Tri-agonist polypeptide compound targeting GLP-1 receptor, glucagon receptor and GIP receptor and use thereof

Abstract

Provided is a tri-agonist polypeptide compound targeting a GLP-1 receptor, a glucagon receptor and a GIP receptor, wherein the polypeptide compound can selectively act on the GLP-1 receptor, the glucagon receptor and the GIP receptor, so as to exert the activities of GLP-1, glucagon and GIP at the same time, and the polypeptide compound has a unique agonistic activity proportion for the three receptors, thus achieving better glucose reduction, weight reduction and lipid regulation effects.

Description

A class of GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds and their applications Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a class of GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds and applications thereof. Background Art

[0002] Obesity and its associated metabolic syndrome have become a global public health concern. The incidence and course of many metabolic syndromes, such as type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and dyslipidemia, are closely linked to obesity. Studies have shown that 80-90% of T2DM patients are overweight or obese. Current medications for treating obesity have limited efficacy, and many have significant side effects.

[0003] Glucagon-like peptide-1 (GLP-1) is a glucose-dependent, hypoglycemic polypeptide hormone secreted by intestinal L cells. It exerts its glucose-lowering effects after specifically binding to the GLP-1 receptor. The primary advantage of GLP-1 is its glucose-dependent incretin secretion, which avoids the risk of hypoglycemia often associated with diabetes treatment. In addition to regulating blood sugar, GLP-1 can also prevent pancreatic β-cell degeneration and stimulate β-cell proliferation and differentiation, potentially addressing the underlying causes of diabetes. Furthermore, GLP-1 inhibits gastric acid secretion, delays gastric emptying, and suppresses appetite, potentially contributing to weight loss. Currently, several long-acting GLP-1 drugs are marketed, such as liraglutide and semaglutide. While GLP-1 drugs are safe for lowering blood sugar, achieving significant weight loss typically requires higher doses. High-dose GLP-1 drugs are prone to gastrointestinal side effects and poor tolerability, resulting in a narrow therapeutic window. Therefore, there remains a need for safer, more tolerable therapeutic agents that effectively reduce weight and control blood sugar.

[0004] Glucagon is a hormone secreted by pancreatic alpha cells. Under stressful conditions such as cold and hunger, it acts on the liver, breaking down glycogen in the liver and raising blood sugar. In addition to its blood sugar-raising effects, glucagon also promotes lipolysis, fat oxidation, and fever (Diabetologia, 2017, 60, 1851-1861). Long-term administration can increase energy metabolism and lead to weight loss. However, these beneficial effects on energy metabolism have been limited by glucagon's inherent blood sugar-raising effects.

[0005] Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide. Like GLP-1, it belongs to the incretin class and plays a key physiological role in the metabolism of blood glucose in the body. GIP exerts its physiological activity in the body by interacting with GIP receptors distributed in pancreatic β cells, adipose tissue, and the central nervous system. Similar to GLP-1, GIP can stimulate pancreatic β cells to secrete insulin, thereby lowering blood glucose, and can protect pancreatic β cells to control glucose metabolism in the body. In addition, GIP can also stimulate GIP receptors in adipose tissue to promote fat metabolism, and GIP also has the effect of suppressing appetite.

[0006] The GLP-1, glucagon, and GIP receptors corresponding to GLP-1, glucagon, and GIP all belong to the GPCR family of receptors and share similar protein structures and binding mechanisms, making it possible to design tri-agonist peptide compounds targeting these three receptors. These tri-agonist peptide compounds can act simultaneously on the GLP-1, glucagon, and GIP receptors, simultaneously exerting the activities of GLP-1, glucagon, and GIP. GLP-1 lowers blood sugar and suppresses appetite; glucagon breaks down fat and reduces weight, but it raises blood sugar, but this is offset by the glucose-lowering activity of GLP-1; and GIP primarily stimulates insulin secretion. The three activities of tri-agonist peptide compounds for the GLP-1, glucagon, and GIP receptors work together, forming a feedback mechanism based on blood sugar levels, enabling blood sugar control, fat breakdown, and weight loss. Tri-agonist peptide compounds for the GLP-1, glucagon, and GIP receptors offer significant advantages over single GLP-1 analogs for the treatment of metabolic diseases such as diabetes and obesity. Summary of the Invention

