycemia caused by insulin resistance. Studies have shown that most T2DM patients are obese, with a high percentage of body fat or abnormal body fat distribution (Chandrasekaran, P. & Weiskirchen, R. The Role of Obesity in Type 2 Diabetes Mellitus - An Overview. International Journal of Molecular Sciences. 2024; 25:1882). Increased obesity is a major factor contributing to the rising prevalence and incidence of T2DM, and there is a complex link and pathophysiological mechanism between obesity and T2DM. Meanwhile, obese individuals have a higher prevalence and incidence of insulin resistance, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), and a range of metabolic abnormalities. Excessive fat accumulation in obese individuals is stored in the liver as triglycerides. Recent studies have shown that adipokines and inflammatory cytokines released from adipose tissue may promote hepatic insulin resistance and influence the progression of NAFLD (Chiang, DJ, Pritchard, MT & Nagy, LEObesity, diabetes mellitus, and liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2011; 300: G697-G702.).

[0003] Weight loss and blood sugar control are considered important measures for treating metabolic diseases such as diabetes, hyperlipidemia, NAFLD, and non-alcoholic steatohepatitis (NASH). Although there are many drugs available for the treatment of diabetes, the coordinated treatment of diabetes and its complications and obesity is still far from meeting clinical needs.

[0004] Glucagon-like peptide-1 receptor (GLP-1R) is considered one of the most effective therapeutic targets for treating type 2 diabetes mellitus (T2DM) and obesity. Single-target GLP-1R agonists were among the earliest drugs developed. Since the launch of exenatide, the first GLP-1 drug, in 2005, GLP-1 drugs have rapidly evolved, with numerous blockbuster drugs emerging. A clinical study of NASH patients treated with the GLP-1R agonist liraglutide (GLP-1RA) for 12 weeks showed increased insulin sensitivity and improved metabolism. Furthermore, clinical results for semaglutide support its indications for treating obesity and diabetes. Nevertheless, the weight loss effects of these single-target GLP-1RA drugs have not yet reached the levels achieved by surgery.

[0005] As research progresses, gastrointestinal hormone receptors are considered to have synergistic advantages in weight loss and blood glucose control. Existing studies have shown that the gastric inhibitory polypeptide receptor (GIPR) plays a crucial role in regulating insulin secretion, blood glucose, and lipid metabolism, and is essential for maintaining normal metabolic function. Compared to single-target GLP-1 receptor agonists, dual-target drugs not only reduce side effects and increase efficacy but also decrease overall side effects. Eli Lilly's GLP-1 / GIP (gastric inhibitory polypeptide) dual-target agonist Tirzepatide has shown significant efficacy in obesity treatment. In the SURMOUNT-1 trial, patients in the 15mg dose group achieved an average weight loss of 20.9% over 72 weeks, approaching the results of bariatric surgery. However, due to insufficient response to the dual GLP-1 / GIP target in some patients, approximately 40% of patients experienced weight loss of <15%, failing to meet the ideal clinical needs (Jastreboff, AM; le Roux, CW; Stefanski, A.; et al. Tirzepatide for Obesity Treatment and Diabetes Prevention. N. Engl. J. Med. 2025, 392: 958-971.).

[0006] Exogenous administration of glucagon (GCG) can reduce the synthesis of triglycerides (TG) in the liver and has a catabolic effect and increases the body's thermogenesis. It can suppress food intake and promote weight loss. GLP-1 / GIP / GCGR (Glucagon Receptor) three-target coordinated therapy is considered to be the development direction of the next generation of weight loss drugs. Based on the combined effects of GLP-1R agonism (lowering glucose and reducing appetite) and GIPR agonism (enhancing insulin sensitivity and reducing food intake), activation of the GCGR target can provide better glycemic control and weight loss through multi-pathway synergy, and increase the therapeutic effect on NAFLD, showing a stronger efficacy (Zhang, J.; Wei, J.; Lai, W.; Sun, J.; Bai, Y.; Cao, H.; Guo, J.; Su, Z. Focus on Glucagon-like Peptide-1 Target: Drugs Approved or Designed to Treat Obesity. Int. J. Mol. Sci. 2025; 26:1651).

[0007] Furthermore, the short half-life and high frequency of administration of peptide drugs urgently need to be addressed, and new management methods are urgently needed for diabetes and its complications, as well as obesity. The development of novel GLP-1R / GIPR / GCGR tri-target agonist drugs with ultra-long-acting, safe, and effective properties will provide an integrated solution for blood glucose management, complication prevention and control, and weight regulation, becoming a revolutionary strategy in the treatment of metabolic diseases. Summary of the Invention

[0008] The purpose of this invention is to provide a polypeptide compound and its application and preparation method. The long-acting polypeptide compound of this invention has a longer half-life than the marketed drugs smegglutide and telpolide, as well as better hypoglycemic and weight-loss effects. It can reverse insulin resistance, regulate lipid metabolism, and has good therapeutic effects on metabolic-related complications such as diabetes, obesity, hyperlipidemia, and non-alcoholic steatohepatitis.

[0009] Specifically, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a polypeptide compound having agonistic activity against GLP-1R, GIPR, and GCGR, or a pharmaceutically acceptable salt thereof or a solvate thereof, said polypeptide compound having a polypeptide moiety and a modification moiety, said polypeptide moiety having a sequence including Y-Aib-EGT-X6-TSD-X 10 -SI-Iva-LDKKAQKAF-X 23 -X24 -WLLEGGPSSGAPPPS (SEQ ID NO.1), wherein the modified portion includes a bridged ring link formed between lysine (Lys, K) at position 17 and lysine (Lys, K) at position 20 in SEQ ID NO.1.

[0011] The bridge ring link of the present invention has the structure shown in formula (b):

[0012]

[0013] The The diagram shows the connection positions of the amino groups on the lysine residues at positions 17 and 20 of the polypeptide moiety.

[0014] Preferably, the structure of the polypeptide compound is shown in formula (a):

[0015]

[0016] Y-Aib-EGT-X6-TSD-X10-SI-lva-LDKKAQKAF-X23-X24-WLLEGGPSSGAPPPS-NH2 Formula (a) (SEQ ID NO. 26),

[0017] Where R is selected from (AEEA). a -γGlu-CO((CH2) b CO2H, ((PEG2) c -γGlu-CO(CH2) d CO2H) or (G e (SG) f -γGlu-CO(CH2) g CO2H).

[0018] Specifically, a is an integer selected from 1 to 6, including 1, 2, 3, 4, 5 or 6.

[0019] Specifically, b is an integer selected from 12 to 20, including 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0020] Specifically, c is an integer selected from 1 to 6, including 1, 2, 3, 4, 5 or 6.

[0021] Specifically, d is an integer selected from 12 to 20, including 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0022] Specifically, e is an integer selected from 0 to 5, including 1, 2, 3, 4 or 5.

[0023] Specifically, f is an integer selected from 1 to 6, including 1, 2, 3, 4, 5 or 6.

[0024] Specifically, g is an integer selected from 12 to 20, including 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0025] Preferably, R is (G e (SG) f -γGlu-CO(CH2) g CO2H), where e is an integer selected from 0 to 5, f is an integer selected from 1 to 6, and g is an integer selected from 12 to 20.

[0026] More preferably, e is 2.

[0027] More preferably, f is 2 or 3.

[0028] More preferably, g is 16, 18 or 20.

[0029] Specifically, R is selected from any of the following:

[0030] 1)GG(SG)2-γGlu-CO(CH2) 16 CO2H,

[0031] 2)GG(SG)2-γGlu-CO(CH2) 18 CO2H,

[0032] 3)GG(SG)2-γGlu-CO(CH2) 20 CO2H,

[0033] 4)GG(SG)3-γGlu-CO(CH2) 16 CO2H,

[0034] 5)GG(SG)3-γGlu-CO(CH2) 18 CO2H,

[0035] 6)GG(SG)3-γGlu-CO(CH2) 20 CO2H.

[0036] Specifically, the two carboxyl groups (-COOH) of the iminodiacetic acid undergo amide condensation reactions with the amino groups (-NH2) of the lysine residues at positions 17 (Lys, K) and 20 (Lys, K), respectively, to form amide bonds (-CO-NH-), thereby forming the following structure:

[0037]

[0038] In some alternative implementations, X6, X 10 X 23 or X 24 Each amino acid can be independent and can be any amino acid, specifically including genetically encoded natural amino acids, non-genetically encoded natural amino acids, and synthetic amino acids.

[0039] Preferably, the natural amino acids encoded by the gene include alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0040] Preferably, the non-gene-encoded natural amino acids include cysteine, hydroxyproline, γ-carboxyglutamic acid, ornithine, and phosphoserine.

[0041] Preferably, the synthetic amino acid includes any amino acid produced through chemical synthesis, specifically including D-isomers of genetically encoded natural amino acids, Aib (α-Aminoisobutyric acid), Abu (α-aminobutyric acid), Tle (tert-butylglycine), Iva (Isovaline), Cba (α-Aminocyclobutanecarboxylic acid), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), or GABA (γ-aminobutyric acid).

[0042] Preferably, X6 is selected from W or F.

[0043] Preferably, X 10 Choose from S, L, or Y.

[0044] Preferably, X 23 Selected from V or T.

[0045] Preferably, X 24 Choose from N or Q.

[0046] Preferably, X10 is L and X24 is N, or X10 is Y and X24 is Q.

