CN116120425A

CN116120425A - GLP-1/GIP receptor dual agonist and application thereof

Info

Publication number: CN116120425A
Application number: CN202211706838.3A
Authority: CN
Inventors: 耿月兵; 韩京; 王伟明
Original assignee: Nanjing Sanuo Biotechnology Co ltd
Current assignee: Nanjing Sanuo Biotechnology Co ltd
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-05-16

Abstract

The invention discloses a GLP-1/GIP receptor dual agonist and application thereof. The amino acid sequence general formula of the GLP-1/GIP receptor double-excited polypeptide compound is as follows: tyr-Xaa ₁ ‑Glu‑Gly‑Thr‑Xaa ₂ ‑Thr‑Asn‑Asp‑Xaa ₃ ‑Ser‑Ile‑Xaa ₄ ‑Leu‑Asp‑Lys‑Ile‑Ala‑Gln‑Xaa ₅ ‑Xaa ₆ ‑Phe‑Val‑Gln‑Trp‑Leu‑Xaa ₇ ‑X _aa8 ‑NH ₂ . The invention also relates to derivatives, long-acting compounds, pharmaceutically acceptable salts, pharmaceutical compositions and medicaments of the polypeptide compounds. The GLP-1/GIP receptor double-excited polypeptide compound has more potential in preparing medicaments for treating metabolic syndrome and other diseases.

Description

GLP-1/GIP receptor dual agonist and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a GLP-1/GIP receptor dual agonist and application thereof.

Background

Diabetes is another metabolic disease with global high incidence, and investigation data of the international diabetes union shows that in 2021, adult diabetes patients of 20-79 years worldwide have reached 5.37 billion, accounting for 10.5% of the world population in this age group. Among them, the most adult diabetics in China. By 2030, the number of diabetics worldwide is expected to increase further, to over 6.43 billion. Diabetes mellitus can be classified into insulin-dependent diabetes mellitus (type 1 diabetes mellitus, T1 DM) and non-insulin-dependent diabetes mellitus (type 2 diabetes mellitus, T2 DM), both types characterized by progressive beta cell failure. T1DM is caused by autoimmune attack on beta cells, resulting in progressive apoptosis of beta cells. Whereas T2DM is mainly chronic hyperglycemia due to insulin resistance and dysfunction of islet beta cells, its pathogenesis is due to a number of factors and is accompanied by a number of complications. In both types of diabetes, the proportion of T2DM is about 90% of the total number of diabetics worldwide. Studies have shown that T2DM is associated with a variety of high risk complications in patients, such as cardiovascular disease, diabetic nephropathy, blindness, etc. Obesity is one of the major causes of triggering T2DM, and the progression from obesity to diabetes is generally: obesity-reduced glucose tolerance- & gtT 2 DM- & gtrefractory hyperglycemia- & gtdiabetic complications. Thus, in addition to lowering blood glucose, weight loss is also a factor to be considered for the treatment of diabetes.

Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells of the terminal jejunum, ileum and colon, and exerts hypoglycemic effects upon specific binding to the GLP-1 receptor. The main advantage of GLP-1 is the glucose-dependent incretin secretion effect, avoiding the risk of hypoglycemia often present in diabetes treatment. In addition to regulating blood glucose, GLP-1 can also prevent pancreatic beta cell degeneration, stimulate beta cell proliferation and differentiation, and can improve diabetes progression from the source. In addition, GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, inhibiting appetite and the like, and has partial weight reduction effect. Several long acting GLP-1 drugs, such as liraglutide, semaglutide and dulaglutine, etc., have been marketed. Although GLP-1 drugs have safe hypoglycemic effect, if better weight loss effect is required, the administration dosage is generally increased, and large-dose GLP-1 drugs are easy to produce gastrointestinal side effects and have poor tolerance, so that the treatment window is narrower. Thus, there remains a need for therapeutic agents that are more safely tolerated, and that are effective in reducing body weight and controlling blood glucose.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid gastrointestinal regulatory peptide, and GLP-1 is an incretin that plays a key physiological role in the metabolism of blood glucose in the body. GIP exerts its physiological activity in vivo through the action of GIP receptors distributed in islet beta cells, adipose tissue and central nervous system. Similar to GLP-1, GIP can stimulate insulin secretion by islet beta cells to lower blood glucose, and can protect islet beta cells to control glucose metabolism in vivo. In addition, GIP also agonizes GIP receptors in adipose tissues to promote fat metabolism, and GIP also has an appetite-suppressing effect. Under normal physiological conditions, GIP and GLP-1 are secreted from the intestinal tract after meal, which can enhance physiological responses to food, including satiety, insulin secretion, and nutritional processing. However, the incretin response of GIP is impaired in T2DM patients. Studies have shown that inhibition of GIP produced in T2DM patients is significantly reduced when blood glucose returns to normal. Therefore, the method for treating T2DM by using GIP can be matched with some clinically effective hypoglycemic drugs to reduce the tolerance of the T2DM patient to GIP, and further utilize the incretin effect of the GIP to obtain more excellent hypoglycemic effect.

The currently reported polypeptide GLP-1/GIP receptor dual agonists are designed based on the sequences of natural GLP-1 and GIP according to the sequence structure, and the published patent documents are as follows: WO2016/111971, CN110684082A, CN111825758A, WO2011/119657, WO2013/164483, etc.

GLP-1 in an amphibian has similar action and effect as human GLP-1, so that structural modification is carried out on the amphibian GLP-1, and a novel GLP-1 medicament with higher efficiency and long-acting hypoglycemic effect is expected to be discovered. XenGLP-1 is a class of animal-derived GLP-1 analogues found in Xenopus laevis, and XenGLP-1 has better hypoglycemic activity and stability than natural GLP-1. XenGLP-1 is a potent agonist of the GLP-1 receptor, however it does not activate the GIP receptor. XenGLP-1 has many of the glucose regulatory effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial antidiabetic properties, including enhanced glucose-dependent insulin synthesis and secretion, slow gastric emptying, reduced food intake and body weight, and promotion of beta cell proliferation and restoration of islet function, among others (biochem. Pharmacol.,2017,142,155-167; FASEB J.,2019,33,7113-7125). These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Currently reported dual GLP-1/GIP receptor agonists generally have GIP receptor agonistic activity similar to or stronger than native GIP, and GLP-1 receptor agonistic activity similar to or weaker than native GLP-1. For example, the GIP receptor agonistic activity of the marketed tirzepatide is similar to that of native GIP, and its GLP-1 receptor agonistic activity is about 13-fold weaker than that of native GLP-1. The agonistic activity of the dual GLP-1/GIP receptor agonist on different receptors and the preparation of metabolic syndrome medicaments and the like thereof still need to be further studied.

Disclosure of Invention

The invention provides a GLP-1/GIP receptor dual agonist and application thereof. The invention is based on XenGLP-1 and GIP sequence designed variant, which retains the therapeutic effect of XenGLP-1 on diabetes and has the beneficial effect of GIP on glucose, lipid metabolism and appetite suppression, thereby generating synergistic effect on glucose, lipid and energy metabolism, and having more potential in preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, hypertension, nonalcoholic steatohepatitis, dyslipidemia, senile dementia and other diseases, than GLP-1 and GIP single receptor agonists.

