A glp-1 / gip receptor dual agonist and uses thereof

Abstract

The application discloses a GLP-1 / GIP receptor dual agonist and application thereof. The amino acid sequence general formula of the GLP-1 / GIP receptor dual agonist polypeptide compound is as follows: Tyr-Xaa1-Glu-Gly-Thr-Xaa2-Thr-Asn-Asp-Xaa3-Ser-Ile-Xaa4-Leu-Asp-Lys-Ile-Ala-Gln-Xaa5-Xaa6-Phe-Val-Gln-Trp-Leu-Xaa7-X aa8 -NH2. The application also relates to derivatives, long-acting compounds, pharmaceutically acceptable salts, pharmaceutical compositions and medicaments of the polypeptide compound. The GLP-1 / GIP receptor dual agonist polypeptide compound has more potential in preparation of drugs for treating metabolic syndrome and other diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a GLP-1 / GIP receptor dual agonist and its applications. Background Technology

[0002] Diabetes is another prevalent metabolic disease worldwide. According to data from the International Diabetes Federation, in 2021, there were 537 million adults aged 20-79 with diabetes globally, accounting for 10.5% of the global population in this age group. China has the largest number of adult diabetes patients. It is projected that by 2030, the number of people with diabetes worldwide will further increase, reaching over 643 million. Diabetes can be divided into insulin-dependent diabetes mellitus (type 1 diabetes, T1DM) and non-insulin-dependent diabetes mellitus (type 2 diabetes, T2DM), both characterized by progressive β-cell depletion. T1DM is caused by an autoimmune attack on β-cells, leading to progressive β-cell apoptosis. T2DM, on the other hand, is mainly caused by insulin resistance and pancreatic β-cell dysfunction, resulting in chronic hyperglycemia. Its pathogenesis is caused by multiple factors and is accompanied by various complications. Of the two types of diabetes, T2DM accounts for approximately 90% of all diabetes cases worldwide. Studies have shown that T2DM is associated with several high-risk complications, such as cardiovascular disease, diabetic nephropathy, and blindness. Obesity is one of the main causes of type 2 diabetes mellitus (T2DM). The progression from obesity to diabetes generally follows this pattern: obesity → impaired glucose tolerance → T2DM → uncontrollable hyperglycemia → diabetic complications. Therefore, in the treatment of diabetes, in addition to lowering blood sugar, weight loss is also a crucial factor to consider.

[0003] Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells in the terminal jejunum, ileum, and colon. It exerts its hypoglycemic effect after specifically binding to the GLP-1 receptor. A major advantage of GLP-1 is its glucose-dependent incretin secretion, avoiding the risk of hypoglycemia often present in diabetes treatment. Besides regulating blood sugar, GLP-1 can also prevent pancreatic β-cell degeneration and stimulate β-cell proliferation and differentiation, thus improving the progression of diabetes at its source. Furthermore, GLP-1 also inhibits gastric acid secretion, delays gastric emptying, and suppresses appetite, resulting in some weight loss effects. Several long-acting GLP-1 drugs are currently available, such as liraglutide, semaglutide, and dulaglutide. Although GLP-1 drugs have a safe hypoglycemic effect, achieving significant weight loss generally requires higher doses. However, high doses of GLP-1 drugs can easily cause gastrointestinal side effects, leading to poor tolerability and a narrow therapeutic window. Therefore, there is still a need for safer and more tolerable treatments that can effectively reduce weight and control blood sugar.

[0004] Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide, belonging to the intestinal hypoglycemic group along with GLP-1, and plays a crucial physiological role in glucose metabolism. GIP exerts its physiological activity through its receptors distributed in pancreatic β-cells, adipose tissue, and the central nervous system. Similar to GLP-1, GIP stimulates pancreatic β-cells to secrete insulin, thereby lowering blood glucose, and can protect pancreatic β-cells, thus controlling glucose metabolism. Furthermore, GIP can stimulate GIP receptors in adipose tissue, promoting fat metabolism, and also has an appetite-suppressing effect. Under normal physiological conditions, GIP and GLP-1 are secreted from the intestines after meals, enhancing physiological responses to food, including satiety, insulin secretion, and nutrient processing. However, the incretin response of GIP is impaired in patients with type 2 diabetes mellitus (T2DM). Studies have shown that the inhibitory effect of GIP produced in T2DM patients is significantly reduced when blood glucose levels return to normal. Therefore, the use of GIP to treat type 2 diabetes mellitus can be combined with some clinically effective hypoglycemic drugs to reduce the tolerance of patients with type 2 diabetes mellitus to GIP, and further utilize the incretin effect of GIP to achieve better hypoglycemic effect.

[0005] Currently reported peptide-based GLP-1 / GIP receptor dual agonists are mainly designed based on the sequences of natural GLP-1 and GIP. Published patent documents include: WO2016 / 111971, CN110684082A, CN111825758A, WO2011 / 119657, WO2013 / 164483, etc.

[0006] GLP-1 in amphibians has similar effects to human GLP-1, so structural modifications to amphibian GLP-1 may lead to the discovery of novel GLP-1 drugs with more efficient and longer-lasting hypoglycemic effects. XenGLP-1 is an animal-derived GLP-1 analogue discovered in the African clawed frog. Compared to natural GLP-1, XenGLP-1 exhibits superior hypoglycemic activity and stability. XenGLP-1 is a highly potent agonist of the GLP-1 receptor; however, it does not activate the GIP receptor. XenGLP-1 exhibits many of the glucose-regulating effects observed in natural GLP-1. Numerous preclinical studies have demonstrated several beneficial anti-diabetic properties of XenGLP-1, including enhanced glucose-dependent insulin synthesis and secretion, slowed gastric emptying, reduced food intake and weight, and promotion of β-cell proliferation and restoration of pancreatic islet function (Biochem. Pharmacol., 2017, 142, 155–167; FASEB J., 2019, 33, 7113-7125). These effects are beneficial not only for diabetic patients but also for obese patients. Currently reported dual GLP-1 / GIP receptor agonists typically possess GIP receptor agonist activity similar to or stronger than natural GIP, and GLP-1 receptor agonist activity similar to or weaker than natural GLP-1. For example, the marketed tirzepatide has GIP receptor agonist activity similar to natural GIP, but its GLP-1 receptor agonist activity is approximately 13 times weaker than natural GLP-1. Further research is needed on the agonistic activity of GLP-1 / GIP receptor dual agonists on different receptors and their application in the preparation of drugs for metabolic syndrome. Summary of the Invention

[0007] This invention provides a dual GLP-1 / GIP receptor agonist and its applications. Based on variants designed from the XenGLP-1 and GIP sequences, this invention retains the therapeutic effects of XenGLP-1 on diabetes while possessing the beneficial effects of GIP on glucose and lipid metabolism and appetite suppression, thus generating a synergistic effect on glucose, lipid, and energy metabolism. This presents greater potential than single GLP-1 and GIP receptor agonists in the development of drugs for treating metabolic syndromes such as diabetes, obesity, hypertension, non-alcoholic steatohepatitis, dyslipidemia, and Alzheimer's disease.

