Peptides with multiple agonist activities and their applications

Abstract

This invention provides a triple agonist peptide compound that activates three receptors: GLP-1R, GIPR, and GCGR. This peptide compound exhibits strong target activation efficacy and excellent hypoglycemic, lipid-lowering, and weight-controlling effects. It also provides the application of this peptide compound in the treatment of metabolic-related diseases, especially type II diabetes and related diseases.

Description

Polypeptides with multiple agonist activities and their applications

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 26, 2023, with application number 202311403282.5 and invention name “Polypeptides with multiple agonistic activities and their applications”, the entire contents of which are incorporated by reference into this application. Technical Field

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

[0003] Incretin is a type of polypeptide hormone secreted from the intestine after food stimulation under normal physiological conditions. Early studies have 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, delaying gastric emptying, and other means.

[0004] Glucagon-like peptide-1 (GLP-1) is an incretin insulin 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 always been a hot research and development 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 also become a hot competitive direction.

[0005] Glucose-dependent insulinotropic polypeptide (GIP) is also an incretin. Composed of 42 amino acids, it is produced by the κ cells of the small intestinal mucosa and primarily acts on the GIP receptor (GIPR) in pancreatic islet cells and adipocytes. It plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β cells in the presence of glucose. GIP also stimulates the uptake and utilization of fatty acids by adipose tissue cells. GIP also has the physiological effects of promoting osteoblast differentiation, inhibiting osteoblast apoptosis, inhibiting bone resorption, and increasing bone mineral density, thus playing a bone-protecting role.

[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 (GCGR) primarily located in the liver and kidneys, stimulating hepatic glycogenolysis, raising blood glucose, activating lipase, and promoting lipolysis. It also inhibits hepatic fat synthesis and enhances fatty acid oxidation. Research results indicate that GCG is effective in reducing food intake, increasing adipose tissue energy expenditure, and reducing body fat mass. The moderate blood glucose-raising effect of GCG can feedback-regulate the effects of insulin and reduce the occurrence of hypoglycemic episodes.

[0007] Glucagon (GCG) helps maintain blood sugar levels by binding to and activating the glucagon receptor on liver cells, triggering the release of glucose stored as glycogen in the liver through a process called glycogenolysis. Studies have shown that while glucagon receptor (GCGR) drugs effectively lower pre- and post-prandial blood sugar and glycated hemoglobin in patients with type 2 diabetes, they are also associated with adverse effects such as increased lipids and liver transaminases. However, the development of GLP-1R and GCGR co-agonists not only lowers blood sugar but also effectively mitigates these adverse effects. Currently, the majority of multi-target agonists under development globally focus on the GLP-1R / GIPR and GLP-1R / GCGR receptors. The successful development of various GLP-1 / GCG and GLP-1 / GIP dual receptor agonists has also stimulated the search for single-molecule agonists that simultaneously activate all three target receptors. Therefore, not only dual-targeting, but also triple-targeting GLP-1R / GIPR / GCGR agonists have the potential to demonstrate even more potent therapeutic effects. In addition, GLP-1R also has the possibility of combining with GCGR, FGF21R, GLP-2R, etc. to form single-molecule dual-receptor and triple-receptor agonists.

[0008] CN104902919A and CN111040022A disclose a series of GLP-1 / GIP / GCG R triple agonist molecules based on the structural modification of exendin-4; CN109071624A discloses a series of molecules composed of cyclic cyclic peptide molecules and long-acting conjugates. In addition, WO2015067716A1, WO2019125929A1 and WO2019125938A1 also disclose some polypeptides with fatty acids connected to the side chains of amino acids at position 17. These polypeptides all show a triple agonist effect of GLP-1 / GIP / GCG receptors and have a long-term potential of once a week. The molecules disclosed in the above patent applications cannot simultaneously have sufficient and appropriate activity for the three targets of GLP-I R / GIP R / GCGR and provide strong blood sugar and blood lipid control capabilities. Therefore, there is still room for improvement in further optimizing the triple agonist molecules.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure provides a novel polypeptide compound with triple agonist activity of GLP-1R / GIPR / GCGR, which has sufficiently high agonist activity against GLP-1R / GIPR / GCGR, shows excellent glucose-control and lipid-lowering effects in vivo, and has broad prospects in the treatment of related metabolic diseases such as type 2 diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease / non-alcoholic steatohepatitis, metabolic syndrome, etc.