[0007] The present invention provides a class of GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds and applications thereof. The triple agonist polypeptide compounds can act on the GLP-1 receptor, glucagon receptor and GIP receptor simultaneously, and can simultaneously exert the activities of GLP-1, glucagon and GIP; they not only have the therapeutic effect of GLP-1 on diabetes, but also have the beneficial effects of glucagon on body weight, energy metabolism and lipid metabolism, and also have the beneficial effects of GIP on sugar and lipid metabolism and appetite suppression, thereby producing a synergistic effect on sugar, lipid and energy metabolism; they have greater potential for use in the preparation of drugs for treating metabolic syndrome, such as diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia and the like.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] A class of GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds, the amino acid sequence of the polypeptide compound is as follows:

[0010] Tyr-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa1-Leu-Asp-Lys-Xaa2-Ala-Gln-Aib -Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2,

[0011] wherein: Xaa1 is selected from Leu or αMeLeu; Xaa2 is selected from Lys or Lys with a modified side chain;

[0012] The side chain of the modified Lys is selected from

[0013] Wherein: n is a natural number, and 16≤n≤20.

[0014] Preferably, n is 16, 18 or 20.

[0015] Preferably, the sequence structure of the tri-agonist polypeptide compound is selected from any one of the amino acid sequences shown in SEQ ID NO: 1-2:

[0016] SEQ ID NO: 1

[0017] SEQ ID NO:2

[0018] The present invention also provides a pharmaceutically acceptable salt of the above-mentioned GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound.

[0019] Furthermore, the pharmaceutically acceptable salt is a salt formed by a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound and one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, hexanoic acid, benzenesulfonic acid, pamoic acid, benzoic acid, salicylic acid, lauric acid, cinnamic acid, propionic acid, dodecyl sulfuric acid, citric acid, ascorbic acid, stearic acid, tartaric acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, and sulfosalicylic acid.

[0020] The present invention also provides a class of medicaments prepared from the above-mentioned GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds, wherein the medicaments include any tablets, capsules, syrups, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions, suppositories or compound preparations mentioned in pharmacy.

[0021] The present invention also provides a pharmaceutical composition prepared from a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, the pharmaceutical composition comprising the class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, and a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprising a pharmaceutically acceptable salt of the class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, and a pharmaceutically acceptable carrier or diluent.

[0022] The present invention also provides use of the aforementioned GLP-1 receptor, glucagon receptor, and GIP receptor tri-agonist polypeptide compound, or a pharmaceutically acceptable salt thereof, or the aforementioned medicament, or the aforementioned pharmaceutical composition, in the preparation of a medicament for treating metabolic diseases or conditions, including diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or dyslipidemia.

[0023] The compound prepared by the present invention has strong agonist activity on the GLP-1 receptor, relatively strong agonist activity on the GIP receptor, and relatively weak agonist activity on the glucagon receptor, but achieves better blood sugar lowering, weight loss and lipid-regulating effects, and has lower gastrointestinal side effects, providing a new idea for the preparation of such GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds.

[0024] Compared with the prior art, the present invention has the following beneficial technical effects:

[0025] (1) The GLP-1 receptor, glucagon receptor, and GIP receptor triple agonist polypeptide compound of the present invention has a significant weight loss and weight gain prevention effect while more effectively lowering blood sugar, and better regulates lipid metabolism;

[0026] (2) The GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound of the present invention has a unique N-terminal 6-10 sequence structure (YTNDV) and a unique in vitro GLP-1 receptor, glucagon receptor and GIP receptor agonist activity ratio. The triple agonist polypeptide compound of the present invention has strong agonist activity on the GLP-1 receptor, relatively strong agonist activity on the GIP receptor, and relatively weak agonist activity on the glucagon receptor, yet brings about significantly improved hypoglycemic, weight loss and lipid metabolism regulation effects, with unexpected beneficial effects;