[0047] In some alternative embodiments, the sequence of the polypeptide moiety differs from any of the following by at most one, at most two, at most three, at most four, or at most five amino acids, wherein the polypeptide moiety comprises any of the following, or the sequence of the polypeptide moiety is selected from any of the following:

[0048] 1) X6 is W, X 10 Let S and X be the values ​​of S and X. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDSSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.2);

[0049] 2) X6 is W, X 10 Let S and X be the values ​​of S and X. 23 Let V, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDSSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.3);

[0050] 3) X6 is W, X 10 Let S and X be the values ​​of S and X. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDSSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.4);

[0051] 4) X6 is W, X 10 Let S and X be the values ​​of S and X. 23 Let T, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDSSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.5);

[0052] 5) X6 is W, X 10 Let L and X be the numbers. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDLSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.6);

[0053] 6) X6 is W, X 10 Let L and X be the numbers. 23 Let V, X 24For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDLSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.7);

[0054] 7) X6 is W, X 10 Let L and X be the numbers. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDLSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.8);

[0055] 8) X6 is W, X 10 Let L and X be the numbers. 23 Let T, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDLSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.9);

[0056] 9) X6 is W, X 10 Let Y and X be the numbers. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDYSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.10);

[0057] 10) X6 is W, X 10 Let Y and X be the numbers. 23 Let V, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDYSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.11);

[0058] 11) X6 is W, X 10 Let Y and X be the numbers. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDYSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.12);

[0059] 12) X6 is W, X 10 Let Y and X be the numbers. 23 Let T, X 24For Q, the sequence of the polypeptide moiety is Y-Aib-EGTWTSDYSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.13);

[0060] 13) X6 is F, X 10 Let S and X be the values ​​of S and X. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDSSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.14);

[0061] 14) X6 is F, X 10 Let S and X be the values ​​of S and X. 23 Let V, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDSSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.15);

[0062] 15) X6 is F, X 10 Let S and X be the values ​​of S and X. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDSSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.16);

[0063] 16) X6 is F, X 10 Let S and X be the values ​​of S and X. 23 Let T, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDSSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.17);

[0064] 17) X6 is F, X 10 Let L and X be the numbers. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDLSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.18);

[0065] 18) X6 is F, X 10 Let L and X be the numbers. 23 Let V, X 24For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDLSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.19);

[0066] 19) X6 is F, X 10 Let L and X be the numbers. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDLSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.20);

[0067] 20)X6 is F, X 10 Let L and X be the numbers. 23 Let T, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDLSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.21);

[0068] 21) X6 is F, X 10 Let Y and X be the numbers. 23 Let V, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDYSI-Iva-LDKKAQKAFVNWLLEGGPSSGAPPPS (SEQ ID NO.22);

[0069] 22) X6 is F, X 10 Let Y and X be the numbers. 23 Let V, X 24 For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDYSI-Iva-LDKKAQKAFVQWLLEGGPSSGAPPPS (SEQ ID NO.23);

[0070] 23) X6 is F, X 10 Let Y and X be the numbers. 23 Let T, X 24 For N, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDYSI-Iva-LDKKAQKAFTNWLLEGGPSSGAPPPS (SEQ ID NO.24);

[0071] 24) X6 is F, X 10 Let Y and X be the numbers. 23 Let T, X 24For Q, the sequence of the polypeptide moiety is Y-Aib-EGTFTSDYSI-Iva-LDKKAQKAFTQWLLEGGPSSGAPPPS (SEQ ID NO.25).

[0072] Preferably, X 10 Let L and X be the numbers. 24 For N, the sequence of the polypeptide moiety is as shown in SEQ ID NO.6, 8, 18 or 20.

[0073] Preferably, X 10 Let Y and X be the numbers. 24 For Q, the sequence of the polypeptide portion is as shown in SEQ ID NO.11, 13, 23 or 25.

[0074] Preferably, X6 is F, X 10 Let Y and X be the numbers. 23 V, X 24 For Q, the sequence of the polypeptide portion is shown in SEQ ID NO.23.

[0075] In some alternative embodiments, the polypeptide compound is selected from any of the following:

[0076] 1) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is

[0077] GG(SG)2-γGlu-CO(CH2) 16 CO2H;

[0078] 2) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is...

[0079] GG(SG)2-γGlu-CO(CH2) 18 CO2H,

[0080] 3) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is...

[0081] GG(SG)2-γGlu-CO(CH2) 20 CO2H,

[0082] 4) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is

[0083] GG(SG)3-γGlu-CO(CH2) 16 CO2H,

[0084] 5) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is

[0085] GG(SG)3-γGlu-CO(CH2) 18 CO2H, or

[0086] 6) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is

[0087] GG(SG)3-γGlu-CO(CH2) 20 CO2H.

[0088] Preferably, the polypeptide compound is selected from any one of the polypeptide compounds represented by formula (aⅠ), (aⅡ), (aⅢ), (aⅣ), (aⅤ), (aⅥ), (aⅦ), or (aⅧ):

[0089]

[0090] Formula (aⅠ), (polypeptide compound L01, SEQ ID NO.27),

[0091]

[0092] Formula (aⅡ), (polypeptide compound L02, SEQ ID NO.28),

[0093]

[0094] Formula (aⅢ), (polypeptide compound L03, SEQ ID NO.29),

[0095]

[0096] Formula (aⅣ), (peptide compound L04, SEQ ID NO.30),

[0097]

[0098] Formula (aⅤ), (peptide compound L05, SEQ ID NO.31),

[0099]

[0100] Formula (aⅥ), (peptide compound L06, SEQ ID NO.32),

[0101]

[0102] Formula (aⅦ), (peptide compound L07, SEQ ID NO.33),

[0103]

[0104] Formula (aⅧ), (peptide compound L08, SEQ ID NO.34).

[0105] More preferably, the polypeptide compound has end modifications, the end modifications including the attachment of an N-terminal protecting group to the free amino terminus of an amino acid and / or the attachment of a C-terminal protecting group to the free carboxyl terminus of an amino acid.

[0106] Preferably, the N-terminal protecting group includes an acyl group (-CO-R1) and an alkoxycarbonyl group or an aryloxycarbonyl group (-CO-O-R1), wherein R1 is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group, or a substituted aromatic group.

[0107] Preferably, the C-terminal (free carboxyl terminus) protecting group includes -NH2, -NHCH3, -N(CH3)2, -NH(ethyl), -N(ethyl)2, -N(methyl)(ethyl), -NH(benzyl), -N(C1-C4 alkyl)(benzyl), -NH(phenyl), -N(C1-C4 alkyl)(phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(isopropyl), O-(sec-butyl), -O-(tert-butyl), -O-benzyl, and -O-phenyl.

[0108] Preferably, the free carboxyl terminus of the 39th amino acid serine (Ser, S) of the polypeptide compound is connected to an amino group (-NH2).

[0109] Preferably, the polypeptide compound is selected from any one of the polypeptide compounds represented by formula (bⅠ), (bⅡ), (bⅢ), (bⅣ), (bⅤ), (bⅥ), (bⅦ), or (bⅧ):

[0110]

[0111] Formula (bⅠ), (polypeptide compound L01-NH2, SEQ ID NO.35),

[0112]

[0113] Formula (bⅡ), (polypeptide compound L02-NH2, SEQ ID NO.36),

[0114]

[0115] Formula (bⅢ), (polypeptide compound L03-NH2, SEQ ID NO.37),

[0116]

[0117] Formula (bⅣ), (polypeptide compound L04-NH2, SEQ ID NO.38),

[0118]

[0119] Formula (bⅤ), (peptide compound L05-NH2, SEQ ID NO.39),

[0120]

[0121] Formula (bⅥ), (peptide compound L06-NH2, SEQ ID NO.40),

[0122]

[0123] Formula (bⅦ), (peptide compound L07-NH2, SEQ ID NO.41),

[0124]

[0125] Formula (bⅧ), (polypeptide compound L08-NH2, SEQ ID NO.42).

[0126] Specifically, the polypeptide compounds of formula (bⅣ) and SEQ ID NO. 38 have the following structures:

[0127]

[0128] Preferably, the polypeptide moiety may also have any one or more of the following modifications: non-natural peptide bonds, acylation, acetylation and other acylation, methylation, glycosylation, phosphorylation, ubiquitination, hydroxylation, carbonylation, alkylation, aliphaticization, hydroxyl modification, PEGylation, and PPGylation.

[0129] Preferably, pharmaceutically acceptable salts of the polypeptide compound include its acetate, hydrochloride, phosphate, or acetic acid salt.

[0130] In a second aspect, the present invention provides a chimeric molecule comprising the aforementioned polypeptide compound and other proteins.

[0131] Preferably, the other proteins include epitope tags or half-life extenders.

[0132] Preferably, the half-life extender comprises an Fc domain and serum albumin.

[0133] Preferably, the other proteins are linked to the free amino terminus and / or free carboxyl terminus of the aforementioned polypeptide compound.

[0134] On the other hand, the present invention provides a pharmaceutical composition comprising the polypeptide compound of the first aspect or a pharmaceutically acceptable salt thereof or a solvate thereof, or the chimeric molecule of the second aspect.

[0135] Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.

[0136] Preferably, the pharmaceutical composition further contains other drugs with hypoglycemic and weight-reducing effects, such as metformin, thiazolidinediones (TZDs, such as pioglitazone), sulfonylureas (such as glimepiride), dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitors, such as sitagliptin), sodium-glucose cotransporter (SGLT-2 inhibitors, such as dapagliflozin), growth differentiation factor 15 (GDF15) regulators, peptide tyrosine tyrosine regulators (PYY), modified or unmodified insulin, and amylin (such as pramlintide).

[0137] On the other hand, the present invention provides the use of the polypeptide compound of the first aspect or a pharmaceutically acceptable salt thereof or a solvate thereof, the chimeric molecule of the second aspect or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to GLP-1R, GIPR and / or GCGR.