[0011] The present disclosure provides a polypeptide compound having GLP-1R / GIPR / GCGR triple agonist activity or a pharmaceutically acceptable salt thereof, which has a structure represented by the general formula (I):

[0012] X1-Aib-QGTFTSDYSI-αMeL-LDK-X 17 -AQ-Aib-AFIEYL-XX1-XX2-XX3-R 1 (Ⅰ)

[0013] in,

[0014] X1 is Y or H;

[0015] X 17 is Ψ;

[0016] XX1 is IA, LE, L d E d , L d E、V d D d or LE d ;

[0017] XX2 is GG, GGG, or GGGG;

[0018] XX3 is PSSGAPPPSKVSRA, PSSGAPPPS, or PSSGA d PPPSKVSRA;

[0019] R 1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof;

[0020] Wherein, Ψ is a Lys with a side chain modified by a structure having 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 , wherein a, b, c are each independently 0 or 1, and a, b, c are not 0 at the same time, and Z is -CO-(CH2) m -R 2, m is an integer between 6 and 24, R 2 is selected from COOH, the amino acid is Glu or γGlu, and U is preferably AEEA-γGlu.

[0021] In some embodiments, the carboxyl end or acyl end of U in formula (II) is connected to the ε-amino group of the side chain of Lys.

[0022] In some embodiments, m in formula (II) is 18.

[0023] In some embodiments, the general formula (II) is AEEA-γG1u-CO(CH2) 18 COOH, preferably, the acetyl group of AEEA is connected to the ε-amino group of the side chain of Lys.

[0024] In some embodiments, the polypeptide compounds disclosed herein are preferably the following compounds or pharmaceutically acceptable salts thereof:

[0025] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the polypeptide compound described in the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier and / or excipient.

[0026] In some embodiments, the pharmaceutical compositions described herein further comprise at least one additional therapeutically active substance.

[0027] In some embodiments, the pharmaceutical composition has a variety of dosage forms, preferably injections, tablets or capsules.

[0028] In some embodiments, the pharmaceutical composition is for oral administration, inhalation administration, or parenteral administration, wherein the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, or intradermal injection.

[0029] The polypeptide compounds herein react with any of several inorganic or organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and common preparation techniques are well known in the art.

[0030] In some embodiments, the present disclosure provides a method for treating a disease, comprising administering to an individual in need thereof a polypeptide compound of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, 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 diseases, metabolic disorders, kidney disease, and the like.

[0031] In some embodiments, the present disclosure provides use of the polypeptide compound described in the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the present disclosure in the preparation of a medicament for treating a disease, 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 related to endocrine diseases, metabolic disorders, kidney disease, etc.

[0032] In some embodiments, the diabetes described in the present disclosure is preferably type II diabetes.

[0033] The polypeptide compounds or pharmaceutically acceptable salts thereof disclosed herein can be synthesized and modified by those skilled in the art using known methods. Preferably, the peptide sequence backbone of the polypeptide compounds disclosed herein can be prepared by solid phase synthesis.

[0034] Compared with existing similar products, the GLP-1 / GIP / GCG receptor triple agonist polypeptide molecule provided by the present disclosure has better GLP-1R, GIPR, and GCGR agonist activity, significantly excellent lipid-lowering and blood sugar-lowering activity, and in vivo efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the mass spectrum of compound 1.

[0036] Figure 2 is a HPLC detection chart of compound 1.

[0037] FIG3 shows the results of the detection of the agonist activity of D21 polypeptide on GLP-1R, GIPR and GCGR at the cellular level.

[0038] FIG4 shows the results of the detection of the agonist activity of D22 polypeptide on GLP-1R, GIPR and GCGR at the cellular level.

[0039] FIG5 shows the results of the detection of the agonist activity of F01-F04 polypeptides on GLP-1R, GIPR and GCGR at the cellular level.

[0040] FIG6 is a blood glucose-time curve of db / db mice after administration of D21 and F01-F04 polypeptides.

[0041] Figure 7 shows the area under the blood glucose-time curve of db / db mice after administration of D21 and F01-F04 peptides. aa P<0.01 vs. vehicle 1; bb P<0.01 vs. vehicle 2.

[0042] FIG8 is a curve showing the body weight changes over time in db / db mice after administration of D21 and F01-F04 polypeptides.

[0043] FIG9 is a curve showing the change in food intake over time in db / db mice after administration of D21 and F01-F04 polypeptides.