[0027] (3) Compared with the reported GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds, the GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds of the present invention have similar GLP-1 receptor agonist activity, similar GIP receptor agonist activity, and significantly weaker glucagon receptor agonist activity. However, the weight loss, lipid metabolism regulation and blood sugar lowering activities of the triple agonist polypeptide compounds of the present invention are significantly improved, and they have greater potential in the treatment of metabolic diseases, providing a new idea for the drug development of such GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds;

[0028] (4) The GLP-1 receptor, glucagon receptor, and GIP receptor tri-agonist polypeptide compound provided by the present invention has stable chemical properties and has pharmacokinetic characteristics that support at least once-weekly dosing; the GLP-1 receptor, glucagon receptor, and GIP receptor tri-agonist polypeptide compound provided by the present invention has a therapeutic effect on metabolic diseases such as T2DM, obesity, and dyslipidemia that is superior to existing GLP-1 marketed drugs and similar drugs under development. Therefore, the GLP-1 receptor, glucagon receptor, and GIP receptor tri-agonist polypeptide compound provided by the present invention is suitable as an active ingredient in drugs for treating metabolic diseases such as diabetes, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and dyslipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 shows the percentage change in body weight of DIO mice after long-term administration of each test substance of the present invention for 21 days. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0031] Unless otherwise defined herein, the scientific and technical terms used in this specification should have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, molecular biology, cell biology, and pharmacology described herein are those well known and commonly used in the art.

[0032] All combinations of the various elements disclosed in the present invention belong to the scope of the present invention.In addition, the scope of the present invention should not be limited by the specific disclosure provided below.

[0033] Furthermore, the amino acids mentioned in the present invention can be abbreviated as follows according to the IUPAC-IUB nomenclature:

[0034] Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V).

[0035] Furthermore, unless otherwise indicated, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0036] Furthermore, the "-NH2" moiety at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0037] Furthermore, in addition to natural amino acids, the sequences of the present invention also use unnatural amino acids, α-aminoisobutyric acid (Aib) and α-methylleucine (αMeLeu).

[0038] Furthermore, the polypeptide compound of the present invention can be synthesized by polypeptide solid phase synthesis or produced by genetic engineering technology.

[0039] In order to illustrate the present invention in more detail, this specification provides the following specific embodiments, but the embodiments of the present invention are not limited thereto.

[0040] Example 1

[0041] Synthesis of the polypeptide compound of SEQ ID NO: 1

[0042] (1) Swelling of resin

[0043] Weigh 0.382 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.262 mmol / g and place it in a 25 mL reactor. Wash the resin once with 7 mL of DCM and methanol alternately, wash the resin twice with 7 mL of DCM, then swell the resin with 7 mL of DCM for 1 hour, and finally wash the resin three times with 7 mL of DMF.

[0044] (2) Removal of Fmoc protecting group from resin

[0045] The swollen resin was transferred to a PSI-200 peptide synthesizer, and 7 mL of 20% piperidine / DMF (v / v) was added and reacted at room temperature for 5 min. The deprotection solution was filtered off, and the resin was washed once with 7 mL of DMF. 7 mL of 20% piperidine / DMF (v / v) deprotection solvent was then added to react with the resin for 15 min. Finally, the resin was washed four times with 7 mL of DMF, each time for 1.5 min, to obtain a Rink resin with the Fmoc protecting group removed.

[0046] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0047] Fmoc-Ser(tBu)-OH (0.4 mmol) was weighed and dissolved in 3 mL of 10% DMF / DMSO (v / v). 2 mL of DIC / HOBt (0.4 mmol / 0.44 mmol) condensing agent was added. After pre-activation for 30 min, the activated amino acid was added to the reactor and shaken at room temperature for 2 h. The reaction solution was filtered and the resin was washed four times with 7 mL of DMF. The Kaiser reagent was used to detect whether the reaction coupling was complete. If not, the reaction was repeated twice.

[0048] (4) Peptide chain extension

[0049] Following the peptide sequence, the above deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids until the peptide chain was complete. DIC / Oxyma (1.2 mmol / 1.2 mmol) was used as a condensing agent for coupling Ile at position 12, and the reaction was shaken at room temperature for 6 hours. The Lys site at position 17, where the side chain was modified, was protected with Fmoc-Lys(Dde)-OH, while the His at the N-terminus was protected with Boc-His(Boc)-OH.