[0008] The technical solution of the present invention is as follows:

[0009] One object of the present invention is to provide a GLP-1 / GIP receptor dual-agonist polypeptide compound having the following general amino acid sequence formula:

[0010] Tyr-Xaa1-Glu-Gly-Thr-Xaa2-Thr-Asn-Asp-Xaa3-Ser-Ile-Xaa4-Leu-Asp-Lys-Ile-Al a-Gln-Xaa5-Xaa6-Phe-Val-Gln-Trp-Leu-Xaa7-X aa8 -NH2(Ⅰ)

[0011] Wherein: Xaa1 is selected from Ala or Aib; Xaa2 is selected from Tyr or Phe; Xaa3 is selected from Tyr or Val; Xaa4 is selected from Tyr or Aib; Xaa5 is selected from Lys or Lys with modified side chains; Xaa6 is selected from Glu or Ala; Xaa7 is selected from Leu or Ile; X aa8 Selected from any natural amino acid or peptide composed of them, or not present;

[0012] Furthermore, the Lys modified by the side chain is selected from...

[0013]

[0014] Where: n is a natural number, and 12≤n≤24.

[0015] Furthermore, n is 14, 16, 18, or 20.

[0016] Furthermore, the sequence of the GLP-1 / GIP receptor dual agonist polypeptide compound is arranged from left to right, with amino acids 6-10 being a combination of 5 amino acids, specifically Tyr-Thr-Asn-Asp-Val or Tyr-Thr-Asn-Asp-Tyr or Phe-Thr-Asn-Asp-Val or Phe-Thr-Asn-Asp-Tyr.

[0017] Furthermore, X aa8 is Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser.

[0018] Furthermore, the amino acid sequence of the polypeptide compound is shown in SEQ ID NO: 16:

[0019] SEQ ID NO:16

[0020] Tyr-Xaa1-Glu-Gly-Thr-Xaa2-Thr-Asn-Asp-Xaa3-Ser-Ile-Xaa4-Leu-Asp-Lys-Ile-Al a-Gln-Xaa5-Xaa6-Phe-Val-Gln-Trp-Leu-Xaa7-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0021] Wherein: Xaa1 is selected from Ala or Aib; Xaa2 is selected from Tyr or Phe; Xaa3 is selected from Tyr or Val; Xaa4 is selected from Tyr or Aib; Xaa5 is selected from Lys or Lys with modified side chains; Xaa6 is selected from Glu or Ala; Xaa7 is selected from Leu or Ile;

[0022] Among them, the Lys with modified side chains are selected from

[0023]

[0024] Where n is a natural number, and 12≤n≤24.

[0025] Furthermore, the sequence structure of the polypeptide compound includes, but is not limited to, the amino acid sequences of SEQ ID NO:1-15 (X in the sequence listing represents Aib):

[0026] SEQ ID NO:1

[0027] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0028] SEQ ID NO:2

[0029] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0030] SEQ ID NO:3

[0031] Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0032] SEQ ID NO:4

[0033] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0034] SEQ ID NO:5

[0035] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Glu-Phe-Val-Gln-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0036] SEQ ID NO:6

[0037]

[0038] SEQ ID NO:7

[0039]

[0040] SEQ ID NO:8

[0041]

[0042] SEQ ID NO:9

[0043]

[0044] SEQ ID NO:10

[0045]

[0046] SEQ ID NO:11

[0047]

[0048] SEQ ID NO:12

[0049]

[0050] SEQ ID NO:13

[0051]

[0052] SEQ ID NO:14

[0053]

[0054] SEQ ID NO:15

[0055]

[0056] Another object of the present invention is to provide a derivative of the above-mentioned GLP-1 / GIP receptor dual agonist polypeptide compound, wherein the derivative is obtained by fusing the above-mentioned dual polypeptide compound with other polypeptide substances; the other polypeptide substances include, but are not limited to, glucagon, gastrin (OXM), tyrosinase (PYY), fibroblast growth factor 21 (FGF21) or amylin.

[0057] Another object of the present invention is to provide a long-acting compound prepared from the above-mentioned GLP-1 / GIP receptor dual agonist polypeptide compound, wherein the long-acting compound is prepared by replacing or cyclizing the above-mentioned dual agonist polypeptide compound with amino acids; or by further modifying it with polyethylene glycol, fusing a long-acting protein fragment, or modifying it with conjugated lipid chains; wherein the long-acting protein fragment includes, but is not limited to, bovine serum albumin (BSA), Fc fusion protein, human chorionic gonadotropin (HCG), or unstructured biodegradable protein polymer (Xten).

[0058] Another object of the present invention is to provide a pharmaceutically acceptable salt of the above-mentioned GLP-1 / GIP receptor dual agonist polypeptide compound.

[0059] Further, the pharmaceutically acceptable salt is a salt formed by a GLP-1 / GIP receptor dual-agonist polypeptide compound and one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, hexanoic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy- 2-Naphthoic acid, camphor sulfonic acid, digluconic acid, nicotinic acid, pyric acid, propionic acid, persulfate, picric acid, 3-phenylpropionic acid, tervaline, itaconic acid, 2-hydroxyethanesulfonic acid, aminosulfonic acid, dodecyl sulfate, trifluoromethanesulfonic acid, naphthalene disulfonic acid, 2-naphthalene sulfonic acid, citric acid, mandelic acid, ascorbic acid, stearic acid, tartaric acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfonic acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphate, glucohepanoic acid, aspartic acid, thiocyanate, or sulfosalicylic acid.

