Polypeptide having multiple agonistic activities and use thereof

Abstract

The present disclosure provides a triple agonist polypeptide compound having an activation effect on three receptors, namely GLP-1R, GIPR, and GCGR. The polypeptide compound has a strong target activation effect, and exhibits excellent effects in lowering blood glucose and blood lipids and controlling body weight. The present disclosure further provides a use of the polypeptide compound in the treatment of metabolic-related diseases, especially type II diabetes and diseases related thereto.

Description

Peptides with multiple agonist activities and their applications

[0001] This application claims priority to Chinese patent application 2025103609819, filed on March 25, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to polypeptide compounds with agonist activity and their applications, specifically to polypeptide compounds with triple agonist activity against glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR), and glucagon receptor (GCGR), and their applications in metabolic-related diseases. Background Technology

[0003] Incretin is a class of polypeptide hormones that are secreted from the intestines after eating in a normal physiological state. Early studies found that it can stimulate pancreatic β-cells to secrete insulin as glucose levels rise after meals, regulate glucose homeostasis, protect pancreatic β-cells, and reduce weight by suppressing appetite and delaying gastric emptying.

[0004] Glucagon-like peptide-1 (GLP-1) is an incretin composed of 36 amino acids. Its main biologically active fragment is a 30-amino acid C-terminal amidated peptide, which stimulates glucose-dependent insulin secretion and has been shown to prevent hyperglycemia in diabetes. GLP-1R (glucagon-like peptide-1 receptor) agonists have been a hot research topic in the field of diabetes and have become the non-insulin diabetes drugs with the highest global market share, including dulaglutide, exenatide, and liraglutide. Therefore, the continued development of iterative products—multi-target agonists of GLP-1R—has become a popular competitive direction.

[0005] Glucose-dependent insulinotropic peptide (GIP) is also an incretin, composed of 42 amino acids, produced by κ cells in the small intestinal mucosa. It primarily acts on GIP receptors (GIPR) in pancreatic islet cells and adipocytes, exerting its physiological effects on glucose homeostasis by stimulating insulin secretion from pancreatic β cells in the presence of glucose. Furthermore, GIP stimulates the uptake and utilization of fatty acids by adipose tissue cells. GIP also promotes osteoblast differentiation, inhibits osteoblast apoptosis, suppresses bone resorption, and increases bone mineral density, thus playing a protective role in bone health.

[0006] Glucagon (GCG) is a 29-amino acid polypeptide expressed and secreted by the proglucagon gene in pancreatic α-cells. It acts on glucagon receptors (GCGRs), primarily located in the liver and kidneys, stimulating glycogenolysis, raising blood glucose levels, activating lipases, promoting lipolysis, while simultaneously inhibiting hepatic fat synthesis and enhancing fatty acid oxidation. Research indicates that GCG has a certain effect on reducing food intake, increasing energy expenditure from adipose tissue, and reducing body fat mass. The appropriate blood glucose-raising effect of GCG can feedback regulate insulin action, reducing the occurrence of hypoglycemic events.

[0007] Glucagon (GCG) helps maintain blood glucose levels by binding to and activating glucagon receptors on hepatocytes, thereby enabling the liver to release glucose stored in glycogen through glycogenolysis. Studies have shown that while glucagon receptor (GCGR) drugs effectively lower pre- and post-meal blood glucose and glycated hemoglobin in patients with type 2 diabetes, they also cause adverse reactions such as increased blood lipids and liver transaminases. When GLP-1R and GCGR are developed as co-agonists, they can not only lower blood glucose but also effectively reduce these adverse reactions. Currently, most multi-target agonists under development globally focus on GLP-1R / GIPR and GLP-1R / GCGR. The successful development of various GLP-1 / GCG and GLP-1 / GIP dual-receptor agonists has also spurred research into single-molecule agonists that simultaneously activate these three target receptors. Therefore, not only dual-target agonists, but also GLP-1R / GIPR / GCGR tri-target agonists may exhibit even stronger therapeutic effects. In addition, GLP-1R has the potential to combine with GCGR, FGF21R, GLP-2R, etc., to form single-molecule dual- or triple-receptor agonists.