[0138] On the other hand, the present invention provides the polypeptide compound of the first aspect or a pharmaceutically acceptable salt thereof or a solvate thereof, the chimeric molecule of the second aspect or the aforementioned pharmaceutical composition, for the prevention and / or treatment of diseases related to GLP-1R, GIPR and / or GCGR.

[0139] On the other hand, the present invention also provides a method for preventing and / or treating diseases related to GLP-1R, GIPR, and / or GCGR: the method comprising administering to a patient in need of such treatment an effective amount of the polypeptide compound of the first aspect or a pharmaceutically acceptable salt thereof or a solvate thereof, the chimeric molecule of the second aspect, or the aforementioned pharmaceutical composition.

[0140] Preferably, the diseases include hyperglycemia, hyperlipidemia, diabetes (including type 1 diabetes and / or type 2 diabetes), obesity, metabolic syndrome, neurodegenerative diseases, impaired glucose tolerance (IGT), non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD).

[0141] Preferably, the disease is diabetes or obesity.

[0142] Preferably, the diabetes includes complications of diabetes.

[0143] On the other hand, the present invention provides a method for preparing the aforementioned polypeptide compound, wherein the preparation method is a chemical synthesis method.

[0144] The beneficial effects achieved by this invention are as follows:

[0145] 1) Compared with existing single-target and dual-target agonists, the novel GLP-1R / GIPR / GCGR tri-target agonist peptide compound provided by this invention has an innovative solid compound structure and more effective and ultra-long-lasting hypoglycemic and weight-loss effects, which is of great significance for the treatment of major chronic diseases such as T2DM, obesity, NAFLD, NASH, and hyperlipidemia, which require long-term medication; 2) The peptide compound provided by this invention has strong agonist activity against GLP-1R and GIPR, which can effectively lower blood glucose after activation; it has relatively weak activity against GCGR, which has a suitable glycemic effect after activation. This combination of different agonist activities of the compound of this invention becomes a key factor in maintaining blood glucose balance; 3) The peptide compound provided by this invention has stable chemical properties and is scientifically inferred to have pharmacokinetic characteristics that support dosing once every two weeks or more in future clinical practice, and has a longer-lasting hypoglycemic and weight-loss effect; 4) The peptide compound provided by this invention has good safety and a large safety window, and its therapeutic effect on metabolic diseases such as T2DM, obesity, NAFLD, NASH, and hyperlipidemia is superior to existing marketed drugs. Attached Figure Description

[0146] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0147] Figure 1 The graph shows the time-glucose results of the OGTT experiment in C57BL / 6J mice after a single dose.

[0148] Figure 2 A graph showing the random blood glucose results after a single dose in ob / ob mice.

[0149] Figure 3 A graph showing the random blood glucose results after multiple administrations to ob / ob mice.

[0150] Figure 4 Figure 1 shows the HOMA-IR, HbA1c, and TG levels in ob / ob mice after multiple administrations; Figure A shows the insulin resistance index (HOMA-IR); Figure B shows the glycated hemoglobin (HbA1c) content; and Figure C shows the triglyceride (TG) content.

[0151] Figure 5 Figure showing the results of long-term weight loss in diet-induced obese mice after administration of medication.

[0152] Figure 6 The results of total fat weight in diet-induced obese mice after long-term administration of the drug. Detailed Implementation

[0153] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided through specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of this disclosure. All other implementations obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of this disclosure.

[0154] This disclosure may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this disclosure may be combined with technical features of any or more other embodiments to obtain further embodiments. This disclosure includes such further embodiments obtained through combination.

[0155] (I) Definitions and Explanations

[0156] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used have meanings commonly understood by those skilled in the art. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. It should also be understood that the terminology used in this disclosure is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0157] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and that used in this disclosure, the use and terminology of this disclosure shall prevail. Section headings used in this disclosure are for organizational purposes only and should not be construed as limiting the subject matter.

[0158] In this disclosure, the conjunction term “and / or” between multiple elements means to include both the meaning of “and” and “or”, for example, the phrase “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0159] In this disclosure, the terms “comprising,” “including,” “having,” and “containing,” and any variations thereof, are intended to cover non-exclusive inclusion. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially composed of.” In this disclosure, the term “containing” indicates that various ingredients may be used together in the mixtures or pharmaceutical compositions of this disclosure. Therefore, the terms “substantially composed of” and “composed of” are included in the term “containing.”

[0160] The range of numbers used in this disclosure should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0161] In this disclosure, the term "about" or "approximately" when applied to one or more target values ​​refers to a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term "approximately" or "about" refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0162] In this disclosure, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0163] In this disclosure, the disclosure of all ranges should be considered as a disclosure of all subranges and all point values ​​within the range. All point values ​​can be combined independently. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range referenced in this application is intended to include all subranges within that range. For example, the disclosure of 0.1-200 should be considered as also disclosing ranges such as 0.1-10, 1-100, 100-200, etc., and also disclosing point values ​​such as 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200.

[0164] The amino acids in the polypeptide compounds disclosed herein contain conventional single-letter or three-letter codes for amino acids, such as: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R). This disclosure also relates to other commonly accepted three-letter codes for amino acids, such as Aib (α-Aminoisobutyric acid, α-aminoisobutyric acid or 2-aminoisobutyric acid), whose structural formula is: And Iva (Isovaline), its structural formula is:

[0165] In this disclosure, the structural formula of the term "iminodiacetic acid (IDA)" is as follows:

[0166] In this disclosure, the two carboxyl groups (-COOH) of iminodiacetic acid react with the amino groups (-NH2) in the lysine (K, Lys) residues respectively, thereby forming a bridged ring connection between the two lysine residues with iminodiacetic acid, the structure of which is shown in formula (b):

[0167]

[0168] Wherein, R can be any group, preferably selected from ((AEEA)a-γGlu-CO((CH2)bCO2H), (PEG2-γGlu-CO(CH2)cCO2H) or (GG(SG)d-γGlu-CO(CH2)eCO2H), preferably, a is an integer selected from 1-6, b is an integer selected from 16-20, c is an integer selected from 16-20, d is an integer selected from 2-3, and e is an integer selected from 16-20.

[0169] In this disclosure, the term "γGlu" or "gammaGlu" refers to the γ-glutamyl group, a chemical group derived from glutamic acid, characterized by the γ-carboxyl group of glutamic acid participating in the formation of peptide bonds or other chemical bonds.

[0170] In this disclosure, the term "PEG2" refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid (CAS No. 134978-97-5).

[0171] In this disclosure, the term "free amino terminus" refers to the amino terminus (N-terminus) of a protein or polypeptide chain that is not linked to other amino acids by peptide bonds.

[0172] In this disclosure, the term "free carboxyl terminus" refers to the carboxyl terminus (C-terminus) of a protein or polypeptide chain that is not linked to other amino acids by peptide bonds.

[0173] In this disclosure, the term "amino acid" refers to a molecule containing both amino and carboxyl functional groups, wherein the amino and carboxyl groups of an α-amino acid are attached to the same carbon atom (α-carbon). The α-carbon may also have one or two additional organic substituents. Amino acids include L- and D isomers and racemic mixtures. Unless otherwise specified, the amino acid residues in the polypeptide sequences of this disclosure are all L-isomers, i.e., L-amino acids. This invention also covers polypeptide compounds that also contain one or more other non-conservative substitutions, provided that such non-conservative substitutions do not significantly affect the desired function and biological activity of the polypeptide compounds of this invention. Conservative amino acid substitutions may be performed at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be altered (deleted, substituted, or replaced) without changing their biological activity, while "essential" amino acid residues are required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Those skilled in the art can identify essential amino acids in the polypeptide compounds of this disclosure using methods known in the art, such as local mutagenesis or polypeptide compound evolution or bioinformatics analysis. The catalytic domains, active sites, or other functional domains of peptide compounds can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key amino acids. Amino acid substitutions can occur in non-conserved regions of amino acids within peptide compounds. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for the peptide compound's activity.

[0174] Those skilled in the art will understand that amino acids in peptide compounds can be altered without adversely affecting the activity and functionality of the peptide compound. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence without adversely affecting the activity and / or three-dimensional structure of the peptide compound. Examples and implementation methods of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar, uncharged amino acid residue can replace another polar, uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Amino acid residues containing basic side chains include lysine, arginine, and histidine; amino acid residues containing acidic side chains and amide side chains include aspartic acid, glutamic acid, asparagine, and glutamine; small aliphatic, nonpolar, or weakly polar side chain amino acid residues include glycine, alanine, threonine, serine, and proline; large aliphatic, nonpolar side chain amino acid residues include leucine, isoleucine, and valine; aromatic amino acid residues include phenylalanine, tryptophan, and tyrosine; and sulfur-containing side chain amino acid residues include cysteine ​​and methionine. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions where one amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided the substitution does not lead to the inactivation of the polypeptide compound's biological activity.

[0175] Those skilled in the art will understand that one or more amino acid residues in a polypeptide compound can be altered (replaced, deleted, truncated, or inserted) at the free amino terminus and / or free carboxyl terminus while retaining its functional activity. Therefore, polypeptide compounds that alter one or more amino acid residues at both ends while retaining their desired functional activity are also within the scope of this invention.

[0176] In this disclosure, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Salts of the polypeptide compounds can be formed, for example, by reacting the compound with a certain amount (e.g., an equal amount) of acid or base in a medium, such as a medium in which the salt precipitates or an aqueous medium (fly-drying after the reaction). Specific salts include those that, within a reliable medical judgment, are suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are proportionate to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0177] In this disclosure, the term "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0178] In this disclosure, the term "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0179] In this disclosure, the term "solvent" refers to the form of the polypeptide compound or its salt of the present invention, typically formed by a solvent decomposition reaction and bound to a solvent. This physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. "Solvent" includes solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates, and methanolates. When the solvent is water, the complex is referred to as a "hydrate". This disclosure covers all solvates of the polypeptide compounds or their salts of the present invention.