[0060] Another object of the present invention is to provide a pharmaceutical preparation made from the above-mentioned GLP-1 / GIP receptor dual agonist polypeptide compound.

[0061] Furthermore, the pharmaceutical preparation includes any pharmaceutically defined tablet, capsule, syrup, tincture, inhaler, spray, injection, film, patch, powder, granule, emulsion, suppository, or compound preparation.

[0062] Another object of the present invention is to provide a pharmaceutical composition prepared from a GLP-1 / GIP receptor dual-agonist polypeptide compound.

[0063] Further, the pharmaceutical composition comprises the GLP-1 / GIP receptor dual agonist polypeptide compound and a carrier or diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt of the GLP-1 / GIP receptor dual agonist polypeptide compound and a carrier or diluent.

[0064] Another object of the present invention is to provide the use of the GLP-1 / GIP receptor dual agonist polypeptide compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical agent thereof, or the pharmaceutical composition thereof in the preparation of a medicament for treating metabolic diseases or conditions, including diabetes, obesity, hypertension, non-alcoholic steatohepatitis, dyslipidemia, or Alzheimer's disease.

[0065] The compounds prepared in this invention exhibit strong agonistic activity against GLP-1 receptors and weaker agonistic activity against GIP receptors, yet achieve better hypoglycemic and weight-loss effects, providing a new approach for the preparation of multiple agonists. The reason this invention achieves these effects is due to the unique N-terminal sequence of the prepared compounds. Specifically, the N-terminal sequence of the compounds utilizes fragments of the N-terminal sequence of XenGLP-1, such as the YTNDV sequence fragments at positions 6-10, the TNDV sequence fragments at positions 7-10, and the YTND sequence fragments at positions 6-9. This results in compounds with different ratios of GLP-1 and GIP receptor agonistic activity (high GLP-1 receptor agonistic activity, weak GIP receptor agonistic activity), while still achieving highly efficient hypoglycemic and weight-loss effects.

[0066] The beneficial technical effects of this invention are as follows:

[0067] (1) This invention designs the 8th amino acid to be Asn, which is completely different from natural GLP-1 and GIP. This mutation results in a unique ratio of GLP-1 and GIP receptor agonist activity in the polypeptide compound, thus leading to superior weight loss and blood sugar lowering activities. In addition, the 6th-10th amino acids in this invention form a combination pattern Tyr(Phe)-Thr-Asn-Asp-Val(Tyr). The amino acids in this region are different from natural GLP-1 and GIP. This mutation leads to a special change in the binding of the polypeptide compound to GLP-1 and GIP receptors, resulting in higher agonist activity of the polypeptide compound to GLP-1 receptors but weaker agonist activity to GIP receptors, thus leading to superior weight loss and blood sugar lowering activities.

[0068] (2) Compared with existing GLP-1 receptor agonists, the GLP-1 / GIP receptor dual agonist polypeptide compound of the present invention has significant weight loss and anti-weight gain effects while more effectively lowering blood glucose, reversing insulin resistance, and regulating lipid metabolism. Compared with existing GLP-1 / GIP receptor dual agonists, the polypeptide compound of the present invention has a unique N-terminal sequence structure and a unique in vitro GLP-1 and GIP receptor agonist activity ratio, thereby bringing about significantly improved weight loss and lipid metabolism regulation effects, and has unexpected beneficial effects compared with existing drugs.

[0069] (3) The polypeptide compounds provided by this invention are chemically stable and have pharmacokinetic characteristics that support once-daily or once-weekly administration. The polypeptide compounds provided by this invention have superior therapeutic effects on metabolic diseases such as type 2 diabetes mellitus (T2DM), obesity, and dyslipidemia compared to existing marketed drugs. Therefore, the polypeptide compounds provided by this invention are suitable as active ingredients in drugs for treating metabolic diseases such as diabetes, obesity, hypertension, non-alcoholic steatohepatitis, and dyslipidemia. Attached Figure Description

[0070] Figure 1 This invention demonstrates the long-lasting hypoglycemic effect of a single dose of each test substance in db / db mice under non-fasting conditions.

[0071] Figure 2 The percentage change in body weight of each test substance of the present invention after 21 days of long-term administration to DIO mice. Detailed Implementation

[0072] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0073] Unless otherwise defined in this specification, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, molecular biology, cell biology, and pharmacology as described herein are well-known and commonly used in the art.

[0074] All combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention should not be limited by the specific disclosures provided below.

[0075] The GLP-1 / GIP receptor dual agonist polypeptide compound of this invention has an Asn at position 8 and / or a combination of 5 amino acids at positions 6-10, specifically Tyr-Thr-Asn-Asp-Val, Tyr-Thr-Asn-Asp-Tyr, Phe-Thr-Asn-Asp-Val, or Phe-Thr-Asn-Asp-Tyr. Any polypeptide obtained by adjusting amino acids at other positions is within the scope of protection of this application.

[0076] Furthermore, the amino acids mentioned in this invention can be abbreviated according to the IUPAC-IUB naming rules as follows:

[0077] 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).

[0078] Furthermore, unless otherwise specified, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0079] Furthermore, the "-NH2" portion at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0080] Furthermore, in addition to natural amino acids, the sequence of this invention also uses a non-natural amino acid, α-aminoisobutyric acid (Aib).

[0081] Furthermore, the polypeptide compounds described in this invention can be synthesized by solid-phase polypeptide synthesis or produced by genetic engineering technology.

[0082] Furthermore, the abbreviations used in this invention are explained as follows: DCM: dichloromethane; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; TFA: trifluoroacetic acid; EDT: 1,2-ethylenedithiol;

[0083] To illustrate the present invention in more detail, the following specific embodiments are provided in this specification, but the embodiments of the present invention are not limited thereto.

[0084] Example 1

[0085] Synthesis of polypeptide compounds with sequence structures such as SEQ ID NO:1

[0086] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0087] That is, Y(Aib)EGTYTNDYSI(Aib)LDKIAQKAFVQWLIAGGPSSGAPPPS-NH2

[0088] (1) Swelling of resin

[0089] Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 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, swell the resin with 7 mL of DCM for 1 h, and finally wash the resin three times with 7 mL of DMF.