[0008] CN104902919A and CN111040022A disclose a series of GLP-1 / GIP / GCGR triple agonist molecules based on the structural modification of exendin-4 (exendin-4); CN109071624A discloses a series of molecules composed of cyclic peptide molecules and long-acting conjugates. In addition, WO2015067716A1, WO2019125929A1, and WO2019125938A1 also disclose some polypeptides with fatty acids linked to the side chain of amino acid 17. These polypeptides all show triple agonist effects on the GLP-1 / GIP / GCG receptors and have the potential for long-acting once-weekly administration. The molecules disclosed in the above patent applications do not simultaneously possess sufficiently suitable activity against all three targets (GLP-1R, GIPR, and GCGR) and provide strong glycemic and lipid-lowering control capabilities; therefore, there is still room for further optimization and improvement of the triple agonist molecules. Summary of the Invention

[0009] This disclosure provides a novel polypeptide compound with triple agonist activity of GLP-1R / GIPR / GCGR, which exhibits high agonist activity against each target of GLP-1R / GIPR / GCGR, demonstrating excellent in vivo glucose and lipid-lowering effects. It has broad prospects in the treatment of type II diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, metabolic syndrome and other related metabolic diseases.

[0010] This disclosure provides a polypeptide compound or a pharmaceutically acceptable salt thereof having GLP-1R / GIPR / GCGR triple agonist activity, having the structure shown in general formula (I):

[0011] X1-Aib-QGTFTSDYSI-αMeL-LDK-X 17 -AQ-Aib-AFIEYL-XX1-XX2-XX3-R 1 (I)

[0012] in,

[0013] X1 is either Y or H;

[0014] X 17 For Ψ;

[0015] XX2 is GG, GGG, or GGGG (SEQ ID NO:12);

[0016] When XX1 is IA, XX3 is PSSGAPPPSKVSEA (SEQ ID NO:7) or PSSGAPPPSEVSEA (SEQ ID NO:8);

[0017] When XX1 is ID or IE, XX3 is PSSGAPPPSKVSRA (SEQ ID NO:9), PSSGAPPPSKVSEA (SEQ ID NO:7), or PSSGAPPPSEVSEA (SEQ ID NO:8);

[0018] R 1 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0019] Wherein, Ψ is Lys whose side chain is modified with the following general formula (II): UZ(II), wherein U is (AEEA and / or amino acid). a -(AEEA and / or amino acids) b -(AEEA and / or amino acids) c Where a, b, and c are each independently 0 or 1, and a, b, and c are not simultaneously 0, and Z is -CO-(CH2). m-R 2 m is an integer between 6 and 24, R 2 The amino acid is COOH, and U is Glu or γGlu, preferably AEEA-γGlu. The amino acid sequence AEEA is designated SEQ ID NO:1 in this disclosure; X1-Aib-QGTFTSDYSI-αMeL-LDK in the structure shown in formula (I) is designated SEQ ID NO:10; and AQ-Aib-AFIEYL in the structure shown in formula (I) is designated SEQ ID NO:11.

[0020] Unless otherwise specified, in the general formulas of all polypeptides disclosed herein, the "-" between each amino acid residue represents an amide bond. All amino acid residues in the general formulas of this disclosure are connected by amide bonds, even if not all are shown with "-".

[0021] In some implementations, XX2 is GG.

[0022] In some implementations, XX1 is IA, and XX3 is PSSGAPPPSKVSEA (SEQ ID NO:7) or PSSGAPPPSEVSEA (SEQ ID NO:8).

[0023] In some embodiments, XX1 is ID or IE, and XX3 is PSSGAPPPSKVSRA (SEQ ID NO:9), PSSGAPPPSKVSEA (SEQ ID NO:7), or PSSGAPPPSEVSEA (SEQ ID NO:8). In some embodiments, XX1 is ID, and XX3 is PSSGAPPPSKVSRA (SEQ ID NO:9). In some embodiments, XX1 is IE, and XX3 is PSSGAPPPSKVSRA (SEQ ID NO:9).

[0024] In some embodiments, the carboxyl or acyl terminus of U in general formula (II) is attached to the ε-amino group of the side chain of Lys having the structural modification of general formula (II).

[0025] In some implementations, m in general formula (II) is 18.

[0026] In some embodiments, the general formula (II) is AEEA-γG1u-CO(CH2). 18 COOH. Preferably, the acetyl group of the AEEA is linked to the ε-amino group of the side chain of Lys having a structure modified with general formula (II).

[0027] In some embodiments, the polypeptide compounds of this disclosure are preferably the following compounds or pharmaceutically acceptable salts thereof:

[0028] In some embodiments, the polypeptide compound of this disclosure is a compound as shown in SEQ ID NO:3 or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, this disclosure provides a pharmaceutical composition comprising the polypeptide compound described herein or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients, diluents, carriers, and / or excipients.

[0030] In some embodiments, the pharmaceutical compositions described herein further comprise at least one other therapeutically active substance.