[0180] In this disclosure, the term "chimeric molecule" refers to a polypeptide compound of the present invention combined with one or more protein components. The protein component may be an epitope tag or a half-life extender. The half-life extender includes an Fc domain and serum albumin.

[0181] As used herein, the term "Fc domain" refers to at least a portion of the C-terminal region of an immunoglobulin heavy chain containing a constant region. It does not possess antigen-binding activity but contains a carbohydrate moiety and binding sites for complement and Fc receptors. Fc domains can originate from humans or other animals, including cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, specifically humans, but not limited to these. Fc domains can be Fc domains derived from human IgG, IgA, IgD, IgE, or IgM, or Fc domains prepared by combination or hybridization thereof. Preferably, they are derived from IgG or IgM. IgG is the most abundant antibody type in the human body, and based on structural differences in its heavy chain, it includes four subclasses: IgG1, IgG2, IgG3, and IgG4.

[0182] As used herein, the term "treatment" includes inhibiting, slowing, stopping, or reversing the progression or severity of existing symptoms or disease. Therefore, treatment includes prevention, treatment, and / or cure. The term "prevention" refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. The PEGylation can be achieved using several PEG linking moieties, including but not limited to hydroxysuccinimide active esters, succinimide propionates, maleimide, vinyl sulfones, or thiols. The PEG polymer can be linked to a peptide compound at a predetermined position or can be randomly linked to a peptide compound. PEGylation can also be mediated by a peptide linker linked to a peptide compound.

[0183] As used in this article, the term "patient" refers to mammals, such as humans.

[0184] As used herein, the term "effective amount" refers to an amount of active ingredient sufficient to significantly improve a condition without causing serious side effects, specifically including "therapeutic effective amounts" and "preventive effective amounts." A "therapeutic effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. A "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, for example, preventing or delaying the occurrence or recurrence of a disease or symptom, or reducing the likelihood of the occurrence or recurrence of a disease or symptom. A fully preventive effective amount does not necessarily occur through the administration of a single dose and can occur only after the administration of a series of doses. Therefore, a preventive effective amount can be administered in one or more applications.

[0185] When the polypeptide compound of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or the chimeric molecule of the second aspect, is administered, a safe and effective amount of the polypeptide compound of the first aspect, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or the chimeric molecule of the second aspect, is administered to a mammal (such as a human) requiring treatment, wherein the dose administered is a pharmaceutically considered effective dose. Of course, the specific dose should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0186] The polypeptide compound described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or the chimeric molecule described in the second aspect, is generally effective over a wide dose range. For example, it is administered once every 1-180 days, preferably every 1-60, 1-30, 1-15, or 1-7 days; the dose administered each time is about 1-500 nmol / kg, preferably 1-300 nmol / kg, preferably 1-150 nmol / kg, preferably 5-100 nmol / kg, preferably 10-50 nmol / kg.

[0187] This invention provides a pharmaceutical composition comprising the polypeptide compound described in the first aspect or its pharmaceutically acceptable salt or solvate thereof, or the chimeric molecule described in the second aspect; and further comprising a pharmaceutically acceptable carrier or excipient. Specifically, it includes carriers capable of reducing drug degradation and loss, and reducing side effects, such as micelles, microemulsions, gels, etc.; excipients refer to materials added to formulate the drug into a suitable dosage form, such as buffers, lyophilization excipients, etc., capable of preparing the polypeptide compound described in the first aspect or its pharmaceutically acceptable salt or solvate thereof, or the chimeric molecule described in the second aspect, into a solution or lyophilized powder for parenteral administration. The lyophilized powder can be reconstituted by adding an appropriate solvent or other pharmaceutically acceptable carrier before use. Liquid formulations are generally buffer solutions, isotonic solutions, and aqueous solutions. Buffer solutions can be phosphate buffers, isotonic solutions can be 0.9% sodium chloride solutions, and aqueous solutions are directly dissolved in purified water.

[0188] Those skilled in the art will understand that the polypeptide compounds or their pharmaceutically acceptable salts or solvates, chimeric molecules, or pharmaceutical compositions described in this invention are suitable for various routes of administration, including oral administration, injection administration, respiratory administration, skin administration, mucosal administration, or rectal administration; wherein, injection administration includes intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection, and mucosal administration includes oral mucosal administration, nasal mucosal administration, ocular mucosal administration, and vaginal mucosal administration. Depending on the route of administration employed, the polypeptide compounds or their pharmaceutically acceptable salts or solvates, chimeric molecules, or pharmaceutical compositions can be formulated into various suitable dosage forms, examples of suitable dosage forms being tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin application, aerosols, nasal sprays, and sterile solutions suitable for injection. The sterile solution for injection contains sterile diluents such as water, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol, methylparaben, phenol, or m-cresol; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, vials, disposable syringes, glass or plastic multi-dose vials, or injection pens.

[0189] The polypeptide compound or its pharmaceutically acceptable salt or solvate thereof described in the first aspect of this invention, the chimeric molecule or the aforementioned pharmaceutical composition described in the second aspect, can be administered alone or in combination with other known drugs for treating or improving similar symptoms, either simultaneously, separately or sequentially. These other drugs include metformin, thiazolidinediones (TZDs, such as pioglitazone), sulfonylureas (such as glimepiride), dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitors, such as sitagliptin), sodium-glucose cotransporter (SGLT-2 inhibitors, such as dapagliflozin), growth differentiation factor 15 (GDF15) regulators, peptide tyrosine tyrosine regulators (PYY), modified or unmodified insulin, and amylin (such as pramlintide).

[0190] Furthermore, the present invention also provides the following applications of the polypeptide compound described in the first aspect of the invention or its pharmaceutically acceptable salt or solvate, the chimeric molecule described in the second aspect, or the aforementioned pharmaceutical composition: prevention of weight rebound after successful weight loss; treatment of diseases or conditions associated with overweight or obesity; treatment of bulimia; treatment of binge eating; treatment of dyslipidemia, atherosclerosis, hypertension, coronary heart disease, and beta-blocker poisoning; non-alcoholic fatty liver disease (including simple fatty liver and its associated cirrhosis); and for inhibiting gastrointestinal motility, for use in conjunction with gastrointestinal surveys using techniques such as X-ray, CT, and NMR scanning.

[0191] Commonly used abbreviations are as follows:

[0192] Fmoc: Fluorenylmethoxycarbonyl, fluorenemethoxycarbonyl

[0193] Fmoc / t-Bu: Fluorenylmethoxycarbonyl / tert-Butyl, fluorenemethoxycarbonyl / tert-butyl

[0194] DMF: N,N-Dimethylformamide, N,N-Dimethylformamide

[0195] HOBt: 1-Hydroxybenzotriazole, 1-hydroxybenzotriazole

[0196] DIC: N,N'-Diisopropylcarbodiimide, N,N'-Diisopropylcarbodiimide

[0197] eq: Equivalent

[0198] DCM: Dichloromethane

[0199] Alloc: Allyloxycarbonyl, allyloxycarbonyl

[0200] OAll: Allyl ester

[0201] Dde: 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl PYBOP: Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

[0202] DIPEA: N,N-Diisopropylethylamine, N,N-Diisopropylethylamine

[0203] MeOH: Methanol

[0204] TFA: Trifluoroacetic acid

[0205] DODT: Dodecanethiol

[0206] CAN: Ceric ammonium nitrate (II) Detailed Technical Solution

[0207] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0208] Materials and Methods:

[0209] The following specific embodiments further illustrate the implementation of the present invention. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0210] Boc-Tyr(tBu)-OH and Fmoc-Aib-OH were purchased from Shanghai Jier. Eicosanoic acid monotert-butyl ester was prepared in-house. The remaining amino acids were purchased from Chengdu Zhengyuan Company, and the condensing agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were of analytical grade. Solvents were purchased from Shanghai Titan Company. Centrifuges were purchased from Lu Xiangyi. A 5.0 cm reversed-phase C18 preparative column (46 mm × 250 mm) was used to purify the peptides. The high-performance liquid chromatograph (HPLC) was a Thermo Fisher Scientific product. Mass spectrometry analysis was performed using a Waters mass spectrometer.

[0211] The experimental results of this invention were analyzed using GraphPad Prism software. Data are expressed as mean ± standard error (Mean ± SEM) and evaluated using t-tests. A statistically significant difference between the peptide compound group and the model group was indicated by *P < 0.05. Western blot results were analyzed using ImageJ image analysis software.

[0212] Example 1: Synthesis of polypeptide compounds

[0213] The polypeptide compounds described in this invention are synthesized using the Fmoc solid-phase polypeptide synthesis method, proceeding from the carboxyl terminus to the amino terminus, with the polypeptide moiety linked sequentially according to its amino acid sequence. For ease of explanation, the synthesis of polypeptide compound L04-NH2 is used as an example below.

[0214]

[0215] Formula (bⅣ), (polypeptide compound L04-NH2, SEQ ID NO.38),

[0216] The polypeptide compound L04-NH2 has the structure shown in formula bⅣ above, and its molecular formula is C 239 H 364 N 56 O 77 Its molecular weight is 5250.63, and its chemical structural formula is as follows:

[0217]

[0218] The synthesis method is as follows:

[0219] Step 1: Synthesize the main peptide resin

[0220] Following the Fmoc / t-Bu strategy, the following main peptide resins were synthesized at a synthetic scale of 0.5 mmol: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-Lys(Alloc)-Ala-Gln(Trt)-Lys(Dde)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu- Glu(OtBu)-Gly -Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM resin.