[0090] (2) Removal of Fmoc protecting groups from resin

[0091] The swollen resin was transferred to a PSI200 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. Then, 7 mL of 20% piperidine / DMF (v / v) deprotection solvent was added and reacted with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF for 1.5 min each time to obtain Rink resin with the Fmoc protecting group removed.

[0092] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0093] Fmoc-Ser(Boc)-OH (0.4 mmol) was dissolved in 3 mL of 10% DMF / DMSO (v / v), and 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 the reaction was carried out at room temperature with shaking for 2 h. After filtering off the reaction solution, the resin was washed 4 times with 7 mL of DMF. Kaiser reagent was used to check whether the reaction coupling was complete. If it was not complete, the coupling was performed twice.

[0094] (4) Elongation of peptide chains

[0095] Following the peptide chain sequence, repeat the above deprotection and coupling steps to sequentially connect the corresponding amino acids until the peptide chain synthesis is complete.

[0096] (5) Peptide cleavage

[0097] The obtained peptide-linked resin was transferred to a round-bottom flask and cleaved with 5 mL of Reagent R (TFA / anisole / phenol / EDT, 90:5:3:2, V / V). The reaction was carried out at a constant temperature of 30°C in an oil bath for 2 h. The cleavage solution was poured into 40 mL of ice-cold ether. After freezing and centrifugation, the crude product was washed three times with 15 mL of ice-cold ether and finally dried with nitrogen to obtain the crude peptide.

[0098] (6) Purification of peptides

[0099] The crude target peptide was dissolved in water, filtered through a 0.25 μm microporous membrane, and then purified using a Shimadzu preparative reversed-phase HPLC system. Chromatographic conditions were: 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: 8 mL / min; detection wavelength: 214 nm. A linear gradient elution (20% B to 70% B / 30 min) was used. The target peak was collected, methanol was removed, and the product was lyophilized to obtain 0.12 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 4112.5. ESI-MS m / z: calculated values ​​[M+3H]3+1371.8, [M+4H]4+1029.1; observed values ​​[M+3H]3+1371.7, [M+4H]4+1029.0.

[0100] Example 2

[0101] Synthesis of the polypeptide compound SEQ ID NO:2

[0102] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0103] That is, the synthesis method of Y(Aib)EGTYTNDVSI(Aib)LDKIAQKAFVQWLIAGGPSSGAPPPS-NH2 is the same as in Example 1. The target peak was collected and lyophilized to obtain 0.11 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4048.5. ESI-MS m / z: calculated values ​​[M+3H]3+1350.5, [M+4H]4+1013.1; observed values ​​[M+3H]3+1350.4, [M+4H]4+1013.0.

[0104] Example 3

[0105] Synthesis of the polypeptide compound SEQ ID NO:3

[0106] Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0107] That is, the synthesis method of Y(Aib)EGTFTNDVSI(Aib)LDKIAQKAFVQWLIAGGPSSGAPPPS-NH2 is the same as in Example 1. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4032.5. ESI-MS m / z: calculated values ​​[M+3H]3+1345.2, [M+4H]4+1009.1; observed values ​​[M+3H]3+1345.0, [M+4H]4+1009.0.

[0108] Example 4

[0109] Synthesis of the polypeptide compound SEQ ID NO:4

[0110] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Glu-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0111] That is, the synthesis method of Y(Aib)EGTYTNDVSI(Aib)LDKIAQKEFVQWLIAGGPSSGAPPPS-NH2 is the same as in Example 1. The target peak was collected and lyophilized to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4106.6. ESI-MS m / z: calculated values ​​[M+3H]3+1369.9, [M+4H]4+1027.7; observed values ​​[M+3H]3+1369.7, [M+4H]4+1027.5.

[0112] Example 5

[0113] Synthesis of the polypeptide compound SEQ ID NO:5

[0114] Tyr-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gl n-Lys-Glu-Phe-Val-Gln-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0115] That is, the synthesis method of Y(Aib)EGTYTNDVSI(Aib)LDKIAQKEFVQWLLAGGPSSGAPPPS-NH2 is the same as in Example 1. The target peak was collected and lyophilized to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4106.6. ESI-MS m / z: calculated values ​​[M+3H]3+1369.9, [M+4H]4+1027.7; observed values ​​[M+3H]3+1369.7, [M+4H]4+1027.5.

[0116] Example 6

[0117] Synthesis of the polypeptide compound SEQ ID NO:6

[0118]

[0119] (1) Swelling of resin

[0120] Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 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, swell the resin with 7 mL of DCM for 1 h, and finally wash the resin three times with 7 mL of DMF.

[0121] (2) Removal of Fmoc protecting groups from resin

[0122] The swollen resin was transferred to a PSI200 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. Then, 7 mL of 20% piperidine / DMF (v / v) deprotection solvent was added and reacted with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF for 1.5 min each time to obtain Rink resin with the Fmoc protecting group removed.

[0123] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0124] Fmoc-Ser(Boc)-OH (0.4 mmol) was dissolved in 3 mL of 10% DMF / DMSO (v / v), and 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 the reaction was carried out at room temperature with shaking for 2 h. After filtering off the reaction solution, the resin was washed 4 times with 7 mL of DMF. Kaiser reagent was used to check whether the reaction coupling was complete. If it was not complete, the coupling was performed twice.

[0125] (4) Elongation of peptide chains

[0126] Following the peptide chain sequence, the above deprotection and coupling steps are repeated sequentially to add the corresponding amino acids until the peptide chain synthesis is complete. The 20-position Lys can be protected using Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH, or Fmoc-Lys(ivDde)-OH, etc. In this example, the Fmoc-Lys(Dde)-OH protection strategy is used, while the N-terminal His is protected using Boc-His(Boc)-OH.

[0127] (5) Modification of Lys sidechains

[0128] After peptide synthesis, 7 mL of 2% hydrazine hydrate / DMF (v / v) was added to selectively remove the Dde protecting group at position 12 (Lys). After Dde protection, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added, and the reaction was shaken for 2 h. Then, the Fmoc protecting group was removed using the same method, followed by the addition of 0.4 mmol of palmitic acid, 0.4 mmol of DIC, and 0.44 mmol of HOBt, and the condensation reaction was carried out for 2 h. After the reaction was complete, the resin was washed four times with 7 mL of DMF.