[0031] In some embodiments, the pharmaceutical composition has multiple dosage forms, preferably injections, tablets, or capsules.

[0032] In some embodiments, the pharmaceutical composition is intended for oral administration, inhalation administration, or parenteral administration, wherein the parenteral administration is selected from intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous, or intradermal injection.

[0033] The polypeptide compounds described herein react with any of several inorganic or organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and their commonly used preparation techniques are well known in the art.

[0034] In some embodiments, this disclosure provides a method for treating a disease, the method comprising administering to an individual in need a polypeptide compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, selected from diabetes, diabetes-related conditions, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases associated with endocrine disorders, metabolic disturbances, kidney disease, etc. In some embodiments, the administration route in the method includes oral administration, inhalation administration, or parenteral administration; preferably, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injection.

[0035] In some embodiments, this disclosure provides the use of the polypeptide compounds described herein or pharmaceutically acceptable salts thereof or pharmaceutical compositions described herein in the preparation of medicaments for treating diseases selected from diabetes, diabetes-related conditions, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases associated with endocrine disorders, metabolic disorders, kidney disease, etc.

[0036] In some embodiments, this disclosure provides the polypeptide compounds described herein or their pharmaceutically acceptable salts or pharmaceutical compositions described herein for the treatment of diseases selected from diabetes, diabetes-related conditions, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases related to endocrine disorders, metabolic disorders, kidney disease, etc.

[0037] In some embodiments, the diabetes described in this disclosure is preferably type II diabetes.

[0038] The polypeptide compounds or their pharmaceutically acceptable salts described in this disclosure can be synthesized and modified by those skilled in the art using known techniques. Preferably, the peptide sequence backbone of the polypeptide compounds described in this disclosure can be prepared by solid-phase synthesis.

[0039] Compared to existing similar products, the GLP-1 / GIP / GCG receptor triple agonist polypeptide molecule disclosed herein exhibits superior GLP-1R, GIPR, and GCGR agonist activities, significantly better lipid-lowering and glucose-lowering activities, and better in vivo efficacy. The GLP-1 / GIP / GCG receptor triple agonist polypeptide molecule disclosed herein demonstrates stronger GLP-1 receptor and GIP receptor activation activity compared to existing polypeptide molecules in the art. Attached Figure Description

[0040] Figure 1 shows the mass spectrum of compound 1 (D25).

[0041] Figure 2 shows the HPLC detection chromatogram of compound 1 (D25).

[0042] Figure 3 shows the results of detecting the agonistic activity of D25 and D26 peptides at the GLP-1R cell level.

[0043] Figure 4 shows the results of detecting the agonistic activity of D25 and D26 peptides at the GIPR cell level.

[0044] Figure 5 shows the results of detecting the agonistic activity of D25 and D26 peptides at the GCGR cell level.

[0045] Figure 6 shows the results of detecting the agonistic activity of the D31 peptide on GLP-1R cells.

[0046] Figure 7 shows the results of the D31 peptide's agonistic activity against GIPR cells.

[0047] Figure 8 shows the results of detecting the agonistic activity of the D31 peptide on GCGR cells.

[0048] Figure 9 shows the results of detecting the agonistic activity of the D32 peptide on GLP-1R cells.

[0049] Figure 10 shows the results of detecting the agonistic activity of the D32 peptide on GIPR cells.

[0050] Figure 11 shows the results of detecting the agonistic activity of the D32 peptide on GCGR cells.

[0051] Figure 12 shows the blood glucose-time curves after db / db mice were administered different doses of D25 and D26 peptides.

[0052] Figure 13 shows the area under the blood glucose-time curves after administration of different doses of D25 and D26 peptides to db / db mice. aa P<0.01 vs. Vehicle).

[0053] Figure 14 shows the body weight-time curves of db / db mice after administration of different doses of D25 and D26 peptides.

[0054] Figure 15 shows the cumulative food intake-time curves after db / db mice were administered different doses of D25 and D26 peptides.

[0055] Figure 16 shows the blood glucose-time curves after db / db mice were administered different doses of D31 and D32 peptides.

[0056] Figure 17 shows the area under the blood glucose-time curves after db / db mice were administered different doses of D31 and D32 peptides (Note: aa P<0.01 vs. Vehicle).

[0057] Figure 18 shows the body weight-time curves of db / db mice after administration of different doses of D31 and D32 peptides.

[0058] Figure 19 shows the cumulative food intake-time curves after db / db mice were administered different doses of D31 and D32 peptides.