[0050] (5) Modification of Lys side chain

[0051] After peptide chain synthesis was complete, 7 mL of 2% hydrazine hydrate / DMF (v / v) was added to selectively remove the Dde protecting group from Lys at position 17. After Dde protection was removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added and the mixture was shaken for 2 h. After Fmoc protection was removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added and the mixture was shaken for 2 h. After Fmoc protection was removed, 0.4 mmol of mono-tert-butyl eicosanedioate, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added and the mixture was shaken for 2 h. After the reaction was complete, the resin was washed four times with 7 mL of DMF.

[0052] (6) Peptide cleavage

[0053] The obtained resin with the peptide was transferred to a round-bottom flask, and the resin was cut with 5 mL of cutting agent Reagent R (TFA / thioanisole / phenol / EDT, 90:5:3:2, V / V). The reaction was kept at 30°C in an oil bath for 2 h. The cutting solution was poured into 40 mL of icy ether. After refrigerated centrifugation, the crude product was washed three times with 15 mL of icy ether and finally dried with nitrogen to obtain the crude peptide.

[0054] (7) Purification of polypeptides

[0055] The crude target peptide was dissolved in water, filtered through a 0.25 μm microporous filter membrane, and then purified using a Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were a C18 reversed-phase preparative column (250 mm × 20 mm, 12 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: methanol (V / V); flow rate of 8 mL / min; detection wavelength of 214 nm. A linear gradient (20% B to 70% B / 30 min) was used for elution, and the target peak was collected. After removing the methanol, lyophilization was performed to obtain 0.20 g of the pure product with a purity greater than 98%. The molecular weight of the target peptide was confirmed by MS. The theoretical relative molecular mass was 4710.4. ESI-MS m / z: calculated value [M+3H] 3+ 1517.1,[M+4H] 4+ 1178.6; observed value [M+3H] 3+ 1517.0,[M+4H] 4+ 1178.4.

[0056] Example 2

[0057] Synthesis of SEQ ID NO:2 polypeptide compound

[0058] The synthesis method is the same as that in Example 1, except for the modification of the Lys side chain, which is as follows: After the peptide chain is synthesized, 7 mL of 2% hydrazine hydrate / DMF (v / v) is added to selectively remove the Dde protecting group of Lys at position 17. After the Dde protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added, and the reaction is carried out under shaking for 2 hours. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added again, and the reaction is carried out under shaking for 2 hours. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC, and 0.44 mmol of HOBt are added, and the reaction is carried out under shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of mono-tert-butyl eicosanedioate, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added and the reaction was allowed to proceed for 2 hours. After completion, the resin was washed four times with 7 mL of DMF. The target peak was collected and lyophilized to obtain 0.21 g of pure product with a purity greater than 98%. The molecular weight of the target peptide was confirmed by MS. The theoretical relative molecular mass was 4855.6. ESI-MS m / z: calculated value [M+3H] 3+ 1619.5,[M+4H] 4+ 1214.9; observed value [M+3H] 3+ 1619.3,[M+4H] 4+ 1214.7.

[0059] Example 3

[0060] Determination of the agonist activity of peptide compounds on GLP-1 receptor, glucagon receptor and GIP receptor

[0061] The receptor agonist effects of peptide compounds were determined using a functional assay that measures the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor, glucagon receptor, or GIP receptor. Cells stably expressing each of the three receptors were plated into T175 culture flasks and grown overnight in culture medium to near confluence. The medium was then removed, and the cells were washed with calcium- and magnesium-free PBS and then protease-treated with Accutase. Detached cells were washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1× HBSS), the cell density was determined, and 25 μL aliquots were dispensed into the wells of a 96-well plate. For measurement, 25 μL of a solution of the test peptide compound in assay buffer was added to the wells and incubated at room temperature for 30 minutes. Cellular cAMP levels were determined using a Cisbio kit based on homogeneous time-resolved fluorescence (HTRF). After adding the HTRF reagent diluted in lysis buffer (kit component), the plate was incubated for 1 hour and then the fluorescence ratio at 665 / 620 nm was measured. The concentration that caused 50% activation of the maximal response (EC 50 ) to quantify the in vitro potency of agonists.