[0129] (6) Peptide cleavage

[0130] The obtained peptide-linked resin was transferred to a round-bottom flask and cleaved with 5 mL of Reagent R (TFA / anisole / phenol / EDT, 90:5:3:2, V / V). The reaction was carried out at a constant temperature of 30°C in an oil bath for 2 h. The cleavage solution was poured into 40 mL of ice-cold ether. After freezing and centrifugation, the crude product was washed three times with 15 mL of ice-cold ether and finally dried with nitrogen to obtain the crude peptide.

[0131] (7) Purification of peptides

[0132] The crude target peptide was dissolved in water, filtered through a 0.25 μm microporous membrane, and then purified using a Shimadzu preparative reversed-phase HPLC system. Chromatographic conditions were: 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: 8 mL / min; detection wavelength: 214 nm. A linear gradient elution (20% B to 80% B / 30 min) was used. The target peak was collected, methanol was removed, and the product was lyophilized to obtain 0.13 g of pure peptide with a purity greater than 98%. The molecular weight of the target peptide was confirmed by MS. The theoretical relative molecular mass was 4480.1. ESI-MS m / z: calculated values ​​[M+3H]3+1494.4, [M+4H]4+1121.0; observed values ​​[M+3H]3+1494.3, [M+4H]4+1120.9.

[0133] Example 7

[0134] Synthesis of the polypeptide compound SEQ ID NO:7

[0135]

[0136] The synthesis method was the same as in Example 6. The target peak was collected and lyophilized to obtain 0.11 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4416.0. ESI-MS m / z: calculated values ​​[M+3H]3+1473.0, [M+4H]4+1105.0; observed values ​​[M+3H]3+1472.9, [M+4H]4+1104.9.

[0137] Example 8

[0138] Synthesis of the polypeptide compound SEQ ID NO:8

[0139]

[0140] The synthesis method was the same as in Example 6. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4400.0. ESI-MS m / z: calculated values ​​[M+3H]3+1467.7, [M+4H]4+1101.0; observed values ​​[M+3H]3+1467.5, [M+4H]4+1100.8.

[0141] Example 9

[0142] Synthesis of the polypeptide compound SEQ ID NO:9

[0143]

[0144] The synthesis method was the same as in Example 6. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4474.1. ESI-MS m / z: calculated values ​​[M+3H]3+1492.4, [M+4H]4+1119.5; observed values ​​[M+3H]3+1492.2, [M+4H]4+1119.3.

[0145] Example 10

[0146] Synthesis of the polypeptide compound SEQ ID NO:10

[0147]

[0148] The synthesis method was the same as in Example 6. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4474.1. ESI-MS m / z: calculated values ​​[M+3H]3+1492.4, [M+4H]4+1119.5; observed values ​​[M+3H]3+1492.2, [M+4H]4+1119.3.

[0149] Example 11

[0150] Synthesis of the polypeptide compound SEQ ID NO:11

[0151]

[0152] (1) Swelling of resin

[0153] Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 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, swell the resin with 7 mL of DCM for 1 h, and finally wash the resin three times with 7 mL of DMF.

[0154] (2) Removal of Fmoc protecting groups from resin

[0155] 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. Then, 7 mL of 20% piperidine / DMF (v / v) deprotection solvent was added and reacted with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF for 1.5 min each time to obtain Rink resin with the Fmoc protecting group removed.

[0156] (3) Synthesis of Fmoc-Ser-Rink amide-MBHA Resin

[0157] Fmoc-Ser(Boc)-OH (0.4 mmol) was dissolved in 3 mL of 10% DMF / DMSO (v / v), and 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 the reaction was carried out at room temperature with shaking for 2 h. After filtering off the reaction solution, the resin was washed 4 times with 7 mL of DMF. Kaiser reagent was used to check whether the reaction coupling was complete. If it was not complete, the coupling was performed twice.

[0158] (4) Elongation of peptide chains

[0159] Following the peptide chain sequence, the above deprotection and coupling steps are repeated sequentially to add the corresponding amino acids until the peptide chain synthesis is complete. The 20-position Lys can be protected using Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH, or Fmoc-Lys(ivDde)-OH, etc. In this example, the Fmoc-Lys(Dde)-OH protection strategy is used, while the N-terminal His is protected using Boc-His(Boc)-OH.

[0160] (5) Modification of Lys sidechains

[0161] After peptide synthesis, 7 mL of 2% hydrazine hydrate / DMF (v / v) was added to selectively remove the Dde protecting group at position 20 (Lys). After Dde protection, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added, and the reaction was carried out with shaking for 2 h. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added again, and the reaction was carried out with shaking for 2 h. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC, and 0.44 mmol of HOBt were added, and the reaction was carried out with shaking for 2 h. After removing the Fmoc protecting group, 0.4 mmol of eicosanoic acid monotert-butyl ester, 0.4 mmol of DIC and 0.44 mmol of HOBt were added, and the condensation reaction was carried out for 2 hours. After the reaction was complete, the resin was washed 4 times with 7 mL of DMF.

[0162] (6) Peptide cleavage

[0163] The obtained peptide-linked resin was transferred to a round-bottom flask and cleaved with 5 mL of Reagent R (TFA / anisole / phenol / EDT, 90:5:3:2, V / V). The reaction was carried out at a constant temperature of 30°C in an oil bath for 2 h. The cleavage solution was poured into 40 mL of ice-cold ether. After freezing and centrifugation, the crude product was washed three times with 15 mL of ice-cold ether and finally dried with nitrogen to obtain the crude peptide.

[0164] (7) Purification of peptides

[0165] The crude target peptide was dissolved in water, filtered through a 0.25 μm microporous membrane, and then purified using a Shimadzu preparative reversed-phase HPLC system. Chromatographic conditions were: 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: 8 mL / min; detection wavelength: 214 nm. A linear gradient elution (20% B to 80% B / 30 min) was used. The target peak was collected, methanol was removed, and the product was lyophilized to obtain 0.19 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 4856.5. ESI-MS m / z: calculated values ​​[M+3H]3+1619.8, [M+4H]4+1215.1; observed values ​​[M+3H]3+1619.5, [M+4H]4+1214.9.

[0166] Example 12

[0167] Synthesis of the polypeptide compound SEQ ID NO:12

[0168]

[0169] The synthesis method was the same as in Example 11. The target peak was collected and lyophilized to obtain 0.14 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4792.4. ESI-MS m / z: calculated values ​​[M+3H]3+1598.5, [M+4H]4+1199.1; observed values ​​[M+3H]3+1598.5, [M+4H]4+1198.9.