[0059] Figure 20 shows the body weight-time curves of DIO mice after administration of D25 and D26 peptides.

[0060] Figure 21 shows the cumulative food intake-time curves after DIO mice were administered D25 and D26 peptides.

[0061] Figure 22 shows the adipose tissue weight at the experimental endpoint after DIO mice were administered D25 and D26 peptides. aa P<0.01 vs. Vehicle).

[0062] Figure 23 shows the body weight-time curves of DIO mice after administration of D31 and D32 peptides.

[0063] Figure 24 shows the cumulative food intake-time curves after DIO mice were administered D31 and D32 peptides.

[0064] Figure 25 shows the adipose tissue weight at the experimental endpoint after DIO mice were administered D31 and D32 peptides. Detailed Implementation

[0065] the term

[0066] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each publication, patent or patent application has been specifically and individually indicated to be incorporated herein by reference.

[0067] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0068] Some embodiments of this disclosure include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0069] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0070] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0071] The polypeptide compounds described in this disclosure exhibit agonistic activity against GLP-1 receptor, GIP receptor, and GCG receptor. The term "agonistic activity" refers to the ability of the polypeptide compounds to stimulate specific receptor cells to produce cAMP. The cells used can be host cells, pancreatic islet cells, adipocytes, hepatocytes, etc., that overexpress GLP-1 receptor, GIP receptor, or GCG receptor, as constructed by those skilled in the art. The receptor agonistic activity can be achieved by stimulating ECMO (electrodeogenesis imperfecta) in receptor cells to produce cAMP. 50 Value as a numerical measure. EC50 The value refers to the drug concentration required to achieve half (50%) of the compound's maximum activity in a specific assay system.

[0072] The term "triple agonist activity" refers to the activity of a polypeptide compound at each of the GIP, GLP-1, and GCG receptors, particularly a balanced and adequate activity at each receptor to provide the agonistic benefit of that receptor while avoiding the undesirable side effects associated with excessively high activity.

[0073] The term "treatment" refers to the suppression, slowing, stopping, or reversal of the progression or severity of an existing condition, disease, disorder, or symptom.

[0074] The term “individual in need” refers to a condition, disease, disorder, or symptom that requires treatment or care, including, for example, those listed in this disclosure, such as humans.

[0075] The term "effective amount" refers to the amount, concentration, or dose of one or more polypeptide compounds or pharmaceutically acceptable salts thereof described herein that provide the desired effect in an individual being diagnosed or treated, after administration of a single or multiple doses. An effective amount can be readily determined by those skilled in the art using known techniques and by observing results obtained under similar conditions. In determining the effective amount for an individual, many factors are considered, including but not limited to: the mammalian species; its size, age, and general health status; the specific disease or condition involved; the degree or severity of the disease or condition; the individual patient's response; the specific polypeptide compound or pharmaceutically acceptable salt thereof administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosage regimen; the use of concomitant drug therapy; and other relevant circumstances.

[0076] "Related conditions" of diabetes include: insulin resistance, glucose intolerance, elevated fasting blood glucose, prediabetes, gestational diabetes, hypertension, dyslipidemia, bone-related conditions, as well as arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, dyslipidemia, high blood pressure, thrombosis, etc.

[0077] The polypeptide compounds or their pharmaceutically acceptable salts described herein can be formulated into pharmaceutical compositions that can be administered orally, by inhalation, or by parenteral administration (e.g., intraperitoneally, intramuscularly, intra-arterially, intravenously, subcutaneously, or intradermally). Such pharmaceutical compositions and their preparation techniques are well known in the art.

[0078] The polypeptide compounds of this disclosure, possessing triple agonist activity, can react with any of a number of inorganic or organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and their commonly used preparation techniques are well known in the art.

[0079] The amino acids in the polypeptide compounds described in this disclosure are derived from natural amino acids or related amino acid variants and / or derivatives. The abbreviations and codes of the natural amino acids follow generally accepted rules familiar to those skilled in the art. For example, the chemical structural formula of Aib, αMeL is as follows:

[0080] The specific meanings of the abbreviations used in this disclosure are as follows:

[0081] Aib: α-amino isobutyric acid

[0082] αMeL: α-methyl leucine

[0083] AEEA: [2-(2-amino-ethoxy)-ethoxy]-acetyl

[0084] cAMP: Cyclic adenosine monophosphate

[0085] Fmoc:fluorenemethyloxycarbonyl

[0086] Boc: tert-Butyloxycarbonyl

[0087] DMF: Dimethylformamide

[0088] HOAt:N-hydroxy-7-azabenzotriazole

[0089] Trt: Triphenylmethyl

[0090] ivDde:1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methyl-butyl

[0091] tBu: tert-butyl

[0092] OtBu: Oxy-tert-butyl

[0093] TFA: Trifluoroacetic acid

[0094] TIS: Triisopropylsilane

[0095] DCM: Dichloromethane

[0096] DIC: N,N-diisopropylcarbodiimide

[0097] Example

[0098] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0099] Example 1: Synthesis of polypeptide compounds

[0100] The intermediates and compounds of this invention can be synthesized using various methods known in the art. The following specific examples illustrate the preparation of the compounds of this invention using chemical synthesis methods. Each specific synthetic step described can employ different combinations of materials and methods to synthesize various corresponding compounds of this invention or their salts. The reagents and raw materials used are readily available to those skilled in the art. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of this invention in any way.

[0101] Material

[0102] All materials and reagents used in this invention were purchased from commercial products. The protected amino acids used in the entire synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, F moc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys( ivDde)-OH, Fmoc-α-Me-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Boc-Tyr(tBu)-OH, Eicosanedioic acid(mon-tBu)-γGlu(α-OtBu)-AEEA-OH.

[0103] The following uses polypeptide compound 1 (D25) as an example to illustrate the synthesis and preparation method of the compounds of this invention (the other compounds are prepared by changing the order of amino acid raw material synthesis).

[0104] (1) Resin pretreatment: Weigh 66.68g of Rink Amide AM Resin and add it to the reactor. Add 900mL of DMF and stir under nitrogen for 30min to allow it to swell. When the resin has swelled to the required level, filter to remove the solvent. Wash the resin three times with 900mL of DMF each time.

[0105] (2) Deprotection: Add 450 mL of 20% piperidine / DMF solvent to the reaction vessel, and stir under nitrogen for 30 min. After the reaction is complete, wash the resin 6 times with 450 mL of DMF each time. After washing, test the resin with ninhydrin reagent; the resin is positive.

[0106] (3) Preparation of amino acid solution: Weigh 18.68g of Fmoc-Ala-OH and 8.16g of HOAt, dissolve them in 450mL of DMF, then add 9.2mL of DIC and mix well.

[0107] (4) Coupling reaction: The prepared amino acid solution was added to the reaction vessel, and the reaction was carried out under nitrogen atmosphere at a controlled temperature of 25–35 °C with stirring. The reaction progress was monitored with ninhydrin reagent, and the reaction was completed when the resin showed a negative result. After the reaction was completed, the resin was washed three times with 450 mL of DMF each time. The above operation was repeated, and the corresponding protective amino acids were coupled sequentially according to the peptide sequence of peptide compound 1 until the peptide backbone was synthesized.

[0108] (5) Removal of the ivDde protecting group at position 17 Lys: Add 150 mL of 8% hydrazine hydrate / DMF solution to the reactor, purge with nitrogen and stir for 1 h. When the reaction time is up, filter to remove the solvent. Repeat the operation once. Then wash the resin 10 times with 150 mL of DMF each time. After washing, test the resin with ninhydrin reagent. The resin is positive.

[0109] (6) Lys side chain modification: Weigh 7.29 g of Eicosanedioic acid (mon-tBu)-γGlu (α-OtBu)-AEEA-OH and 1.15 g of HOSu, dissolve them in 150 mL of DMF, then add 1.55 mL of DIC, activate for 5 h, and then add to the reaction vessel. Control the temperature at 25-35℃ and stir under nitrogen. Monitor the reaction progress with ninhydrin reagent. The reaction is complete when the resin shows a negative result. After the reaction is complete, wash the resin 4 times with DMF, 3 times with DCM, and 3 times with methyl tert-butyl ether, 300 mL each time. Dry the resin and set aside for later use.

[0110] (7) Peptide resin cleavage: Prepare a cleavage solution according to TFA / TIS / H2O / DTT = 90 / 2.5 / 2.5 / 5, then add the dried peptide resin to the cleavage solution and stir for 2.5 h. When the reaction is complete, filter, concentrate the filtrate to 1 / 2 of the original volume, then add it to 5 times the volume of methyl tert-butyl ether to precipitate, filter under vacuum, wash the filter cake 4 times with methyl tert-butyl ether, and dry under vacuum to obtain the crude product.

[0111] (8) Purification and refining: The crude product was purified by C18 reversed-phase preparative chromatography system to obtain a refined product of polypeptide compound 1 with a purity of not less than 90%. The mass spectrometry and HPLC detection results are shown in Figure 1 and Figure 2.