The technical scheme of the invention is as follows:

it is an object of the present invention to provide GLP-1/GIP receptor dual agonistic polypeptide compounds having the amino acid sequence of formula:

Tyr-Xaa ₁ -Glu-Gly-Thr-Xaa ₂ -Thr-Asn-Asp-Xaa ₃ -Ser-Ile-Xaa ₄ -Leu-Asp-Lys-Ile-Al a-Gln-Xaa ₅ -Xaa ₆ -Phe-Val-Gln-Trp-Leu-Xaa ₇ -X _aa8 -NH ₂ (Ⅰ)

wherein: xaa ₁ Selected from Ala or Aib; xaa ₂ Selected from Tyr or Phe; xaa ₃ Selected from Tyr or Val; xaa ₄ Selected from Tyr or Aib; xaa ₅ Selected from Lys or Lys with modified side chain; xaa ₆ Selected from Glu or Ala; xaa ₇ Selected from Leu or Ile; x is X _aa8 Selected from any natural amino acid or peptide fragment consisting thereof, or is absent;

further, the side chain modified Lys is selected from

Wherein: n is a natural number, and n is more than or equal to 12 and less than or equal to 24.

Further, n is 14, 16, 18 or 20.

Further, the sequence of the GLP-1/GIP receptor dual-agonist polypeptide compound is ordered from left to right, and the 6 th to 10 th amino acids are combinations 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.

Further, X _aa8 Is Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser.

Further, the amino acid sequence of the polypeptide compound is shown in SEQ ID NO:16, as shown in:

SEQ ID NO:16

Tyr-Xaa ₁ -Glu-Gly-Thr-Xaa ₂ -Thr-Asn-Asp-Xaa ₃ -Ser-Ile-Xaa ₄ -Leu-Asp-Lys-Ile-Al a-Gln-Xaa ₅ -Xaa ₆ -Phe-Val-Gln-Trp-Leu-Xaa ₇ -Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

wherein: xaa ₁ Selected from Ala or Aib; xaa ₂ Selected from Tyr or Phe; xaa ₃ Selected from Tyr or Val; xaa ₄ Selected from Tyr or Aib; xaa ₅ Selected from Lys or Lys with modified side chain; xaa ₆ Selected from Glu or Ala; xaa ₇ Selected from Leu or Ile;

wherein the side chain modified Lys is selected from

Wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 24.

Further, the sequence structure of the polypeptide compound includes, but is not limited to, the amino acid sequences of SEQ ID NOs 1-15 (Aib is represented by X in the sequence Listing):

SEQ ID NO:1

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-NH ₂

SEQ ID NO:2

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-NH ₂

SEQ ID NO:3

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-NH ₂

SEQ ID NO:4

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-NH ₂

SEQ ID NO:5

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-NH ₂

SEQ ID NO:6

SEQ ID NO:7

SEQ ID NO:8

SEQ ID NO:9

SEQ ID NO:10

SEQ ID NO:11

SEQ ID NO:12

SEQ ID NO:13

SEQ ID NO:14

SEQ ID NO:15

another object of the present invention is to provide a derivative of the above GLP-1/GIP receptor dual-agonism polypeptide compound, which is obtained by fusing the above dual-polypeptide compound with other polypeptide substances; such other polypeptide substances include, but are not limited to, glucagon, oxyntomodulin (OXM), tyrosin (PYY), fibroblast growth factor 21 (FGF 21), or amylin.

It is still another object of the present invention to provide a long-acting compound prepared from the above GLP-1/GIP receptor dual agonist polypeptide compound, wherein the long-acting compound is obtained by amino acid substitution or cyclization of the above dual agonist polypeptide compound; or further modifying polyethylene glycol, fusing long-acting protein fragments, and modifying the acid-proof chain of the conjugate lipid; such long-acting protein fragments include, but are not limited to, bovine Serum Albumin (BSA), fc fusion proteins, human Chorionic Gonadotrophin (HCG), or unstructured biodegradable protein polymers (Xten).

It is still another object of the present invention to provide a pharmaceutically acceptable salt of the above GLP-1/GIP receptor dual agonist polypeptide compound.

Further, the pharmaceutically acceptable salt is a salt of a GLP-1/GIP receptor dual agonist polypeptide compound with one of the following compounds; the following compounds include hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectate acid, butyric acid, caproic 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, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid or sulfosalicylic acid.

It is still another object of the present invention to provide a pharmaceutical agent prepared from the above GLP-1/GIP receptor dual agonist polypeptide compound.