[0044] FIG10 is a curve showing the body weight-time changes of DIO mice after administration of different doses of D21 polypeptide.

[0045] Figure 11 shows the area under the body weight-time curve of DIO mice after administration of different doses of D21 polypeptide. aa P<0.01 vs. solvent 1, bb P<0.01 vs. vehicle 2, ** P<0.01vs.LY3437943.

[0046] FIG12 is a curve showing the change in food intake over time after administration of different doses of D21 polypeptide to DIO mice.

[0047] Figure 13 shows the abdominal fat content of DIO mice after administration of different doses of D21 polypeptide at the end of the experiment. aa P<0.01 vs. solvent 1, bb P<0.01 vs. vehicle 2, * P<0.05vs.LY3437943. DETAILED DESCRIPTION

[0048] the term

[0049] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0050] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0051] Certain embodiments of the present disclosure include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or the use of range descriptions are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions using ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0052] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations 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.

[0053] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0054] The polypeptide compounds disclosed herein have agonist activity on GLP-1 receptors, GIP receptors, and GCG receptors. The "agonist activity" mentioned herein means that the polypeptide compounds can stimulate specific receptor cells to produce cAMP. The cells used can be host cells overexpressing GLP-1 receptors, GIP receptors, or GCG receptors constructed by those skilled in the art, or pancreatic tissue cells, adipocytes, hepatocytes, etc. The receptor agonist activity can be achieved by using ECs that stimulate receptor cells to produce cAMP. 50 The value is used as a measure. 50 The value refers to the drug concentration required to achieve half of the maximal activity (50% activity) of the compound in a specific assay system.

[0055] The term "triple agonist activity" refers to a polypeptide compound that has activity at each of the GIP receptor, GLP-1 receptor, and GCG receptor, and in particular has balanced and sufficient activity at each receptor to provide an agonist benefit at that receptor while avoiding undesirable side effects associated with too high activity.

[0056] The terms "treat," "treat," "treat," and the like refer to inhibiting, slowing, stopping, or reversing the progression or severity of an existing condition, disease, disorder, or symptom.

[0057] The term "subject in need thereof" refers to a mammal, such as a human, having a condition, disease, disorder or symptom in need of treatment or therapy, including, for example, those listed in the present disclosure.

[0058] The term "effective amount" means the amount, concentration or dosage of one or more polypeptide compounds of the present disclosure or a pharmaceutically acceptable salt thereof that provides the desired effect in an individual being diagnosed or treated, following single or multiple doses of administration to an individual in need thereof. An effective amount is readily determined by one skilled in the art using known techniques and by observing the results obtained under similar circumstances. In determining an effective amount for an individual, many factors are considered, including but not limited to the species of mammal; its size, age and general health; the specific disease or condition involved; the extent or severity of the disease or condition; the response of the individual patient; the specific polypeptide compound or a pharmaceutically acceptable salt thereof administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0059] The "related diseases" of diabetes include: insulin resistance, glucose intolerance, elevated fasting blood sugar, prediabetes, gestational diabetes, hypertension, dyslipidemia, bone-related diseases, etc., as well as arteriosclerosis, coronary heart disease, peripheral arterial disease, stroke, dyslipidemia, elevated blood pressure, thrombosis, etc.

[0060] The polypeptide compounds described herein or pharmaceutically acceptable salts thereof can be formulated into pharmaceutical compositions that can be administered orally, by inhalation, or by parenteral administration (e.g., intraperitoneally, intramuscularly, intraarterially, intravenously, subcutaneously, or intradermally). Such pharmaceutical compositions and their preparation techniques are well known in the art.

[0061] The polypeptide compounds with triple agonist activity disclosed herein can react with any of a variety of inorganic or organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and their common preparation techniques are well known in the art.