[0221] 1.1 Weigh 0.89 g of Rink Amide AM resin (loading capacity 0.56 mmol / g, Xi'an Lanxiao), add it to the reaction column, and swell it in 15 mL of DMF for 30 min. After removing the DMF by filtration, wash the resin thoroughly twice with DMF. Then add 1.587 g (6 eq) of Fmoc-Ser(tBu)-OH and 0.672 g (7.2 eq) of HOBt. Add an appropriate amount of DMF, stir evenly under nitrogen, add 0.83 mL (7.8 eq) of DIC, and react for 2 h. Filter to remove the reaction solution, wash three times with DMF, add acetic anhydride / pyridine (7:6, v / v) for blocking for 4 h, filter to remove the blocking solution, and wash six times with DMF to obtain Fmoc-Ser(tBu)-Rink Amide AM resin.

[0222] 1.2 Using the synthetic product from step 1.1 as a carrier, HOBt and DIC as coupling agents, and DMF as a solvent, the Fmoc group was removed using a 20% Piperidine / DMF solution (twice, for 5 min and 7 min). The coupling effect was monitored using ninhydrin (2,2-Dihydroxyindane-1,3-dione) during the coupling process.

[0223] Carry out the condensation reaction from C-terminal to N-terminal: Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly -OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Gln( Trt)-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Iva-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)- OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH. In the above synthesis reaction, the amount of amino acid fed is 5 equivalents (5 eq) of the target product. The above synthesis reaction yields: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-Lys(Alloc)-Ala-Gln(Trt)-Lys(Dde)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM resin.

[0224] 1.3 To remove Dde, 20 mL of 2% hydrazine hydrate DMF solution was added to the synthetic product from step 1.2. The mixture was stirred under nitrogen atmosphere for 3 min. After the reaction, the reaction solution was removed by filtration. This process was repeated 3 times. Then, the reaction solution was removed by filtration and washed 6 times with DMF. A sample was taken for monitoring with ninhydrin hydrate. A positive test indicates the successful preparation of the main chain peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-Lys(Alloc)-Ala-Gln(Trt)-Lys-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu- Glu(OtBu)-Gly -Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM resin.

[0225] Step 2, Couple the "Modifier Part" Structure:

[0226] Coupling of Fmoc-Ida(OAll)-OH: Add Fmoc-Ida(OAll)-OH (5 eq, 0.988 g), HOBt (6 eq, 0.405 g), and an appropriate amount of DMF to the synthetic product from step 1 above. Stir under nitrogen until homogeneous, then add DIC (6.5 eq, 0.51 mL) and stir under nitrogen for 2 hours. The coupling effect is tested by ninhydrin hydrate; the reaction ends when the solution is colorless and transparent. Then, filter to remove the reaction solution, wash three times with DMF, and remove the Fmoc group twice with a 20% piperidine / DMF solution (5 min and 7 min). After Fmoc removal, wash six times with DMF. The sample is then tested by ninhydrin hydrate; a positive result indicates the next coupling step is desired.

[0227] Repeating the above operation, sequentially coupling Fmoc-Gly-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu-OtBu, and tert-butyl eicosanoate, yields: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)- Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-Lys(Alloc) -Ala-Gln(Trt)-Lys(Ida(OAll)-Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH2) 18 CO2-tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu- Glu (OtB u)-Gly- Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink AmideAM resin was used. The synthesized product was washed three times with DMF and three times with DCM before use.

[0228] Step 3, forming a ring:

[0229] 3.1: De-Alloc and OAll

[0230] Add 0.5 mL (12 eq) of morpholine to the synthetic product from step 2, using DCM as solvent, and stir under nitrogen atmosphere until homogeneous. Weigh 0.173 g of Pd(PPh3)4 (0.3 eq) and add it to the reaction column. Rinse the column with DCM, stir under nitrogen atmosphere, and react for 1 h. After the reaction is complete, filter to remove the reaction solution and wash 6 times with DCM. The sample was monitored with ninhydrin hydrate. A positive test result indicates the formation of the peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-Lys-Ala-Gln(Trt)-Lys(Ida-Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH2) 18 CO2-tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu- Glu(OtBu)-Gly -Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM resin.

[0231] 3.2: Circulation

[0232] Add coupling agents to the synthetic product from step 3.1. Weigh out PYBOP (5 eq, 1.3 g) and HOBT (6 eq, 0.405 g) and add them to the reaction column. Use DMF as solvent, stir under nitrogen atmosphere until homogeneous, add DIPEA (7.5 eq, 0.64 mL), and react for 2 h. Monitor the coupling effect with ninhydrin during the cyclization process. A negative test indicates complete reaction; a positive test allows for delaying the reaction time or changing the coupling system until cyclization is complete.

[0233] After the reaction was complete, the resin was washed three times with DMF, three times with DCM, and shrank twice with MeOH. Vacuum drying yielded 3.2 g of fully protected peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys(Boc)-C[Lys-Ala-Gln(Trt)-Lys](Ida-Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu(OtBu)-CO(CH2) 18 CO2-tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu- Glu(OtBu)-Gly -Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM resin.

[0234] Step 4: Remove fully protecting groups from peptide resin (cleavage)

[0235] TFA, DODT, m-cresol, and H2O were prepared in a volume ratio of 92.5:2.5:2.5:2.5 and frozen for 2 hours before use. Then, lysis buffer was added to the dried product prepared in step 3 at a ratio of 10 mL per gram of the product synthesized in step 3, and the mixture was heated to room temperature and allowed to undergo lysis reaction for 3 hours.

[0236] After lysis, the filtrate was collected by filtration and washed three times with a small amount of lysis buffer. All filtrates were combined and concentrated under reduced pressure to about 1 / 4 of the original volume. Then, the filtrate was slowly poured into ice-cold methyl tert-butyl ether with stirring, and the residue in the flask was washed into the methyl tert-butyl ether with a small amount of lysis buffer. The mixture was allowed to stand for more than 2 hours until precipitation was complete. The supernatant was removed, the precipitate was centrifuged, and washed three times with methyl tert-butyl ether. The solid obtained by centrifugation was dried under nitrogen to obtain the crude product, which weighed 1.26 g.

[0237] Step 5: Purification of crude compound

[0238] The crude product obtained in step 4 was dissolved in a solution of CAN:H2O = 1:2 (v / v), and passed through a 5.0 cm reverse-phase flow meter. 18Preparative HPLC purification was performed on a packed 46 mm x 250 mm column. Starting with 45% ACN / H₂O (containing 1% trifluoroacetic acid), the column was eluted for 60 minutes at a gradient elution rate (increasing the ACN proportion at a rate of 0.33% / min) and a flow rate of 10 mL / min. The fraction containing the peptide was collected, yielding a sample with an HPLC purity greater than 90%. The HPLC purification was repeated once, starting with a 31% ACN / 20 mM NaH₂PO₄ aqueous solution adjusted to pH 6.5 with 1 M NaOH solution. Gradient elution was performed, increasing the ACN proportion at a rate of 0.33% / min and a flow rate of 10 mL / min. After elution for 60 minutes, the fraction containing the peptide was collected, freeze-dried, and 200 mg of peptide was obtained with a purity greater than 98.14%, and an overall yield of 15.8%.

[0239] Step 6: Product Confirmation

[0240] The isolated peptide was identified by liquid chromatography-mass spectrometry (LC-MS) and determined to be a polypeptide compound L04-NH2, [M+H]. + The calculated value is 5250.63, [M+3H] + The measured value is 1751.21.

[0241] In addition, the following polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L05-NH2, L06-NH2, L07-NH2, L08-NH2, L09-NH2, and L10-NH2 were synthesized according to the above method for subsequent experimental verification:

[0242]

[0243] Formula (bⅠ), (polypeptide compound L01-NH2, SEQ ID NO.35),

[0244]

[0245] Formula (bⅡ), (polypeptide compound L02-NH2, SEQ ID NO.36),

[0246]

[0247] Formula (bⅢ), (polypeptide compound L03-NH2, SEQ ID NO.37),

[0248]

[0249] Formula (bⅤ), (peptide compound L05-NH2, SEQ ID NO.39),

[0250]

[0251] Formula (bⅥ), (peptide compound L06-NH2, SEQ ID NO.40),

[0252]

[0253] Formula (bⅦ), (peptide compound L07-NH2, SEQ ID NO.41),

[0254]

[0255] Formula (bⅧ), (peptide compound L08-NH2, SEQ ID NO.42)

[0256]

[0257] Formula (bIX), (peptide compound L09-NH2, SEQ ID NO.43),

[0258]

[0259] Formula (bX), (polypeptide compound L10-NH2, SEQ ID NO.44).

[0260] The results of the liquid chromatography-mass spectrometry (LC-MS) identification of L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, L06-NH2, L07-NH2, L08-NH2, L09-NH2, and L10-NH2 are shown in Table 1.

[0261] Table 1. Amino acid sequence listing and LC-MS / MS identification results of compounds L01-NH2 to L10-NH2

[0262]

[0263]

[0264] Example 2: Agonistaltic activity of peptide compounds on GLP-1R, GIP, and GCGR receptors

[0265] 1. Test drug

[0266] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, L06-NH2, L07-NH2, L08-NH2, L09-NH2, L10-NH2 and the positive control drug: Tirzepatide (purchased from Hangzhou Gutuo Biotechnology Co., Ltd., catalog number: GT-L007).