Further, the medicament comprises any one of a tablet, a capsule, a syrup, a tincture, an inhalant, a spray, an injection, a film, a patch, a powder, a granule, an emulsion, a suppository or a compound preparation.

It is yet another object of the present invention to provide a pharmaceutical composition prepared from a GLP-1/GIP receptor dual agonist polypeptide compound.

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

It is a further object of the present invention to provide the use of said GLP-1/GIP receptor double-agonism polypeptide compound or a pharmaceutically acceptable salt of said GLP-1/GIP receptor double-agonism polypeptide compound or said medicament or said pharmaceutical composition for the manufacture of a medicament for the treatment of a metabolic disease or disorder; the metabolic disease or disorder includes diabetes, obesity, hypertension, nonalcoholic steatohepatitis, dyslipidemia, or senile dementia.

The compound prepared by the invention has strong agonistic activity to GLP-1 receptor and weaker agonistic activity to GIP receptor, but realizes better hypoglycemic and weight-reducing effects, thus providing a new idea for preparing multiple agonists. The invention can realize the effect because the special N-terminal sequence of the prepared compound, in particular, the N-terminal sequence of the compound adopts partial N-terminal sequence fragments of XenGLP-1, such as YTIDV sequence fragments of 6-10 amino acid sequences, TNDV sequence fragments of 7-10 positions and YTOD sequence fragments of 6-9 positions, so that the prepared compound has different GLP-1 receptor and GIP receptor agonist activity proportions (high GLP-1 receptor agonist activity and weak GIP receptor agonist activity), but realizes high-efficiency blood glucose reduction and weight reduction.

The beneficial technical effects of the invention are as follows:

(1) According to the invention, the 8 th amino acid is Asn, the amino acid is completely different from natural GLP-1 and GIP, and the position mutation can lead the polypeptide compound to have a unique GLP-1 and GIP receptor agonistic activity proportion, so that better weight losing and blood glucose reducing activities are brought. In addition, the 6-10 th amino acid is a combination mode Tyr (Phe) -Thr-Asn-Asp-Val (Tyr), the amino acid in the region is different from natural GLP-1 and GIP, the mutation in the position can lead to the special change of the combination of the polypeptide compound and the GLP-1 and GIP receptor, the agonistic activity of the polypeptide compound on the GLP-1 receptor is higher, but the agonistic activity on the GIP receptor is weaker, and better weight losing and blood glucose reducing activities are brought.

(2) Compared with the GLP-1 receptor agonist on the market, the GLP-1/GIP receptor double-excited polypeptide compound has the advantages of reducing blood sugar more effectively, simultaneously having obvious weight reduction and weight increase prevention effects, reversing insulin resistance and regulating lipid metabolism; compared with the GLP-1/GIP receptor dual agonists on the market, the polypeptide compound provided by the invention has a unique N-terminal sequence structure and a unique in-vitro GLP-1 and GIP receptor agonistic activity proportion, thereby bringing about remarkably improved weight loss and lipid metabolism regulation effects, and having unexpected beneficial effects compared with the existing medicines.

(3) The polypeptide compound provided by the invention has stable chemical properties and has pharmacokinetic characteristics for supporting once-daily or once-weekly administration; the polypeptide compound provided by the invention has better treatment effect on metabolic diseases such as T2DM, obesity, dyslipidemia and the like than the existing medicines on the market. Therefore, the polypeptide compound provided by the invention is suitable for being used as an active ingredient of medicines for treating metabolic diseases, such as diabetes, obesity, hypertension, nonalcoholic steatohepatitis, dyslipidemia and the like.

Drawings

FIG. 1 shows the long-acting hypoglycemic effect of a single administration of each subject of the invention in the non-fasted state of db/db mice.

Figure 2 shows the percent change in body weight of each subject of the invention over 21 days of prolonged DIO mice administration.

Detailed Description

The present invention will be described in detail below with reference to the drawings and examples.

Unless defined otherwise herein, scientific and technical terms used in this specification 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, pharmacology, according to the invention, are well known and commonly used in the art.