[0170] Example 13

[0171] Synthesis of the polypeptide compound SEQ ID NO:13

[0172]

[0173] The synthesis method was the same as in Example 11. The target peak was collected and lyophilized to obtain 0.14 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4776.4. ESI-MS m / z: calculated values ​​[M+3H]3+1593.1, [M+4H]4+1195.1; observed values ​​[M+3H]3+1592.9, [M+4H]4+1194.9.

[0174] Example 14

[0175] Synthesis of the polypeptide compound SEQ ID NO:14

[0176]

[0177] The synthesis method was the same as in Example 11. The target peak was collected and lyophilized to obtain 0.13 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4850.5. ESI-MS m / z: calculated values ​​[M+3H]3+1617.8, [M+4H]4+1213.6; observed values ​​[M+3H]3+1617.6, [M+4H]4+1213.4.

[0178] Example 15

[0179] Synthesis of the polypeptide compound SEQ ID NO:15

[0180]

[0181] The synthesis method was the same as in Example 11. The target peak was collected and lyophilized to obtain 0.16 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass was 4850.5. ESI-MS m / z: calculated values ​​[M+3H]3+1617.8, [M+4H]4+1213.6; observed values ​​[M+3H]3+1617.6, [M+4H]4+1213.4.

[0182] Test example:

[0183] The agonistic activity of the polypeptide compounds prepared in the above examples on human GLP-1 receptor and GIP receptor was determined. The specific test process and results are as follows:

[0184] (1) Determination of the agonistic effect of polypeptide compounds

[0185] The agonistic effect of peptide compounds on receptors was determined by a functional assay, which measured the cAMP response in HEK-293 cell lines stably expressing human GLP-1 or GIP receptors. Specifically, cells stably expressing either receptor were aliquoted into T175 culture flasks and grown overnight in DMEM / 10% FBS until near confluence. The culture medium was then removed, and the cells were washed with calcium- and magnesium-free PBS, followed by protease treatment with Accutase. The detached cells were washed and resuspended in assay buffer (20 mM HEPES, 0.1% BSA, 2 mM IBMX, 1×HBSS) while determining cell density. 25 μL aliquots were dispensed into the wells of 96-well plates. For measurement, 25 μL of the test peptide compound in assay buffer was added to each well, and the plates were incubated at room temperature for 30 minutes. The cAMP content of the cells was determined using a Cisbio kit based on homogeneous time-resolved fluorescence (HTRF). After adding HTRF reagent diluted in lysis buffer (kit components), incubate the plate for 30 minutes, then measure the fluorescence ratio at 665 / 620 nm. The fluorescence is then measured by detecting the concentration that elicits the 50% activation threshold (EC50) that produces the maximum response. 50 This is used to quantify the in vitro efficacy of agonists.

[0186] The detection data (nM) in the embodiments of the present invention are shown in Table 1 below. Although a certain number of significant figures are used to describe the detection data, it should not be assumed that the data has been determined to be an exact number of significant figures.

[0187] Table 1: Agonistaltic activity of peptide compounds on human GLP-1 and GIP receptors

[0188]

[0189]

[0190] As shown in Table 1, the polypeptide compounds prepared in Examples 1-15 of this invention exhibit agonistic activity against both GLP-1 and GIP receptors, indicating that these polypeptide compounds meet the characteristics of dual agonists described in this patent. It is noteworthy that some polypeptide compounds (such as SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:12) possess stronger GLP-1 receptor agonistic activity than GLP-1 and Tirzepatide. The GIP receptor agonistic activity of the polypeptide compounds of this invention is weaker than that of GIP and Tirzepatide. For example, the GIP receptor agonistic activity of SEQ ID NO:12 is approximately 102 times lower than that of natural GIP and approximately 84 times lower than that of Tirzepatide, indicating that the agonistic activity of the polypeptide compounds of this invention against GIP receptors is weaker than that of GIP and Tirzepatide, and also indicating that the ratio of agonistic activity of the polypeptide compounds of this invention against GLP-1 and GIP receptors differs from that of Tirzepatide.

[0191] (2) Stability of polypeptide compounds to DPP-IV and NEP enzymes

[0192] The polypeptide compounds prepared in Examples 1-15 were co-incubated with purified human DPP-IV or NEP enzyme at 37°C for 0, 2, 4, and 8 hours. The peak area of ​​the residual sample in the solution at each time point was determined by HPLC, and the half-life of the sample was calculated. The results are shown in Table 2.

[0193] Table 2: Half-life of polypeptide compounds in DPP-IV or NEP enzyme systems (in hours)

[0194]

[0195]

[0196] As shown in Table 2, the polypeptide compounds prepared in Examples 1-15 of this invention have a half-life of more than 8 hours in both DPP-IV enzyme solution and NEP enzyme solution system, indicating that they can effectively withstand the degradation of DPP-IV and NEP enzymes.

[0197] (3) Effects of polypeptide compounds on blood glucose in diabetic model mice (db / db mice)

[0198] Male db / db mice were randomly divided into four groups of six. The control group received a subcutaneous injection of physiological saline (10 mg / kg). The treatment groups were divided into four subgroups. Mice had free access to food and water during the experiment. Mice were subcutaneously injected with 25 nmol / kg of liraglutide, semaglutide, or the polypeptide compounds prepared in Examples 12-13, respectively, while not fasting. Blood glucose levels in each group were measured using a glucometer at 0 h before administration and at 2, 6, 24, 34, and 48 h after administration.

[0199] like Figure 1 The results showed that in db / db mice, the hypoglycemic effect of liraglutide lasted only about 6 hours, and had completely ceased after 24 hours. Semaglutide showed a good hypoglycemic effect within 24 hours, but its effect significantly decreased after 48 hours. SEQ ID NO:12 (Example 12) and SEQ ID NO:13 (Example 13) both peptide compounds showed excellent hypoglycemic effects throughout the entire 48-hour experimental period, and their hypoglycemic effect did not decrease after 48 hours, indicating that they have a longer-lasting hypoglycemic effect than semaglutide. Semaglutide is a marketed once-weekly GLP-1 receptor agonist. The longer-lasting hypoglycemic effect of SEQ ID NO:12 and SEQ ID NO:13 compared to semaglutide also supports their potential for development as a once-weekly or even longer-term dosing regimen.