[0062] The amino acids in the polypeptide compound sequences described herein are derived from natural amino acids or related amino acid variants and / or derivatives. The abbreviations and codes for the natural amino acids are based on common rules well known to those in the industry. For example, the chemical structure of Aib, αMel is as follows:

[0063] The abbreviations used in this disclosure have the following meanings:

[0064] Aib:α-amino isobutyric

[0065] αMel:α-methyl leucine

[0066] AEEA: [2-(2-amino-ethoxy)-ethoxy]-acetyl

[0067] cAMP: cyclic adenosine monophosphate

[0068] Fmoc: Fluorenylmethoxycarbonyl

[0069] Boc: tert-Butyloxycarbonyl

[0070] DMF:Dimethylformamide

[0071] HOAt: N-hydroxy-7-azabenzotriazole

[0072] Trt: Trityl

[0073] ivDde:1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methyl-butyl

[0074] tBu: tert-butyl

[0075] OtBu: tert-butyloxy

[0076] TFA: trifluoroacetic acid

[0077] TIS: Triisopropylsilane

[0078] DCM: dichloromethane

[0079] DIC: N,N-diisopropylcarbodiimide

[0080] Example

[0081] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are intended only to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0082] Example 1: Synthesis of polypeptide compounds

[0083] The intermediates and compounds of the present invention can be synthesized by various methods known in the art. The following specific examples illustrate the preparation of the compounds of the present invention using chemical synthesis methods. Each specific synthetic step described can be combined with different materials and methods to synthesize a variety of corresponding compounds of the present invention or salts thereof. The reagents and raw materials used are readily available to those of ordinary skill in the art. In particular, the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention in any way.

[0084] Material

[0085] The materials and reagents used in the present 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, Fmo-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc- oc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(i vDde)-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.

[0086] The following uses polypeptide compound 1 (D21) as an example to illustrate the synthesis and preparation methods of the compounds of this invention (the remaining compounds F01-F04 and D22 were prepared by replacing the synthetic sequence of amino acid raw materials).

[0087] (1) Resin Pretreatment: Weigh 23.44 g of Rink Amide AM Resin into a reactor, add 200 mL of DMF, and stir under nitrogen to allow the mixture to swell for 30 min. When the mixture is fully swollen, remove the solvent by filtration and wash the resin three times with 150 mL of DMF each time.

[0088] (2) Deprotection: Add 200 mL of 20% piperidine / DMF solvent to the reaction vessel and stir under nitrogen for 30 min. After the reaction, wash the resin six times with 150 mL of DMF each time. After washing, remove the resin and test it with ninhydrin reagent. The resin should be positive.

[0089] (3) Preparation of amino acid solution: Weigh 7.00 g of Fmoc-Ala-OH and 3.06 g of HOAt and dissolve them in 150 mL of DMF. Then add 3.5 mL of DIC and mix well.

[0090] (4) Coupling reaction: Add the prepared amino acid solution to the reactor, control the temperature at 25-35°C, and stir the reaction under nitrogen. Monitor the reaction progress with ninhydrin reagent. The reaction is complete when the resin shows a negative color. After the reaction is complete, wash the resin with DMF three times, 150 mL each time. Repeat the above steps and sequentially couple the corresponding protected amino acids according to the peptide sequence of polypeptide compound 1 until the peptide backbone is synthesized.

[0091] (5) Removal of the ivDde protecting group at the 17-position Lys side chain: 500 mL of 8% hydrazine hydrate / DMF solution was added to the reaction vessel and stirred under nitrogen for 1 h. When the reaction was complete, the solvent was removed by filtration. This process was repeated once. The resin was then washed 10 times with 300 mL of DMF each time. After washing, the resin was tested with ninhydrin reagent, which was positive.

[0092] (6) Lys side chain modification: Weigh 16.47 g of Eicosanedioic acid (mon-tBu)-γGlu (α-OtBu)-AEEA-OH and 2.72 g of HOSu, dissolve them in 250 mL of DMF, then add 3.5 mL of DIC. After activation for 5 h, add them to the reactor and control the temperature at 25-35°C. Stir and react under nitrogen. Monitor the reaction progress with ninhydrin reagent until the resin shows a negative reaction, indicating that the reaction is complete. After the reaction is complete, wash the resin with DMF 4 times, DCM 3 times, and methyl tert-butyl ether 3 times, each time 300 mL. Dry the resin and set aside for use.

[0093] (7) Peptide resin cleavage: A cleavage solution was prepared according to TFA / TIS / H2O / DTT = 90 / 2.5 / 2.5 / 5. The dried peptide resin was then added to the cleavage solution and stirred for 2.5 h. When the reaction was complete, the mixture was filtered and the filtrate was concentrated to 1 / 2 of its original volume. The mixture was then added to 5 volumes of methyl tert-butyl ether for precipitation. The mixture was filtered with suction and the filter cake was washed four times with methyl tert-butyl ether. The crude product was obtained after vacuum drying.

[0094] (8) Purification: The crude product was purified by C18 reverse phase preparative chromatography to obtain a purified product of polypeptide compound 1 with a purity of not less than 90%.