All combinations of the various elements disclosed herein are within the scope of the invention. Furthermore, the scope of the invention should not be limited by the specific disclosure provided below.

The 8 th position of the amino acid sequence of the GLP-1/GIP receptor double-excited polypeptide compound is Asn, and/or the 6 th to 10 th positions are 5 amino acid combinations, specifically Tyr-Thr-Asn-Asp-Val or Tyr-Thr-Asn-Asp-Tyr or Phe-Thr-Asn-Asp-Val or Phe-Thr-Asn-Asp-Tyr, and any polypeptide obtained by adjusting amino acids at other positions is within the protection scope of the application.

Further, the amino acids mentioned in the present invention may be abbreviated as follows according to the naming convention of IUPAC-IUB:

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).

Further, unless explicitly indicated, all amino acid residues in the polypeptides of the invention are preferably in the L configuration.

Further, "-NH on the C-terminus of the sequence ₂ "part indicates an amide group (-CONH) at the C-terminus ₂ )。

Further, in addition to natural amino acids, unnatural amino acids, alpha-aminoisobutyric acid (Aib) are used in the sequences of the invention.

Furthermore, the polypeptide compound can be synthesized by a polypeptide solid-phase synthesis method or can be produced by a genetic engineering technology.

Further, abbreviations used in the present invention are explained as follows: DCM: dichloromethane; DMF: n, N-dimethylformamide; DMSO; dimethyl sulfoxide; TFA: trifluoroacetic acid; EDT:1, 2-ethanedithiol;

the following specific embodiments are provided in order to illustrate the present invention in more detail, but the aspects of the present invention are not limited thereto.

Example 1

Synthesis of polypeptide compound with sequence structure shown as SEQ ID NO. 1

That is, Y (Aib) EGTYTNDYSI (Aib) LDKIAQKAFVQWLIAGGPSSGAPPPS-NH ₂

(1) Swelling of the resin

0.262g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382mmol/g was weighed into a 25mL reactor, the resin was alternately washed 1 time with 7mL of DCM and methanol, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally the resin was washed 3 times with 7mL of DMF.

(2) Removal of Fmoc protecting groups from resin

Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 5min, filtering off the deprotection solution, washing the resin once by 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) for reaction with the resin for 15min, and washing the resin for 4 times by 7mL of DMF for 1.5min each time to obtain the Rink resin without Fmoc protecting groups.

(3) Synthesis of Fmoc-Ser-Rink amide-MBHAresin

Fmoc-Ser (Boc) -OH (0.4 mmol) was weighed, dissolved in 3mL 10% DMF/DMSO (v/v), 2mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, pre-activated for 30min, the activated amino acid was added to the reactor, the reaction was allowed to react for 2h with shaking at room temperature, the reaction solution was filtered off, the resin was washed 4 times with 7mL DMF, and the Kaiser reagent was used to check if the reaction coupling was complete, if not, 2 times.

(4) Extension of peptide chain

And (3) according to the sequence of the peptide chain, repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain is synthesized.

(5) Cleavage of polypeptides

The obtained resin connected with the polypeptide is transferred into a round bottom bottle, 5mL of a cutting agent Reagent R (TFA/benzyl sulfide/phenol/EDT, 90:5:3:2, V/V) is used for cutting the resin, the resin is reacted for 2 hours in an oil bath at the constant temperature of 30 ℃, the cutting fluid is poured into 40mL of glacial ethyl ether, the crude product is washed 3 times by 15mL of glacial ethyl ether after refrigerated centrifugation, and finally the crude peptide is obtained by drying with nitrogen.