[0200] (4) Effects of peptide compounds on blood glucose and body weight in diet-induced obese (DIO) mice

[0201] Male C57BL / 6J mice, weighing approximately 22g, were used to establish the DIO mouse model. The model was established by feeding mice with Research Diets' D12492 high-fat diet for 18 weeks. Before drug administration, the DIO mice were randomly divided into six groups of six mice each, based on their body weight: a saline group (blank control), a positive control group (semaglutide and tirzepatide), and a test sample group (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13). Each group of mice received a subcutaneous injection of saline (10 mg / L) every two days.

[0202] The following drugs were administered: semaglutide (10 nmol / kg), tirzepatide (10 nmol / kg), SEQ ID NO:11 (10 nmol / kg), SEQ ID NO:12 (10 nmol / kg), and SEQ ID NO:13 (10 nmol / kg). The administration period was 21 days. Mouse weight changes were recorded daily, and body fat mass was measured using nuclear magnetic resonance (NMR) before and at the end of the experiment. After the experiment, mice in each group were sacrificed, and liver tissue was collected to measure liver triglyceride (TG) and total cholesterol (TC) levels. Serum was also collected, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, and total cholesterol levels were measured.

[0203] Table 3: Changes in body weight and body fat in DIO mice during a 3-week drug administration period

[0204] Blank control (saline group) -2.1±0.9 -0.8±0.2 Semaglutide (10 nmol / kg) <![CDATA[-15.6±1.0 *** ]]> <![CDATA[-20.3±3.4 *** ]]> Tirzepatide (10 nmol / kg) <![CDATA[-20.4±1.5 *** ]]> <![CDATA[-29.3±3.1 *** ]]> SEQ ID NO:11 (10 nmol / kg) <![CDATA[-25.2±0.8 ***,### ]]> <![CDATA[-35.9±4.8 ***,### ]]> SEQ ID NO:12 (10 nmol / kg) <![CDATA[-38.2±2.0 ***,### ]]> <![CDATA[-49.9±3.7 ***,### ]]> SEQ ID NO:13 (10 nmol / kg) <![CDATA[-34.6±1.7 ***,### ]]> <![CDATA[-48.1±5.5 ***,### ]]>

[0205] ***: P < 0.001 compared with the blank control group; ###: P < 0.001 compared with the semaglutide and tirzepatide groups (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0206] like Figure 2 The results in Table 3 show that the polypeptide compound of the present invention, when administered continuously to DIO mice for 3 weeks, significantly reduced the weight and body fat content of the mice. Furthermore, the weight-loss and body fat-reducing effects of the polypeptide compound of the present invention were significantly stronger than those of the positive control drugs semaglutide and tirzepatide. Notably, the GIP receptor agonist activity of SEQ ID NO:11 was approximately 7 times stronger than that of SEQ ID NO:12 and approximately 4.4 times stronger than that of SEQ ID NO:13; however, the weight-loss effect of SEQ ID NO:11 was significantly lower than that of SEQ ID NO:12 and SEQ ID NO:13.

[0207] Table 4: Liver triglyceride (TG) and total cholesterol (TC) levels in DIO mice after 3 weeks of treatment

[0208] Blank control (saline group) 9.4±0.4 99.4±7.7 Semaglutide (10 nmol / kg) <![CDATA[7.1±0.2 *** ]]> <![CDATA[68.1±4.7 *** ]]> Tirzepatide (10 nmol / kg) <![CDATA[6.9±0.1 *** ]]> <![CDATA[63.3±2.4 *** ]]> SEQ ID NO:11 (10 nmol / kg) <![CDATA[6.2±0.3 ***,### ]]> <![CDATA[49.9±5.2 ***,### ]]> SEQ ID NO:12 (10 nmol / kg) <![CDATA[5.2±0.4 ***,### ]]> <![CDATA[40.2±2.8 ***,### ]]> SEQ ID NO:13 (10 nmol / kg) <![CDATA[5.8±0.4 ***,### ]]> <![CDATA[45.2±2.2 ***,### ]]>

[0209] ***: P < 0.001 compared with the blank control group; ###: P < 0.001 compared with the semaglutide and tirzepatide groups (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0210] Table 5: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in DIO mice after 3 weeks of treatment

[0211] Blank control (saline group) 665±152 632±104 Semaglutide (10 nmol / kg) <![CDATA[297±13 *** ]]> <![CDATA[250±62 *** ]]> Tirzepatide (10 nmol / kg) <![CDATA[210±22 *** ]]> <![CDATA[219±30 *** ]]> SEQ ID NO:11 (10 nmol / kg) <![CDATA[175±39 ***,### ]]> <![CDATA[169±28 ***,### ]]> SEQ ID NO:12 (10 nmol / kg) <![CDATA[150±33 ***,### ]]> <![CDATA[127±42 ***,### <!-- 16 -->]]> SEQ ID NO:13 (10 nmol / kg) <![CDATA[163±28 ***,### ]]> <![CDATA[154±49 ***,### ]]>

[0212] ***: P < 0.001 compared with the blank control group; ###: P < 0.001 compared with the semaglutide and tirzepatide groups (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0213] As shown in Tables 4 and 5, the polypeptide compounds prepared in the embodiments of the present invention, when administered continuously to DIO mice for 3 weeks, significantly reduced the levels of liver triglycerides and total cholesterol, and significantly reduced the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Furthermore, the polypeptide compounds of the present invention showed significantly stronger effects than the positive control drugs semaglutide and tirzepatide, indicating that the polypeptide compounds of the present invention have a promising prospect for treating non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0214] Table 6: Serum triglyceride (TG) and total cholesterol (TC) levels in DIO mice after 3 weeks of treatment

[0215] Blank control (saline group) 9.7±1.8 1.6±0.1 Semaglutide (10 nmol / kg) <![CDATA[7.8±0.3 *** ]]> <![CDATA[1.1±0.2 *** ]]> Tirzepatide (10 nmol / kg) <![CDATA[6.7±0.6 *** ]]> <![CDATA[1.0±0.1 *** ]]> SEQ ID NO:11 (10 nmol / kg) <![CDATA[5.6±0.3 **,### ]]> <![CDATA[0.8±0.2 ***,### ]]> SEQ ID NO:12 (10 nmol / kg) <![CDATA[5.1±0.4 **,### ]]> <![CDATA[0.6±0.1 ***,### ]]> SEQ ID NO:13 (10 nmol / kg) <![CDATA[5.3±0.4 ***,### ]]> <![CDATA[0.7±0.1 ***,### ]]>

[0216] *** Compared with the blank control group, P<0.001; ### Compared with the semaglutide and tirzepatide groups, P<0.001 (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0217] As shown in Table 6, the polypeptide compound of the present invention, when administered to DIO mice for 3 consecutive weeks, can significantly reduce the serum triglyceride and total cholesterol levels in mice. Furthermore, the polypeptide compound of the present invention has a significantly stronger effect on reducing serum lipid (triglyceride and cholesterol) levels than the positive control drugs semaglutide and tirzepatide.