[0062] The test data (nM) in the examples of this patent application are shown in Table 1 below. Although the test data are stated with a certain number of significant figures, it should not be considered that the data have been determined to be accurate to the number of significant figures.

[0063] Table 1: EC values ​​of peptide compounds for human GLP-1 receptor, glucagon receptor and GIP receptor 50 Value (expressed in nM)

[0064] As shown in Table 1, the agonist activity of SEQ ID NO: 1 and SEQ ID NO: 2 on the GLP-1 receptor is slightly stronger than that of natural GLP-1 (about 2-4 times stronger) and retatrutide (Cell Metabolism, 2022, 34, 1234-1247, a clinically developed GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound, about 2-4 times stronger); the agonist activity of SEQ ID NO: 1 and SEQ ID NO: 2 on the glucagon receptor is significantly weaker than that of natural glucagon (about 259-282 times weaker) and retatrutide (about 248-270 times weaker); the agonist activity of SEQ ID NO: 1 and SEQ ID NO: 2 on the GIP receptor is slightly weaker than that of natural GIP (about 13-14 times weaker) and retatrutide (about 7-8 times weaker). It shows that the polypeptide compound of the present invention has strong GLP-1 receptor agonist activity, weak glucagon receptor agonist activity and good GIP receptor agonist activity, and has a special agonist activity ratio for GLP-1 receptor, GIP receptor and glucagon receptor. The agonist activity ratio for GLP-1 receptor, GIP receptor and glucagon receptor is significantly different from that of similar clinical drugs under development, and also meets the characteristics of the triple agonist polypeptide compound described in this patent.

[0065] Example 4

[0066] Pharmacokinetic properties of peptide compounds in rats

[0067] Rats were administered 50 nmol / kg of the drug by subcutaneous (sc) injection, and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36, and 48 hours post-dose. After protein precipitation with acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-lives were calculated using WinonLin 5.2.1 (non-compartmental model) (Table 2).

[0068] Table 2: Pharmacokinetic profile of peptide compounds in rats

[0069] As shown in the results in Table 2, the in vivo half-life of the polypeptide compound of the present invention is significantly prolonged, which is superior to the marketed once-weekly Semaglutide and the clinically investigated similar drug Retatrutide (Cell Metabolism, 2022, 34, 1234-1247), indicating that the polypeptide compound of the present invention has pharmacokinetic characteristics that support at least once-weekly dosing.

[0070] Example 5

[0071] Effects of peptide compounds on blood lipids and body weight in diet-induced obese (DIO) mice

[0072] Male C57BL / 6J mice, weighing approximately 22 g, were fed a high-fat diet (D12492) from Research Diets for 18 weeks to establish the DIO mouse model. Prior to dosing, DIO mice were randomly divided according to body weight into a saline group (blank control), a positive control group (semaglutide, retatrutide (a clinical-stage GLP-1 receptor, glucagon receptor, and GIP receptor tri-agonist peptide compound; Cell Metabolism, 2022, 34, 1234-1247)), and a test sample group (SEQ ID NO: 1, SEQ ID NO: 2). Each group of mice received a subcutaneous injection of saline (10 mg / kg), semaglutide (10 nmol / kg), retatrutide (10 nmol / kg), SEQ ID NO: 1 (10 nmol / kg), and SEQ ID NO: 2 (10 nmol / kg) every two days for a 21-day dosing cycle. The mice's body weight was recorded daily, and body fat was measured using nuclear magnetic resonance (NMR) before and after the experiment. At the end of the experiment, mice in each group were sacrificed, and liver tissue was collected to measure liver triglyceride (TG) and total cholesterol (TC) levels. Blood was also collected for serum preparation, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and total cholesterol (TC) levels were measured.