[0095] Table 1 exemplifies the experimental results of polypeptide compounds 1-5.

[0096] Table 1 List of synthetic peptide compounds and molecular weight

[0097] Example 2: Testing of the agonist activity of the test polypeptide molecules on GLP-1R, GIPR and GCGR at the cellular level

[0098] 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 agonist activity at the GIP / GLP-1 / GCG receptors, and Tirzepatide is a polypeptide with dual agonist activity at the GIP / GLP-1 receptors. The structure of the LY3437943 polypeptide is shown below:

[0099] Y-Aib-QGTFTSDYSI-αMel-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYL-LEGGPSSGAPPPS-NH2.

[0100] 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 can promote the increase of intracellular cAMP expression levels. cAMP interacts with the CRE site on the luciferase reporter gene to activate the expression of luciferase. The chemiluminescent substrate in the Luciferase Assay System generates fluorescence. The fluorescence signal response level is measured using a microplate reader to determine the agonist activity of each test peptide on the corresponding receptor. The specific steps are as follows:

[0101] 1) Preparation of cell suspension: Collect cells in good growth state and in the logarithmic growth phase, resuspend in assay medium (DMEM + 0.25% FBS), and count the cell density;

[0102] 2) Cell plating: Cell suspension was plated at 2×10 4 The cells were seeded at a density of 10 cells / well in a 96-well white flat-bottom plate;

[0103] 3) Add different concentrations of the test polypeptide molecules;

[0104] 4) Place in a 37°C, 5% CO2 incubator and incubate for 4 hours;

[0105] 5) After the incubation, take out the 96-well white flat-bottom plate and add Luciferase Assay System detection reagent, gently shake on a microplate thermostat at room temperature for 10 minutes;

[0106] 6) The microplate reader measures the luminescence value of each sample concentration;

[0107] 7) Using GraphPad Prism 8.0 software, the logarithm of the test sample concentration was plotted as the abscissa and the luminescence value as the ordinate. Nonlinear regression (curve fit) analysis was performed on the data to obtain the EC value of each test sample. 50 value.

[0108] The results are shown in Figures 3, 4, 5 and Table 2.50 Comparison of the values ​​shows that in the GLP-1R, GIPR, and GCGR activation test systems:

[0109] (1) For D21 and D22 molecules: the activities of D21 and D22 are weaker than LY3437943, and the activity of D21 is stronger than D22;

[0110] (2) For F01-F04 molecules: ① In the GLP-1R activation test system, according to the EC50 values, the agonist activity F04>F03>F01>F02. Among them, F04 has the best activity, stronger than LY3437943; F03 has good activity, comparable to LY3437943; F01 is second, and F02 has the weakest activity. Among the four molecules, F02 has an activity comparable to tilpoitide, and the remaining molecules are stronger than tilpoitide; ② In the GLP-1R activation test system, according to the EC50 values, the agonist activity F04>F01>F03>F02. Among them, F04 and F01 have good activity, better than LY3437943; F03 is second, and F02 has the weakest activity. Among the four molecules, F02 was weaker than tilpoxetine, while the remaining molecules were stronger than or equivalent to tilpoxetine. ③ In the GCGR activation test system, according to the EC50 values, the agonist activity of F01>F04>F02>F03. The agonist activity of the four molecules was weaker than that of LY3437943, among which F01 and F04 had better activity, F02 was slightly weaker, and F03 was the weakest.

[0111] Table 2. Activation EC of GLP-1R, GIPR and GCGR by the tested peptide molecules 50 value

[0112] Example 3: Hypoglycemic efficacy of D21 and F01-F04 peptides in db / db type II diabetic mice

[0113] Animals: 10-week-old db / db type II diabetic male mice.