[0218] (5) Effects of peptide compounds on glycated hemoglobin (HbA1c) and fasting blood glucose in db / db mice

[0219] Male db / db mice were randomly divided into three groups of six: a saline group (blank control), a positive control group (semaglutide and tirzepatide), and a test sample group (SEQ ID NO:12, SEQ ID NO:13). After one week of acclimatization, blood was collected from the tail to measure initial HbA1c and fasting blood glucose levels before treatment. Mice in each group were subcutaneously injected every two days with saline (10 mg / kg), semaglutide (10 nmol / kg), tirzepatide (10 nmol / kg), SEQ ID NO:12 (10 nmol / kg), and SEQ ID NO:13 (10 nmol / kg) for 35 days. After treatment, mice were fasted overnight, and fasting blood glucose levels were measured. Simultaneously, blood was collected to measure HbA1c (%).

[0220] Table 7: Changes in HbA1c (%) in db / db mice during a 35-day dosing period

[0221] Blank control (saline group) 5.6±0.3 7.1±0.6 Semaglutide (10 nmol / kg) 5.8±0.4 <![CDATA[6.1±0.3 *** ]]> Tirzepatide (10 nmol / kg) 5.5±0.2 <![CDATA[6.0±0.3 *** ]]> SEQ ID NO:12 (10 nmol / kg) 5.6±0.2 <![CDATA[5.1±0.4 ***,## ]]> SEQ ID NO:13 (10 nmol / kg) 5.6±0.4 <![CDATA[5.2±0.3 ***,## ]]>

[0222] *** Compared with the blank control group, P<0.001; ## Compared with the semaglutide and tirzepatide groups, P<0.01 (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0223] As shown in Table 7, the polypeptide compound of the present invention, when administered continuously to db / db mice for 35 days, can significantly inhibit the increase of HbA1c levels in mice. Furthermore, after treatment, the HbA1c levels in the polypeptide compound group of the present invention were significantly lower than those in the positive controls semaglutide and tirzepatide, indicating that the polypeptide compound of the present invention has a good glycemic control effect.

[0224] Table 8: Changes in fasting blood glucose in db / db mice during a 35-day dosing period

[0225]

[0226]

[0227] *** Compared with the blank control group, P<0.001; ###Compared with the semaglutide and tirzepatide groups, P<0.001 (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 6 mice in each group.

[0228] As shown in Table 8, the polypeptide compound prepared in the embodiments of the present invention, when administered continuously for 35 days in db / db mice, can significantly reduce the fasting blood glucose level in db / db mice, indicating that the polypeptide compound of the present invention has excellent glycemic control effect, and the glycemic control effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs semaglutide and tirzepatide.

[0229] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A GLP-1 / GIP receptor dual-agonist polypeptide compound, characterized in that, The sequence structure of the polypeptide compound is selected from any of the amino acid sequences shown in SEQ ID NO:11-13: SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 2. A derivative of the GLP-1 / GIP receptor dual-agonist polypeptide compound of claim 1, characterized in that, The derivative is obtained by fusing the GLP-1 / GIP receptor dual agonist polypeptide compound of claim 1 with other polypeptide substances; the other polypeptide substances include, but are not limited to, glucagon, gastrin, tyrosinase, fibroblast growth factor 21 or amylin.

3. A pharmaceutically acceptable salt of the GLP-1 / GIP receptor dual agonist polypeptide compound of claim 1.

4. A pharmaceutically acceptable salt of the GLP-1 / GIP receptor dual-agonist polypeptide compound according to claim 3, characterized in that, The pharmaceutically acceptable salt is a salt formed by a GLP-1 / GIP receptor dual-agonist polypeptide compound and one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, pectic acid, butyric acid, hexanoic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, lauric acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, and dioxins. Glucuronic acid, nicotinic acid, pyric acid, propionic acid, persulfate, picric acid, 3-phenylpropionic acid, pentanoic acid, itaconic acid, 2-hydroxyethanesulfonic acid, aminosulfonic acid, dodecyl sulfate, trifluoromethanesulfonic acid, naphthalene disulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, stearic acid, tartaric acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfonic acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphate, glucohepanoic acid, aspartic acid, thiocyanate.

5. A pharmaceutical preparation made from the GLP-1 / GIP receptor dual-agonist polypeptide compound of claim 1, characterized in that, The pharmaceutical preparation includes any pharmaceutically defined tablet, capsule, syrup, tincture, inhaler, spray, injection, film, patch, powder, granule, emulsion, suppository, or compound preparation.

6. A pharmaceutical composition prepared from a GLP-1 / GIP receptor dual-agonist polypeptide compound, characterized in that, The pharmaceutical composition comprises the GLP-1 / GIP receptor dual agonist polypeptide compound of claim 1, and further comprises a carrier; or the pharmaceutical composition comprises a pharmaceutically acceptable salt of the GLP-1 / GIP receptor dual agonist polypeptide compound of any one of claims 3-4, and further comprises a carrier.

7. Use of a pharmaceutically acceptable salt of the GLP-1 / GIP receptor dual agonist polypeptide compound of claim 1, or the GLP-1 / GIP receptor dual agonist polypeptide compound of any one of claims 3-4, or the pharmaceutical agent of claim 5, or the pharmaceutical composition of claim 6, in the preparation of a medicament for treating metabolic diseases or conditions; characterized in that, The metabolic diseases or conditions mentioned are diabetes, obesity, hypertension, non-alcoholic steatohepatitis, or dyslipidemia.

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