(6) Purification of polypeptides

Dissolving the target polypeptide crude product in water, filtering with 0.25 μm microporous membrane, and purifying with island jin preparation type reversed phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.20 mm,12 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20% B-70% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity greater than 98%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4112.5.ESI-MS M/z calculated [ M+3H] ₃ +1371.8,[M+4H] ₄ +1029.1; observed value [ M+3H] ₃ +1371.7,[M+4H] ₄ +1029.0。

Example 2

Synthesis of polypeptide compound of SEQ ID No. 2

That is, Y (Aib) EGTYTNDVSI (Aib) LDKIAQKAFVQWLIAGGPSSGAPPPS-NH ₂ The synthesis method is the same as that of example 1, 0.11g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4048.5.ESI-MS M/z calculated [ M+3H] ₃ +1350.5,[M+4H] ₄ +1013.1; observed value [ M+3H] ₃ +1350.4,[M+4H] ₄ +1013.0。

Example 3

Synthesis of polypeptide compound of SEQ ID No. 3

That is, Y (Aib) EGTFTNDVSI (Aib) LDKIAQKAFVQWLIAGGPSSGAPPPS-NH ₂ The synthesis method is the same as that of example 1, 0.13g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4032.5.ESI-MS M/z calculated [ M+3H] ₃ +1345.2,[M+4H] ₄ +1009.1; observed value [ M+3H] ₃ +1345.0,[M+4H] ₄ +1009.0。

Example 4

Synthesis of polypeptide compound of SEQ ID No. 4

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-NH ₂

That is, Y (Aib) EGTYTNDVSI (Aib) LDKIAQKEFVQWLIAGGPSSGAPPPS-NH ₂ The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain pure0.12g of the product with a purity of more than 98%, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4106.6.ESI-MS M/z calculated [ M+3H] ₃ +1369.9,[M+4H] ₄ +1027.7; observed value [ M+3H] ₃ +1369.7,[M+4H] ₄ +1027.5。

Example 5

Synthesis of polypeptide compound of SEQ ID No. 5

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-NH ₂

That is, Y (Aib) EGTYTNDVSI (Aib) LDKIAQKEFVQWLLAGGPSSGAPPPS-NH ₂ The synthesis method is the same as that of example 1, 0.12g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4106.6.ESI-MS M/z calculated [ M+3H] ₃ +1369.9,[M+4H] ₄ +1027.7; observed value [ M+3H] ₃ +1369.7,[M+4H] ₄ +1027.5。

Example 6

Synthesis of polypeptide compound of SEQ ID No. 6

(1) Swelling of the resin

(2) Removal of Fmoc protecting groups from resin

(3) Synthesis of Fmoc-Ser-Rink amide-MBHAresin

(4) Extension of peptide chain

And (3) according to the sequence of the peptide chain, repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain is synthesized. Wherein, the 20-position Lys can be Fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH or Fmoc-Lys (ivDde) -OH, etc. Fmoc-Lys (Dde) -OH protection strategy was used in this example, while Boc-His (Boc) -OH was used for the N-terminal His.

(5) Modification of Lys side chains

After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove Dde protecting group of Lys at 12 th site, after the Dde protecting group is removed, 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt are added to carry out oscillation reaction for 2h. Then, after Fmoc protecting group was removed by the same method as described above, 0.4mmol of palmitic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2 hours, and after completion of the reaction, the resin was washed with 7mL of DMF for 4 times.

(6) Cleavage of polypeptides

(7) Purification of polypeptides

Dissolving the target polypeptide crude product in water, filtering with 0.25 μm microporous membrane, and purifying with island jin preparation type reversed phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.20 mm,12 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate was 8 mL-min; the detection wavelength was 214nm. Eluting with linear gradient (20% B-80% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity greater than 98%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4480.1.ESI-MS M/z calculated [ M+3H] ₃ +1494.4,[M+4H] ₄ +1121.0; observed value [ M+3H] ₃ +1494.3,[M+4H] ₄ +1120.9。

Example 7

Synthesis of polypeptide compound of SEQ ID No. 7

The synthesis method is the same as that of example 6, 0.11g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4416.0.ESI-MS M/z calculated [ M+3H] ₃ +1473.0,[M+4H] ₄ +1105.0; observed value [ M+3H] ₃ +1472.9,[M+4H] ₄ +1104.9。