[0267] 2. Test cell lines

[0268] HEK-293 cell lines that stably express human GLP-1R, GCGR, or GIPR.

[0269] 3. Experimental Methods

[0270] In cell models overexpressing GIPR, GCGR, and GLP-1R respectively (cell lines constructed in our laboratory), the agonistic activities of the test drug for GIPR, GCGR, and GLP-1R were measured. For the GCGR agonistic assay, natural GCG was used as the positive control, and for the GIPR and GLP-1R agonistic assays, telpolide was used as the positive control.

[0271] After intracellular GIPR, GCGR, and GLP-1R are activated, the second messenger cAMP is produced. In this experiment, the Cisbio cAMP kit was used to detect cAMP and calculate the EC50 value of agonistic activity.

[0272] First, a cAMP standard curve was constructed. cAMP standards were prepared using Stimulation Buffer 1 containing 0.5 mM IBMX and 0.1% casein, starting at 712 nM and serially diluted 4-fold to establish 10 concentration points. Then, the serially diluted cAMP standards were added to reaction plates (GRE384sw), 5 μL per well (the same volume of Stimulation Buffer 1 was added to the negative control). cAMP Eu Cryptate antibody and cAMP-d2 antibody working solutions were prepared using lysis and detection buffer, with 5 μL added to each well (the same volume of lysis and detection buffer was used instead of cAMP-d2 antibody working solution for the negative control). The plates were sealed and incubated at room temperature in the dark for 1 hour. The fluorescence emission at two wavelengths, 665 nm and 620 nm, is read on the microplate reader, and the ratio of the emission at 665 nm to that at 620 nm is calculated. The cAMP standard curve can then be plotted using the concentration and the ratio.

[0273] Next, the agonistic activity of the compounds was detected. Cells overexpressing GIPR, GCGR, or GLP-1R were thawed, cultured, digested, and seeded in 384-well plates. A 2x series of test drug concentrations were prepared using Stimulation Buffer 1 containing 0.5 mM IBMX and 0.1% casein, starting at 400 nM and serially diluted 5-fold, for a total of 9 concentrations. 5 μl of the test drug was added to each well. For the negative control, the same volume of Stimulation Buffer 1 containing 0.5 mM IBMX and 0.1% casein was added. The plates were sealed and incubated at room temperature for 30 min. Simultaneously, cAMP EuCryptate antibody and cAMP-d2 antibody working solutions were prepared using lysis buffer and detection buffer. After incubation, 5 μl of the antibody working solution was added to each well (for the negative control, the same volume of lysis and detection buffer was used instead of the cAMP-d2 antibody working solution). The plates were sealed and incubated at room temperature in the dark for 1 h.

[0274] Finally, in terms of compatibility Fluorescence emission at two wavelengths, 665 nm and 620 nm, was read on an ELISA reader, and the ratio of emission at 665 nm to that at 620 nm was calculated. The actual cAMP level (nM) of each sample well was calculated using the cAMP standard curve. Then, the percentage of activation at different concentrations was calculated using the formula %Activity = (cAMP level of testing sample - average cAMP level of low control) / (average cAMP level of high control - average cAMP level of low control) * 100%. The EC50 value was calculated by fitting the model using the "log(agonist) vs. response - Variable slope" model in GraphPad Prism 7.0.

[0275] 4. Experimental Results

[0276] The results are shown in Table 2. The EC50 values ​​of the three replicates of the positive control compounds for GLP-1R, GCGR, or GIPR were all on the same order of magnitude, indicating that the experimental system was stable and reliable. Peptide compounds L01-NH2 to L08-NH2 exhibited strong agonistic activity against GLP-1R and GIPR, effectively lowering blood glucose upon activation; they had relatively weak activity against GCGR, but showed a suitable glycemic effect upon activation. This combination of different agonistic activities among the peptide compounds is a key factor in maintaining blood glucose homeostasis. Among them, peptide compound L04-NH2 showed the best agonistic activity against all three receptors.

[0277] Peptide compounds L06-NH2, L07-NH2, and L08-NH2 have similar structures and target agonist activities to peptide compound L04-NH2. Therefore, their activity evaluation will be conducted using peptide compound L04-NH2 as a representative.

[0278] Table 2. EC50 values ​​(in nM) of peptide compounds against human GLP-1 receptor, GCG receptor and GIP receptor.

[0279]

[0280]

[0281] Example 3: Glucose tolerance test after a single dose of a polypeptide compound

[0282] 1. Test drug

[0283] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, and control polypeptide compound 21# (compound 21 described in patent 202480001470.1, whose structure is Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)). 18 CO2H)AFVQWLLEGGPSSGAPPPS-NH2, as shown in SEQ ID NO.45) and the positive control drug semaglutide (Bide Pharmaceuticals, BD00800278).

[0284] 2. Test animals

[0285] Eight-week-old male C57BL / 6J mice.

[0286] 3. Experimental Methods

[0287] After 7 days of acclimatization, male C57BL / 6J mice were randomly divided into 8 groups (L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, semaglutide group, 21# group, and PBS control group) based on similar blood glucose levels (assessed from blood samples taken from the tail tip).

[0288] Mice in each group were subcutaneously injected with either the test drug or PBS at a dose of 50 nmol / kg. Mice were fasted overnight at 17:30 daily, and 16 hours later, they were administered 0.4 g / ml glucose by gavage at a dose of 5 μL / g. Blood glucose levels were measured at 24h, 72h, 144h, 240h, and 288h after oral glucose administration, at 0min, 15min, 30min, 60min, 90min, and 120min. Blood glucose levels were recorded, and time-glucose curves were plotted. The area under the curve (AUC) for blood glucose changes at different time points was calculated for each mouse in each group and compared with the untreated PBS control group.

[0289] 4. Experimental Results

[0290] like Figure 1 As shown, at 24 hours post-administration, the test drugs significantly improved glucose tolerance and lowered blood glucose levels compared to PBS. At 72 and 144 hours post-administration, peptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, and the control peptide compound 21# still exhibited good efficacy, while semaglutide had lost its hypoglycemic effect. At 240 hours post-administration, peptide compounds L01-NH2, L02-NH2, L05-NH2, and L05-NH2, as well as the control peptide compound 21#, still showed good efficacy. 3-NH2, L04-NH2, and L05-NH2 maintained good efficacy and significantly improved glucose tolerance in mice compared to PBS, while the control peptide compound 21# lost its hypoglycemic effect. 288 hours after administration, compared to PBS, the glucose clearance of peptide compounds L01-NH2, L02-NH2, L03-NH2, and L05-NH2 had returned to normal levels, especially in the L04-NH2 group, where glucose levels still showed a decreasing trend.

[0291] In other words, although the tested drugs all have good effects in improving glucose tolerance, semaglutide cannot exert a long-term hypoglycemic effect; while the polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, and L05-NH2 of the present invention have significant advantages in terms of efficacy and duration of efficacy (long-term effect), especially polypeptide compound L04-NH2, which has the best long-term hypoglycemic effect.

[0292] Example 4: Effect of a single dose of a polypeptide compound on the control of random blood glucose in diabetic mice.

[0293] 1. Test drug

[0294] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2 and the positive control drugs: semaglutide and telpolide.

[0295] 2. Test animals

[0296] 6-7 week old ob / ob mice.

[0297] 3. Experimental Methods

[0298] ob / ob mice were randomly divided into 8 groups of 10 mice each, based on similar blood glucose levels (assessed from blood samples taken from the tail tip). Each group of mice was subcutaneously injected with the corresponding compound. The experimental groups are shown in Table 3.

[0299] Table 3. Grouping of single-dose experiments in ob / ob mice

[0300] Group Animal numbers compound Dosage (nmol / kg) route of administration PBS control group 10 PBS - subcutaneous injection Semaglutide group 10 Semaglutide 100 subcutaneous injection Telpo peptide group 10 Telpoeptide 100 subcutaneous injection <![CDATA[L01-NH2 group]]> 10 <![CDATA[Polypeptide compound L01-NH2]]> 100 subcutaneous injection <![CDATA[L02-NH2 group]]> 10 <![CDATA[Polypeptide compound L02-NH2]]> 100 subcutaneous injection <![CDATA[L03-NH2 group]]> 10 <![CDATA[Polypeptide compound L03-NH2]]> 100 subcutaneous injection <![CDATA[L04-NH2 group]]> 10 <![CDATA[Polypeptide compound L04-NH2]]> 100 subcutaneous injection <![CDATA[L05-NH2 group]]> 10 <![CDATA[Polypeptide compound L05-NH2]]> 100 subcutaneous injection

[0301] Random blood glucose levels in mice were measured and recorded at 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after drug administration.

[0302] 4. Experimental Results

[0303] like Figure 2 As shown, compared with the PBS control group, semaglutide had a significant hypoglycemic effect on day 1, but after 24 hours, the blood glucose level of mice in the semaglutide group rose significantly, and after 72 hours, there was no significant difference compared with the PBS control group. Overall, the hypoglycemic effect was poor and the long-term effect was insufficient.

[0304] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, and L05-NH2 of the present invention maintained a significant hypoglycemic effect for 168 hours, and after 72 hours, they began to show a hypoglycemic advantage compared to telpoide, proving that the polypeptide compounds of the present invention have better long-lasting effects, among which polypeptide compound L04-NH2 is superior.

[0305] Example 5: Therapeutic effect of repeated administration of polypeptide compounds on diabetic mice

[0306] 1. Test drug

[0307] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2 and the positive control drug: telpolide.

[0308] 2. Test animals

[0309] 10-week-old ob / ob mice.