[0112] Table 1 shows the measured molecular weights of peptide compounds 1-4.

[0113] Table 1. List of synthesized polypeptide compounds and their molecular weights

[0114] The peak parameter information in the HPLC results shown in Figure 2 is shown in the table below.

[0115] Table 2. HPLC Detection Results

[0116] Example 2: Assay of the cellular agonistic activity of the test peptide molecules against GLP-1R, GIPR, and GCGR

[0117] All materials and reagents used in this test were purchased from commercial products. LY3437943 (see, for example, CN111491658A, Example 12) is a polypeptide with triple agonistic activity of GIP / GLP-1 / GCG receptors, and Tirzepatide is a polypeptide with dual agonistic activity of GIP / GLP-1 receptors. The structure of the LY3437943 polypeptide is shown below:

[0118] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYL-LEGGPSSGAPPPS-NH2 (SEQ ID NO: 6).

[0119] The reporter gene cell lines GLP1R / CRE-Luc / HEK293, GIPR / CRE-Luc / HEK293, and GCGR / CRE-Luc / HEK293 were purchased from Nanjing Kebai Biotechnology Co., Ltd. When the polypeptide molecules bind to the receptors on the transgenic cells, they promote an increase in intracellular cAMP expression levels. cAMP then interacts with the CRE site on the luciferase reporter gene, initiating luciferase expression. Luciferase acts on... The chemiluminescent substrate in the Luciferase Assay System generates fluorescence. The agonistic activity of each test peptide for its corresponding receptor is determined by detecting the fluorescence signal response level using a microplate reader. The specific procedure is as follows:

[0120] 1) Cell suspension preparation: Collect cells in good growth condition and in logarithmic growth phase, resuspend them in analytical medium (DMEM + 0.25% FBS), and count the cell density;

[0121] 2) Cell plating: The cell suspension was plated at 2.1 × 10⁻⁶. 4 Seed at a density of 1 cell / well in 96-well white flat plates;

[0122] 3) Add different concentrations of the test polypeptide molecules;

[0123] 4) Incubate at 37℃ with 5% CO2 for 4 hours;

[0124] 5) After incubation, remove the 96-well white flat plate and add [the following ingredient] to each well. The Luciferase Assay System test reagent was gently shaken for 10 minutes at room temperature on a microplate shaker.

[0125] 6) The microplate analyzer measures the luminescence value of the sample at each concentration;

[0126] 7) Using GraphPad Prism 8.0 software, plot the logarithm of the sample concentration on the x-axis and the luminescence value on the y-axis, and perform nonlinear regression (curve fit) analysis on the data to obtain the EC50 of each sample. 50 value.

[0127] The results are shown in Figures 3 to 11 and Table 3. The EC values ​​of each tested peptide were analyzed. 50 Numerical comparisons show that in the GLP-1R, GIPR, and GCGR activation experimental systems:

[0128] (1) For D25 and D26 molecules: (i) In the GLP-1R activation experimental system, according to EC 50 Values, D25 and D26 activities were comparable, and both were stronger than LY3437943 and telpoeptide; (ii) in the GIPR activation experimental system, according to EC 50 Values, D25 and D26 are essentially equivalent to the activities of LY3437943 and telpolide (EC). 50 (i) All are between 0.005 nM and 0.01 nM); (iii) In the GCGR activation experimental system, according to EC 50 The activity of D26 was slightly better than that of D25, and the agonist activity of both molecules was weaker than that of LY3437943.

[0129] (2) For the D31 molecule: (i) In the GLP-1R activation experimental system, according to EC 50 Value, D31 activity is comparable to LY3437943, and stronger than telpoeptide; (ii) in the GIPR activation experimental system, according to EC 50 Value, D31 has comparable activity to LY3437943 and telpoeptide; (iii) In the GCGR activation experimental system, according to EC 50 The D31 activity was weaker than that of LY3437943.

[0130] (3) For the D32 molecule: (i) In the GLP-1R activation experimental system, according to EC 50Value, D32 activity is slightly weaker than LY3437943, but stronger than telpoeptide; (ii) in the GIPR activation experimental system, according to EC 50 Value, D32 has comparable activity to LY3437943 and telpoeptide; (iii) In the GCGR activation experimental system, according to EC 50 The D32 activity was weaker than that of LY3437943.

[0131] Table 3. Activation EC50 of GLP-1R, GIPR, and GCGR by the tested peptide molecules 50 value Note: For the above experiments, n≥2, the values ​​shown are the average values ​​of the experiments.