[0073] Table 3: Changes in body weight and body fat in DIO mice during the 3-week dosing period *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0074] As shown in Figure 1 and Table 3, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the body weight and body fat content of DIO mice after continuous administration for 3 weeks. The weight-reducing and body fat-reducing effects of the polypeptide compounds of the present invention are significantly stronger than those of the positive control drugs semaglutide and retatrutide. It is worth noting that the GLP-1 receptor agonist activity of retatrutide is similar to that of natural GLP-1, the glucagon receptor agonist activity is similar to that of natural glucagon, and the GIP receptor agonist activity is similar to that of natural GIP. Therefore, it can be seen that the GLP-1 receptor agonist activity of retatrutide is similar to that of SEQ ID NO: 1 and SEQ ID NO: 2, and its GIP receptor agonist activity is only about 7-8 times stronger than that of SEQ ID NO: 1 and SEQ ID NO: 2. However, the glucagon receptor agonist activity of retatrutide is significantly stronger than that of SEQ ID NO: 1 and SEQ ID NO: 2. However, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention show significantly better weight-reducing and body fat-reducing activities than retatrutide.

[0075] Table 4: Liver triglyceride (TG) and total cholesterol (TC) contents in DIO mice after 3 weeks of treatment *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0076] Table 5: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in DIO mice after 3 weeks of treatment *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0077] As shown in Tables 4 and 5, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the examples of the present invention can significantly reduce the liver triglyceride and total cholesterol levels of DIO mice, and significantly reduce the serum alanine aminotransferase and aspartate aminotransferase levels of the mice after continuous administration for 3 weeks. In addition, the effects of the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention are significantly stronger than those of the positive control drugs semaglutide and retatrutide, indicating that the polypeptide compounds of the present invention have good prospects for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0078] Table 6: Serum triglyceride (TG) and total cholesterol (TC) levels in DIO mice after 3 weeks of treatment *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0079] As shown in the results in Table 6, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the serum triglyceride and total cholesterol levels of DIO mice after continuous administration for 3 weeks. The effect of the polypeptide compounds of the present invention in reducing serum lipid (triglyceride and cholesterol) levels is significantly stronger than that of the positive control drugs semaglutide and retatrutide.

[0080] Example 6

[0081] Effects of polypeptide compounds on glycated hemoglobin (HbA1c) and blood glucose in db / db mice

[0082] Male db / db mice were randomly divided into groups of 6 per group. They were divided into a normal saline group (blank control group), a positive control group (semaglutide and retatrutide), and a test sample group (SEQ ID NO: 1 and SEQ ID NO: 2). After one week of adaptive feeding, blood was collected from the tail to measure the initial HbA1c value and fasting blood glucose value before the start of treatment. Each group of mice was subcutaneously injected with normal saline (10 mg / kg), semaglutide (10 nmol / kg), retatrutide (10 nmol / kg), SEQ ID NO: 1 (10 nmol / kg), and SEQ ID NO: 2 (10 nmol / kg) once every two days for a dosing period of 35 days. After the end of treatment, the mice were fasted overnight to measure the fasting blood glucose value, and blood was taken to measure the HbA1c (%) value.

[0083] Table 7: Changes in HbA1c (%) in db / db mice during a 35-day dosing period *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0084] As shown in Table 7, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the HbA1c values ​​of db / db mice after continuous administration for 35 days. In addition, the HbA1c values ​​of the mice in the polypeptide compound group after treatment were significantly lower than those of the positive controls semaglutide and retatrutide, indicating that the polypeptide compounds of the present invention have a good blood sugar control effect.

[0085] Table 8: Fasting blood glucose changes in db / db mice during the 35-day dosing period *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice in each group.

[0086] As shown in Table 8, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the Examples of the present invention can significantly reduce the fasting blood glucose levels of db / db mice when administered continuously for 35 days, indicating that the polypeptide compounds of the present invention have excellent blood glucose control effects, and the blood glucose control effects of the polypeptide compounds of the present invention are significantly stronger than those of the positive control drugs semaglutide and retatrutide.