[0310] 3. Experimental Methods

[0311] ob / ob mice were randomly divided into 7 groups of 8 mice each, based on similar blood glucose levels (assessed from blood samples taken from the tail tip). Each group of mice was administered the corresponding compound subcutaneously on days 0, 6, 12, and 18, with the experimental groups shown in Table 4.

[0312] Table 4. Grouping of ob / ob mice in multiple-dose experiments

[0313] Group Animal numbers compound Dosage (nmol / kg) route of administration PBS control group 8 PBS - subcutaneous injection Telpo peptide group 8 Telpoeptide 100 subcutaneous injection <![CDATA[L01-NH2 group]]> 8 <![CDATA[Polypeptide compound L01-NH2]]> 100 subcutaneous injection <![CDATA[L02-NH2 group]]> 8 <![CDATA[Polypeptide compound L02-NH2]]> 100 subcutaneous injection <![CDATA[L03-NH2 group]]> 8 <![CDATA[Polypeptide compound L03-NH2]]> 100 subcutaneous injection <![CDATA[L04-NH2 group]]> 8 <![CDATA[Polypeptide compound L04-NH2]]> 100 subcutaneous injection <![CDATA[L05-NH2 group]]> 8 <![CDATA[Polypeptide compound L05-NH2]]> 100 subcutaneous injection

[0314] After the first administration, blood glucose levels in mice were measured and recorded every 3 days. On day 24 post-treatment, blood was collected to measure glycated hemoglobin (HbA1c), and insulin resistance-related indicators and serum triglyceride (TG) levels were also measured. The examination methods are as follows:

[0315] Fasting blood glucose (FBG): Fasting for 12 hours before measurement, blood is drawn from the tail vein the next morning, and FPG is measured using a blood glucose meter and test strips.

[0316] Fasting insulin (FINS): Fasting for 12 hours before measurement, blood was collected from the tail vein the next morning, centrifuged at 3000 r / min for 15 min to collect serum, and FINS was measured using an ELISA kit.

[0317] Calculate the insulin resistance index (HOMA-IR) of the steady-state model: HOMA-IR = FPG × FINS / 22.5.

[0318] Glycated hemoglobin (HbA1c): At the end of the experiment, blood was collected by pricking the tail vein of mice, and HbA1c (%) was detected using a glycated hemoglobin detection kit.

[0319] Triglycerides (TG): At the end of the experiment, blood was collected by puncturing the tail vein of mice, and serum TG levels were detected using a kit.

[0320] 4. Experimental Results

[0321] (I) Effects of repeated administration of polypeptide compounds on random blood glucose in ob / ob mice

[0322] like Figure 3 As shown, compared with the PBS control group, treatment with telpolide at a dose of 100 nmol / kg via subcutaneous injection once every 6 days had a hypoglycemic effect. However, the blood glucose control effect was poor on the 6th day, and the blood glucose fluctuation was large, indicating that telpolide has insufficient long-term effect and cannot control blood glucose stably in the long term.

[0323] Compared with telpolide, at the same dosage, the peptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, and L05-NH2 showed significant overall hypoglycemic effects, achieving a good hypoglycemic effect by day 3. They maintained a significant hypoglycemic effect throughout the experiment and were able to keep the random blood glucose of mice at a stable level with minimal fluctuations.

[0324] The above results demonstrate that peptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, and L05-NH2 have good effects in lowering blood glucose and maintaining blood glucose stability, with peptide compound L04-NH2 showing the best effect.

[0325] (II) Effects of repeated administration of peptide compounds on HOMA-IR, HbA1c and TG in ob / ob mice

[0326] like Figure 4 As shown in A, compared with the PBS control group, the polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, and L05-NH2 of the present invention, after multiple administrations to ob / ob mice, can significantly reduce HOMA-IR values ​​and have obvious insulin sensitizing effects.

[0327] Meanwhile, the polypeptide compounds L02-NH2, L03-NH2, L04-NH2, and L05-NH2 of the present invention all exhibited a significant inhibitory effect on the increase of HbA1c levels. Figure 4 B) indicates that it has a good glycemic control effect. Among them, the polypeptide compound L04-NH2 has a significantly stronger effect on HOMA-IR and HbA1c in ob / ob mice than the positive control drug telpolide, and has the best effect.

[0328] Figure 4 Results showed that, compared with the PBS control group, the polypeptide compounds L02-NH2 and L04-NH2 of the present invention significantly reduced the TG levels in ob / ob mice after multiple administrations, demonstrating excellent lipid control effects, and were significantly stronger than the positive control drug telpolide.

[0329] Example 6: Effect of repeated administration of peptide compounds on weight loss in diet-induced obese mice.

[0330] 1. Test drug

[0331] The polypeptide compounds L04-NH2 and L05-NH2 and the positive control drug telpolide.

[0332] 2. Test animals

[0333] Ten-week-old male C57BL / 6J mice.

[0334] 3. Experimental Methods

[0335] Ten-week-old C57BL / 6J mice were acclimatized for one week. Mice in the normal diet group were fed standard feed and deionized water, while mice in the model group were fed a high-fat diet (60% HFD) to induce a high-fat diet model. After 12 weeks of continuous feeding, the model group mice were randomly divided into four groups: L04-NH2 group, L05-NH2 group, telpolide group, and PBS control group. Eight mice were in each group. Administered the drug once every 6 days at a dose of 30 nmol / kg. Samples were collected on day 21. Mouse weight was recorded daily, and total body fat was collected and weighed at the experimental endpoint.

[0336] 4. Experimental Results

[0337] (I) Effects of polypeptide compounds on body weight in obese mice

[0338] like Figure 5 As shown, after induction of the model with a high-fat diet, the body weight of the model group was significantly higher than that of the normal diet group. During the treatment period, at the same dose and frequency, peptide compounds L04-NH2, L05-NH2, and telpolide all significantly reduced the body weight of obese mice. However, telpolide showed a regular fluctuation in the body weight of obese mice in each treatment cycle, with a plateau phase two days after each administration. In contrast, peptide compounds L04-NH2 and L05-NH2 maintained a continuous downward trend in the body weight of obese mice without a plateau phase.

[0339] In summary, telpolide did not have a long-lasting effect on weight loss in obese mice, and the weight fluctuated significantly during treatment. In contrast, the peptide compounds L04-NH2 and L05-NH2 showed better weight loss effects than telpolide and supported long-term administration. Among them, peptide compound L04-NH2 had the best long-term weight loss effect.

[0340] (II) Effects of polypeptide compounds on overall fat weight in obese mice

[0341] like Figure 6 As shown, compared with the normal diet group, the model group mice had a significantly increased total fat weight. Compared with PBS, peptide compounds L04-NH2, L05-NH2 and telpoeptide could reduce the total fat weight of obese mice to varying degrees, and had a certain therapeutic effect on obese mice. Moreover, peptide compounds L04-NH2 and L05-NH2 were significantly better than telpoeptide in reducing the total fat weight of obese mice, with peptide compound L04-NH2 having the best effect.

[0342] Example 7: PK pharmacokinetic characteristics of a single subcutaneous injection of a polypeptide compound

[0343] 1. Test drug

[0344] The polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, L06-NH2, L07-NH2, L08-NH2 and the positive control drug telpolide.

[0345] 2. Test animals

[0346] 8-week-old male SD rats.

[0347] 3. Experimental Methods

[0348] Thirty-six well-fed SD rats were randomly divided into nine groups of four (half male and half female) according to their body weight. The test drug was administered as a single subcutaneous injection to each rat at a dose of 315 μg / kg. Blood samples were collected using EDTA-K2 anticoagulant tubes at the following time points: before administration (0 h) and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, and 240 h after administration. The separated plasma was centrifuged at 1800 g for 10 min at 4 °C and stored at -80 °C.

[0349] A method for analyzing the concentration of compounds in the plasma of SD rats was established using UPLC-MS / MS to determine the drug concentration of the compounds in the plasma. Data processing was performed using WinNonlin 8.1 software to calculate pharmacokinetic parameters.

[0350] 4. Experimental Results

[0351] As shown in Table 5, compared with telpolide, the peptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, L06-NH2, L07-NH2, and L08-NH2, administered subcutaneously once, were absorbed more slowly in rats, with shorter time to peak concentration (T0). max The average half-life (T0) is approximately 24 hours, significantly longer than the 8 hours of telpolide; and the half-lives (T0) of the polypeptide compounds L01-NH2, L02-NH2, L03-NH2, L04-NH2, L05-NH2, L06-NH2, L07-NH2, and L08-NH2 are also significantly longer. 1 / 2 The half-life of the polypeptide compound L04-NH2 of this invention is significantly higher than that of telpolide (10.8h), which is longer than that of telpolide.

[0352] Table 5. Results of in vivo pharmacokinetic experiments in SD rats

[0353] Grouping compound Half-life (h) Peak arrival time (h) Telpo peptide group Telpoeptide 10.8±1.2 8.0±0.68 <![CDATA[L01-NH2 group]]> <![CDATA[Polypeptide compound L01-NH2]]> 12.8±1.8 24.0±1.4 <![CDATA[L02-NH2 group]]> <![CDATA[Polypeptide compound L02-NH2]]> 18.8±1.9 24.0±0.7 <![CDATA[L03-NH2 group]]> <![CDATA[Polypeptide compound L03-NH2]]> 22.3±1.5 24.0±1.9 <![CDATA[L04-NH2 group]]> <![CDATA[Polypeptide compound L04-NH2]]> 26.7±2.3 24.0±1.7 <![CDATA[L05-NH2 group]]> <![CDATA[Polypeptide compound L05-NH2]]> 19.9±1.8 24.0±1.2 <![CDATA[L07-NH2 group]]> <![CDATA[Polypeptide compound L06-NH2]]> 26.3±1.6 24.0±1.9 <![CDATA[L08-NH2 group]]> <![CDATA[Polypeptide compound L07-NH2]]> 27.1±2.0 24.0±1.7 <![CDATA[L09-NH2 group]]> <![CDATA[Polypeptide compound L08-NH2]]> 27.7±1.6 24.0±2.0

Claims

1. A polypeptide compound having agonistic activity against GLP-1R, GIPR, and GCGR, or a pharmaceutically acceptable salt thereof or a solvate thereof, said polypeptide compound having a polypeptide moiety and a modification moiety, said polypeptide moiety comprising the sequence Y-Aib-EGT-X6-TSD-X 10 -SI-Iva-LDKKAQKAF-X 23 -X 24 -WLLEGGPSSGAPPPS (SEQ ID NO.1), the modified portion comprising a bridged ring link formed between lysine at position 17 and lysine at position 20.