[0132] Example 3: Hypoglycemic efficacy of D25 and D26 peptides in db / db type II diabetic model mice

[0133] Animals: 8-week-old male db / db type II diabetic mice

[0134] The specific in vivo efficacy experiments of hypoglycemic drugs are as follows:

[0135] On the day of the experiment, db / db mice were grouped (N=7) according to their body weight and blood glucose levels into four groups: Vehicle group, LY3437943 group (10 nmol / kg), telpolide (TR) group (30 nmol / kg), D25 group (10 nmol / kg), D25 group (30 nmol / kg), D26 group (10 nmol / kg), and D26 group (30 nmol / kg). Each group of mice received a single subcutaneous injection of the corresponding test peptide. Blood glucose levels were measured at tail tip blood samples at 1, 2, 8, 12, 24, 48, 72, 96, 120, and 144 hours after administration. Body weight and food intake were measured 24 hours before administration and at 24, 48, 72, 96, 120, and 144 hours after administration.

[0136] As shown in Figures 12 and 13, compared with the Vehicle group, all peptides significantly reduced blood glucose levels in db / db mice. At the same dose (10 nmol / kg), D25, D26, and LY3437943 showed comparable effects. Increasing the dose of D25 and D26 further enhanced their hypoglycemic effects, with D25 (30 nmol / kg) and D26 (30 nmol / kg) showing significantly better hypoglycemic effects than LY3437943 (10 nmol / kg). As shown in Figures 14 and 15, compared with the Vehicle group, all peptides reduced the body weight and suppressed the appetite of db / db mice, with D25 (30 nmol / kg) and D26 (30 nmol / kg) showing the best results. These results indicate that D25 and D26 have significant in vivo hypoglycemic capabilities.

[0137] Example 4: Hypoglycemic efficacy of D31 and D32 peptides in db / db type II diabetic model mice

[0138] Animals: 8-week-old male db / db type II diabetic mice

[0139] The specific in vivo efficacy experiments of hypoglycemic drugs are as follows:

[0140] On the day of the experiment, db / db mice were grouped (N=8) according to their body weight and blood glucose levels into four groups: Vehicle group, LY3437943 group (10 nmol / kg), telpolide (TR) group (30 nmol / kg), D31 group (10 nmol / kg), D32 group (10 nmol / kg), and D32 group (30 nmol / kg). Each group of mice received a single subcutaneous injection of the corresponding test peptide. Blood glucose levels were measured at tail tip blood samples at 1, 2, 8, 12, 24, 48, 72, 96, 120, and 144 hours after administration. Body weight and food intake were measured 24 hours before administration and at 24, 48, 72, 96, 120, and 144 hours after administration.

[0141] As shown in Figures 16 and 17, compared with the Vehicle group, all peptides significantly reduced blood glucose levels in db / db mice. At the same dose (10 nmol / kg), D31, D32, and LY3437943 showed comparable hypoglycemic effects, with D31 (30 nmol / kg) showing significantly better hypoglycemic effects than LY3437943 (10 nmol / kg). As shown in Figures 18 and 19, compared with the Vehicle group, all peptides reduced body weight and food intake in db / db mice. At the same dose (10 nmol / kg), D31, D32, and LY3437943 showed comparable weight-loss effects; D31 (30 nmol / kg) and D32 (30 nmol / kg) showed significantly better weight-reducing effects than LY3437943 (10 nmol / kg). These results indicate that D31 and D32 have significant in vivo hypoglycemic capabilities.

[0142] Example 5: Efficacy test of D25 and D26 peptides on weight loss and lipid reduction in DIO mice

[0143] Animals: 27-week-old male DIO mice with a C57 background

[0144] The specific in vivo efficacy experiments for weight loss and lipid-lowering drugs are as follows:

[0145] Male DIO mice with a C57 background, aged 27 weeks, were divided into four groups (N=6): Vehicle, D25 (8 nmol / kg), D26 (8 nmol / kg), and LY3437943 (8 nmol / kg). Animals in each group received a subcutaneous injection of the corresponding polypeptide molecule every 3 days for 30 days. Body weight and food intake were measured every 3 days.

[0146] As shown in Figure 20, compared with the Vehicle group, all peptides significantly reduced the body weight of DIO mice. At the same dose (8 nmol / kg), D25, D26, and LY3437943 showed comparable weight-reducing effects. As shown in Figures 21 and 22, compared with the Vehicle group, all peptides suppressed appetite and reduced abdominal fat weight in mice. These results indicate that D25 and D26 have significant weight-reduction and fat-reduction capabilities.