[0087] Example 7

[0088] Gastrointestinal side effects of polypeptide compounds

[0089] Male Sprague-Dawley rats (200-250 g) were randomly divided into groups and housed individually in cages. Four days prior to the experiment, each group received a kaolin diet (Research Diets) in addition to their standard diet. The kaolin diet was placed in a separate compartment of the food hopper to allow the rats to habituate to the presence of the kaolin diet in their cages. Rats were fasted for 12 hours prior to the experiment. At 0 hours, each group received an intraperitoneal injection of 10% DMSO / water (blank), 3 mg / kg cisplatin (control group for model success), and 10 nmol / kg, 50 nmol / kg, and 100 nmol / kg of semaglutide, retatrutide, SEQ ID NO: 1, and SEQ ID NO: 2. Subsequently, each group was rapidly administered pre-weighed standard diet and kaolin diet. The amount of standard diet and kaolin diet consumed by each group over a 24-hour period was recorded. The intensity of the compound-induced side effects was determined based on the amount of standard diet and kaolin diet consumed.

[0090] Table 9: Food intake of SD rats in 24 hours: normal diet and kaolin *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 rats in each group.

[0091] Table 10: Food intake of SD rats in 24 hours: normal diet and kaolin *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 rats in each group.

[0092] Table 11: Food intake of SD rats in 24 hours: normal diet and kaolin *** : P < 0.001 compared with the blank control group; ### : P < 0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 rats in each group.

[0093] As shown in the results of Tables 9 to 11, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the examples of the present invention have a good effect of inhibiting rat feeding at doses of 10 nmol / kg, 50 nmol / kg, and 100 nmol / kg, which are significantly better than the positive controls semaglutide and retatrutide. However, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the examples of the present invention did not cause rats to eat kaolin at doses of 10 nmol / kg, 50 nmol / kg, and 100 nmol / kg, and the kaolin intake of the polypeptide compound group prepared in the examples of the present invention was similar to that of the blank group. At doses of 50 nmol / kg and 100 nmol / kg, the kaolin intake of rats in the SEQ ID NO: 1 and SEQ ID NO: 2 groups was significantly lower than the kaolin intake of rats in the positive control semaglutide and retatrutide groups. This shows that the polypeptide compounds prepared in the examples of the present invention did not cause gastrointestinal side effects in rats, and the gastrointestinal side effects were lower than those of the positive controls semaglutide and retatrutide.

Claims

1. A class of GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compounds, characterized in that: The amino acid sequence formula of the polypeptide compound is: Tyr-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa1-Leu-Asp-Lys-Xaa2-Ala-Gln-Aib-Ala- Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2, in: Xaa1 is selected from Leu or αMeLeu; Xaa2 is selected from Lys or Lys with a modified side chain; The side chain of the modified Lys is selected from Wherein: n is a natural number, and 16≤n≤20.

2. A GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound according to claim 1, characterized in that: The n is 16, 18 or 20.

3. A GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound according to claim 1, characterized in that: The sequence structure of the polypeptide compound is selected from any one of the amino acid sequences shown in SEQ ID NO: 1-2: SEQ ID NO: 1 SEQ ID NO:2 4. A pharmaceutically acceptable salt of the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to any one of claims 1 to 3.

5. The pharmaceutically acceptable salt of a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to claim 4, characterized in that: The pharmaceutically acceptable salt is a salt formed by a GLP-1 receptor, a glucagon receptor and a GIP receptor tri-agonist polypeptide compound and one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, caproic acid, benzenesulfonic acid, pamoic acid, benzoic acid, salicylic acid, lauric acid, cinnamic acid, propionic acid, dodecyl sulfuric acid, citric acid, ascorbic acid, stearic acid, stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, and sulfosalicylic acid.

6. A pharmaceutical agent prepared by the GLP-1 receptor, glucagon receptor and GIP receptor triple agonist polypeptide compound according to any one of claims 1 to 3, characterized in that: The medicament includes any tablet, capsule, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound preparation mentioned in pharmacy.

7. A pharmaceutical composition prepared from a triple agonist polypeptide compound of GLP-1 receptor, glucagon receptor and GIP receptor, characterized in that: The pharmaceutical composition comprises a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds as described in any one of claims 1-3, and a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds as described in any one of claims 4-5, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or diluent.

8. Use of a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to any one of claims 1 to 3, or a class of pharmaceutically acceptable salts of a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to any one of claims 4 to 5, or a class of medicaments according to claim 6, or a pharmaceutical composition according to claim 7 in the preparation of drugs for treating metabolic diseases or disorders.

9. The use according to claim 8, characterized in that The metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.

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