2. The polypeptide compound of claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the bridged ring link has the structure shown in formula (b): in The positions shown are the attachment sites to the amino groups on the lysine residues at positions 17 and 20 of the polypeptide moiety. in, R is selected from (AEEA) a -γGlu-CO(CH2) b CO2H, (PEG2) c -γGlu-CO(CH2) d CO2H or G e (SG) f -γGlu-CO(CH2) g CO2H; Where a is an integer selected from 1 to 6, b is an integer selected from 12 to 20, c is an integer selected from 1 to 6, d is an integer selected from 12 to 20, e is an integer selected from 0 to 5, f is an integer selected from 1 to 6, and g is an integer selected from 12 to 20. Preferably, e is 2; Preferably, f is 2 or 3; Preferably, g is 16, 18, or 20; Preferably, the structure of the polypeptide compound is shown in formula (a): (SEQ ID NO.26), More preferably, R is selected from any of the following: 1)GG(SG)2-γGlu-CO(CH2) 16 CO2H, 2)GG(SG)2-γGlu-CO(CH2) 18 CO2H, 3)GG(SG)2-γGlu-CO(CH2) 20 CO2H, 4)GG(SG)3-γGlu-CO(CH2) 16 CO2H, 5)GG(SG)3-γGlu-CO(CH2) 18 CO2H, 6)GG(SG)3-γGlu-CO(CH2) 20 CO2H。 3. The polypeptide compound as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, X6, X 10 X 23 or X 24 Each amino acid is independently selected from genetically encoded natural amino acids, non-genetically encoded natural amino acids, and synthetic amino acids. Preferably, the natural amino acids encoded by the gene include alanine, valine, glycine, leucine, glutamine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, methionine, serine, threonine, cysteine, proline, isoleucine, histidine, and arginine. Preferably, the non-gene-encoded natural amino acids include cystine, hydroxyproline, γ-carboxyglutamic acid, ornithine, and phosphoserine; Preferably, the synthetic amino acids include D-isomers of genetically encoded natural amino acids, Aib, Abu, Tle, Iva, Cba, and Tic. Preferably, X6 is selected from W or F; Preferably, X 10 Selected from S, L, or Y; Preferably, X 23 Selected from V or T; Preferably, X 24 Selected from N or Q; Preferably, X10 is L and X24 is N, or X10 is Y and X24 is Q.

4. The polypeptide compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof or a solvate thereof, wherein the sequence of the polypeptide moiety differs from any one of the sequences shown in SEQ ID NO. 2-25 by at most 1, at most 2, at most 3, at most 4 or at most 5 amino acids, or the sequence of the polypeptide moiety comprises or is any one of the sequences shown in SEQ ID NO. 2-25; Preferably, X 10 Let L and X be the numbers. 24 For N, the sequence of the polypeptide moiety is as shown in SEQ ID NO. 6, 8, 18 or 20; Preferably, X 10 Let Y and X be the numbers. 24 For Q, the sequence of the polypeptide moiety is as shown in SEQ ID NO. 11, 13, 23 or 25; Preferably, X6 is F, X 10 Let Y and X be the numbers. 23 Let V, X 24 For Q, the sequence of the polypeptide portion is shown in SEQ ID NO.

23.

5. The polypeptide compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof or a solvate thereof, wherein the polypeptide compound is selected from any one of the following: 1) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is shown in formula (a) below, wherein, R is GG(SG)2-γGlu-CO(CH2) 16 CO2H, (SEQ ID NO.26), 2) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is shown in formula (a), wherein R is GG(SG)2-γGlu-CO(CH2). 18 CO2H, (SEQ ID NO.26), 3) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is as shown in the aforementioned formula (a), wherein R is GG(SG)2-γGlu-CO(CH2). 20 CO2H, SEQ ID NO.26), 4) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is shown in formula (a), wherein R is GG(SG)3-γGlu-CO(CH2). 16 CO2H, SEQ ID NO.26), 5) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is shown in formula (a), wherein R is GG(SG)3-γGlu-CO(CH2). 18 CO2H, SEQ ID NO.26), 6) The polypeptide compound has a polypeptide moiety and a modification moiety, the sequence of the polypeptide moiety includes or is as shown in any one of SEQ ID NO. 2-25, and the structure of the polypeptide compound is shown in formula (a), wherein R is GG(SG)3-γGlu-CO(CH2). 20 CO2H; SEQ ID NO.26), Preferably, the polypeptide compound is selected from any one of the polypeptide compounds represented by formula (aⅠ), (aⅡ), (aⅢ), (aⅣ), (aⅤ), (aⅥ), (aⅦ), or (aⅧ): (Polypeptide compound L01, SEQ ID NO.27) (Polypeptide compound L02, SEQ ID NO.28) (Polypeptide compound L03, SEQ ID NO.29) (Polypeptide compound L04, SEQ ID NO.30) (Polypeptide compound L05, SEQ ID NO.31) (Polypeptide compound L06, SEQ ID NO.32) (Polypeptide compound L07, SEQ ID NO.33) (Polypeptide compound L08, SEQ ID NO.34).

6. The polypeptide compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof or a solvate thereof, wherein the polypeptide compound has a terminal modification comprising attaching an N-terminal protecting group to the free amino terminus of an amino acid and / or attaching a C-terminal protecting group to the free carboxyl terminus of an amino acid; Preferably, the N-terminal protecting group includes -CO-R1, alkoxycarbonyl, or -CO-O-R1, wherein, R1 is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group, or a substituted aromatic group. Preferably, the C-terminal protecting group includes -NH2, -NHCH3, -N(CH3)2, -NH(ethyl), -N(ethyl)2, -N(methyl)(ethyl), -NH(benzyl), -N(C1-C4 alkyl)(benzyl), -NH(phenyl), -N(C1-C4 alkyl)(phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(isopropyl), O-(sec-butyl), -O-(tert-butyl), -O-benzyl, and -O-phenyl; Preferably, -NH2 is attached to the free carboxyl terminus of the 39th S position of the polypeptide compound; Preferably, the polypeptide compound is selected from any one of the following: Preferably, the polypeptide compound is selected from any one of the polypeptide compounds represented by formula (bⅠ), (bⅡ), (bⅢ), (bⅣ), (bⅤ), (bⅥ), (bⅦ), or (bⅧ): (Polypeptide compound L01-NH2, SEQ ID NO.35) (Polypeptide compound L02-NH2, SEQ ID NO.36) (Polypeptide compound L03-NH2, SEQ ID NO.37) (Polypeptide compound L04-NH2, SEQ ID NO.38) (Polypeptide compound L05-NH2, SEQ ID NO.39) (Polypeptide compound L06-NH2, SEQ ID NO.40) (Polypeptide compound L07-NH2, SEQ ID NO.41) (Polypeptide compound L08-NH2, SEQ ID NO.42); Preferably, the pharmaceutically acceptable salt includes its acetate, hydrochloride, phosphate, or acetic acid salt.

7. A chimeric molecule comprising the polypeptide compound and other proteins according to any one of claims 1-6; Preferably, the other proteins include epitope tags or half-life extenders; Preferably, the half-life extender comprises an Fc domain and serum albumin; Preferably, the other proteins are linked to the free amino terminus and / or free carboxyl terminus of the amino acid of the polypeptide compound.

8. A pharmaceutical composition comprising a polypeptide compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or a solvate thereof, or a chimeric molecule of claim 7; Preferably, the composition further contains a pharmaceutically acceptable carrier or excipient; Preferably, the composition further comprises another therapeutic agent; Preferably, the therapeutic agent comprises: Thiazolidinediones, sulfonylureas, dipeptidyl peptidase-4 inhibitors, sodium-glucose cotransporters, growth differentiation factor 15 regulators, peptide tyrosine tyrosine regulators, modified or unmodified insulin, and amylin, or one or more of these. Preferably, the therapeutic agent comprises one or more of the following: metformin, pioglitazone, glimepiride, sitagliptin, dapagliflozin, and pramlintide.

9. The use of the polypeptide compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or a solvate thereof, the chimeric molecule of claim 7 or the pharmaceutical composition of claim 8 in the preparation of a medicament for the prevention and / or treatment of diseases related to GLP-1R, GIPR and / or GCGR; Preferably, the diseases include hyperglycemia, hyperlipidemia, diabetes, obesity, metabolic syndrome, neurodegenerative diseases, impaired glucose tolerance, non-alcoholic steatohepatitis and / or non-alcoholic fatty liver disease. Preferably, the disease is diabetes or obesity; Preferably, the diabetes includes type 1 diabetes and / or type 2 diabetes; Preferably, the diabetes includes complications of diabetes.

10. A method for preparing the polypeptide compound according to any one of claims 1-6, wherein the method is a chemical synthesis method.

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