[0147] Example 6: Efficacy test of D31 and D32 peptides on weight loss and lipid reduction in DIO mice

[0148] Animals: 27-week-old male DIO mice with a C57 background

[0149] The specific in vivo efficacy experiments for weight loss and lipid-lowering drugs are as follows:

[0150] Male DIO mice with a C57 background, aged 27 weeks, were divided into four groups (N=8): Vehicle group, LY3437943 group (8 nmol / kg), telpolide (TR) group (8 nmol / kg), D31 group (8 nmol / kg), and D32 group (8 nmol / kg). Animals in each group received the corresponding polypeptide molecule subcutaneously every 3 days for 30 days. Body weight and food intake were measured every 3 days.

[0151] As shown in Figure 23, compared with the Vehicle group, all peptides in each group could reduce the body weight of DIO mice. At the same dose (8 nmol / kg), D31, D32, and LY3437943 showed comparable weight-reducing effects, significantly better than telpolide. As shown in Figures 24 and 25, compared with the Vehicle group, all peptides in each group could suppress appetite and reduce abdominal fat weight in mice. These results indicate that D31 and D32 both have significant weight-reduction and fat-reduction capabilities.

Claims

1. A polypeptide compound or a pharmaceutically acceptable salt thereof having GLP-1R / GIPR / GCGR triple agonist activity, having the following general formula (Ⅰ): X1-Aib-QGTFTSDYSI-αMeL-LDK-X 17 -AQ-Aib-AFIEYL-XX1-XX2-XX3-R 1 (Ⅰ) in, X1 is either Y or H; X 17 For Ψ; XX2 is GG, GGG, or GGGG (SEQ ID NO:12); When XX1 is IA, XX3 is PSSGAPPPSKVSEA (SEQ ID NO:7) or PSSGAPPPSEVSEA (SEQ ID NO:8); When XX1 is ID or IE, XX3 is PSSGAPPPSKVSRA (SEQ ID NO:9), PSSGAPPPSKVSEA (SEQ ID NO:7), or PSSGAPPPSEVSEA (SEQ ID NO:8); R 1 It is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof; Wherein, Ψ is Lys whose side chain is modified with the following general formula (II): UZ(II), wherein U is (AEEA and / or amino acid). a -(AEEA and / or amino acids) b -(AEEA and / or amino acids) c Where a, b, and c are each independently 0 or 1, and a, b, and c are not simultaneously 0, and Z is -CO-(CH2). m -R 2 m is an integer between 6 and 24, R 2 The amino acid is COOH, and the amino acid is Glu or γGlu.

2. The polypeptide compound or its pharmaceutically acceptable salt as claimed in claim 1, wherein XX1 is 1A and XX3 is SEQ ID NO:7 or SEQ ID NO:8; or XX1 is ID or IE, and XX3 is SEQ ID NO:9, SEQ ID NO:7, or SEQ ID NO:

8.

3. The polypeptide compound or its pharmaceutically acceptable salt as claimed in claim 1 or 2, wherein the carboxyl or acyl terminus of U in general formula (II) is connected to the ε-amino group of the side chain of Lys having the structure modified by general formula (II); preferably, the carboxyl terminus of U is connected to the ε-amino group of the side chain of Lys having the structure modified by general formula (II).

4. The polypeptide compound or its pharmaceutically acceptable salt as described in any one of claims 1-3, wherein m is 18.

5. The polypeptide compound or its pharmaceutically acceptable salt as described in any one of claims 1-4, wherein the general formula (II) is AEEA-γG1u-CO(CH2). 18 COOH.

6. The polypeptide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, wherein the polypeptide compound is selected from the following compounds or pharmaceutically acceptable salts thereof SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:

5.

7. A pharmaceutical composition comprising a polypeptide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6, and pharmaceutically acceptable excipients, diluents, carriers, and / or excipients.

8. The pharmaceutical composition of claim 7, further comprising at least one other therapeutically active substance.

9. A method of treating a disease, the method comprising administering to an individual in need an effective amount of any one of claims 1-6, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 7, wherein the disease is selected from diabetes, diabetes-related conditions, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases associated with endocrine disorders, metabolic disorders, kidney disease, etc., preferably, wherein the diabetes is type II diabetes.

10. Use of the polypeptide compound or pharmaceutically acceptable salt thereof of any one of claims 1-6 or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating a disease selected from diabetes, diabetes-related conditions, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases related to endocrine disorders, metabolic disorders, kidney disease, etc., preferably, the diabetes is type II diabetes.

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