[0114] The in vivo glucose-lowering ability test is as follows:

[0115] On the day of the experiment, db / db mice (N=7) were grouped according to body weight and blood glucose levels into vehicle 1, vehicle 2, LY3437943 (8 nmol / kg), F01 (8 nmol / kg), F02 (8 nmol / kg), F03 (8 nmol / kg), F04 (8 nmol / kg), and D21 (4 nmol / kg). Each group of mice received a single subcutaneous injection of the corresponding test polypeptide, and tail tip blood was collected for blood glucose measurement at 1, 2, 4, 6, 12, 24, 30, 48, 54, 72, 78, and 96 hours after administration. Body weight and food intake were measured 24 hours before administration and 24, 48, 72, and 96 hours after administration. As shown in Figures 6-7, compared to the vehicle groups, all peptides significantly reduced blood glucose levels in db / db mice, with F03 (8 nmol / kg), D21 (4 nmol / kg), and LY3437943 (8 nmol / kg) showing comparable effects. As shown in Figures 8-9, compared to the vehicle groups, all peptides reduced body weight and suppressed appetite in db / db mice. These results demonstrate that F01-F04 and D21 exhibit significant glucose-lowering properties in vivo. F03 (8 nmol / kg), D21 (4 nmol / kg), and LY3437943 (8 nmol / kg) exhibited comparable glucose-lowering efficacy, indicating that the D21 peptide possesses superior glucose-lowering properties.

[0116] Example 4: D21 polypeptide efficacy test on weight loss and lipid reduction in DIO mice

[0117] Animals: 20-week-old male DIO mice on a C57 background

[0118] The in vivo lipid-lowering ability test is as follows:

[0119] 20-week-old male DIO mice on a C57 background were divided into groups (N=8): vehicle 1, vehicle 2, LY3437943 (8 nmol / kg), D21 (8 nmol / kg), D21 (15 nmol / kg), and D21 (30 nmol / kg). Each group received the corresponding peptide via subcutaneous injection every three days for 30 days, with body weight and food intake measured every three days. As shown in Figures 10-11, compared to the corresponding vehicle group, each peptide group reduced DIO mouse body weight. D21 (8 nmol / kg) was comparable to LY3437943 (8 nmol / kg), with D21 (30 nmol / kg) showing the greatest effect. As shown in Figures 12-13, compared to the corresponding vehicle groups, each peptide group was able to suppress the appetite of mice and reduce abdominal fat content in mice. D21 (8 nmol / kg) and LY3437943 (8 nmol / kg) had comparable effects. These results demonstrate that D21 has a significant lipid-lowering ability in vivo. The same dose (8 nmol / kg) is comparable to LY3437943 (8 nmol / kg), and increasing its dose can achieve significantly better efficacy than LY3437943 (8 nmol / kg).

Claims

1. A polypeptide compound having GLP-1R / GIPR / GCGR tri-agonist activity or a pharmaceutically acceptable salt thereof, which has the following general formula (I): X1-Aib-QGTFTSDYSI-αMeL-LDK-X 17 -AQ-Aib-AFIEYL-XX1-XX2-XX3-R 1 (Ⅰ) in, X1 is Y or H; X 17 is Ψ; XX1 is IA, LE, L d E d , L d E.V. d D d or LE d ; XX2 is GG, GGG or GGGG; XX3 is PSSGAPPPSKVSRA, PSSGAPPPS, or PSSGA d PPPSKVSRA; R 1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof; Wherein, Ψ is a Lys with a side chain modified by a structure having 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 , wherein a, b, c are each independently 0 or 1, and a, b, c are not 0 at the same time, and Z is -CO-(CH2) m -R 2 , m is an integer between 6 and 24, R 2 Selected from COOH, the amino acid is Glu or γGlu.

2. The polypeptide compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the carboxyl end or acyl end of U in the general formula (II) is connected to the ε-amino group of the side chain of Lys.

3. The polypeptide compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein m is 18.

4. The polypeptide compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the general formula (II) is AEEA-γG1u-CO(CH2) 18 COOH.

5. The polypeptide compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the polypeptide compound is selected from the following:

6. A pharmaceutical composition comprising the polypeptide compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, diluent, carrier and / or excipient.

7. The pharmaceutical composition of claim 6, further comprising at least one further therapeutically active substance.

8. A method for treating a disease, the method comprising administering an effective amount of a polypeptide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or a pharmaceutical composition according to any one of claims 6 to 7 to an individual in need thereof, wherein the disease is selected from diabetes, diabetes-related disorders, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases associated with endocrine diseases, metabolic disorders, kidney diseases, etc. Preferably, the diabetes is type II diabetes.

9. Use of the polypeptide compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to any one of claims 6 to 7 in the preparation of a medicament for treating a disease, wherein the disease is selected from diabetes, diabetes-related disorders, obesity, Alzheimer's disease, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia, metabolic syndrome, and bone diseases related to endocrine diseases, metabolic disorders, kidney diseases, etc. Preferably, the diabetes is type II diabetes.

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