Example 8

Synthesis of polypeptide compound of SEQ ID No. 8

The synthesis method is the same as that of example 6, 0.13g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4400.0.ESI-MS M/z calculated [ M+3H] ₃ +1467.7,[M+4H] ₄ +1101.0; observed value [ M+3H] ₃ +1467.5,[M+4H] ₄ +1100.8。

Example 9

Synthesis of polypeptide compound of SEQ ID No. 9

The synthesis method is the same as that of example 6, and target peak jelly is collectedThe dry product is 0.13g, the purity is more than 98 percent, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4474.1.ESI-MS M/z calculated [ M+3H] ₃ +1492.4,[M+4H] ₄ +1119.5; observed value [ M+3H] ₃ +1492.2,[M+4H] ₄ +1119.3。

Example 10

Synthesis of polypeptide compound of SEQ ID No. 10

The synthesis method is the same as that of example 6, 0.13g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4474.1.ESI-MS M/z calculated [ M+3H] ₃ +1492.4,[M+4H] ₄ +1119.5; observed value [ M+3H] ₃ +1492.2,[M+4H] ₄ +1119.3。

Example 11

Synthesis of polypeptide compound of SEQ ID NO. 11

(1) Swelling of the resin

(2) Removal of Fmoc protecting groups from resin

Transferring the swelled resin into a PSI-200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 5min, filtering off the deprotection solution, washing the resin once by 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) for reaction with the resin for 15min, and washing the resin 4 times by 7mL of DMF for 1.5min each time to obtain the Rink resin without Fmoc protecting groups.

(3) Synthesis of Fmoc-Ser-Rink amide-MBHAresin

(4) Extension of peptide chain

(5) Modification of Lys side chains

After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove Dde protecting group of Lys at 20 th position, after the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt are added, and oscillation condensation reaction is carried out for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-AEEA-OH,0.4mmol DIC and 0.44mmol HOBt were added again and the reaction was performed by shaking for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-Glu-OtBu,0.4mmol DIC and 0.44mmol HOBt were added and the reaction was performed by shaking for 2h. After removal of Fmoc protecting groups, 0.4mmol of mono-tert-butyl eicosadioate, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation for 2h, and after completion of the reaction the resin was washed 4 times with 7mL of DMF.

(6) Cleavage of polypeptides

(7) Purification of polypeptides

Dissolving the crude polypeptide in water, filtering with 0.25 μm microporous membrane, and adding Shimadzu preparation type reverse reactionAnd (5) purifying by a phase HPLC system. Chromatographic conditions were C18 reverse phase preparation column (250 mm. Times.20 mm,12 μm); mobile phase a:0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20% B-80% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product with purity greater than 98%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4856.5.ESI-MS M/z calculated [ M+3H] ₃ +1619.8,[M+4H] ₄ +1215.1; observed value [ M+3H] ₃ +1619.5,[M+4H] ₄ +1214.9。

Example 12

Synthesis of polypeptide compound of SEQ ID NO 12

The synthesis method is the same as that of example 11, 0.14g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4792.4.ESI-MS M/z calculated [ M+3H] ₃ +1598.5,[M+4H] ₄ +1199.1; observed value [ M+3H] ₃ +1598.5,[M+4H] ₄ +1198.9。

Example 13

Synthesis of polypeptide compound of SEQ ID NO 13

The synthesis method is the same as that of example 11, 0.14g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4776.4.ESI-MS M/z calculated [ M+3H] ₃ +1593.1,[M+4H] ₄ +1195.1; observed value [ M+3H] ₃ +1592.9,[M+4H] ₄ +1194.9。

Example 14

Synthesis of polypeptide compound of SEQ ID No. 14

The synthesis method is the same as that of example 11, 0.13g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4850.5.ESI-MS M/z calculated [ M+3H] ₃ +1617.8,[M+4H] ₄ +1213.6; observed value [ M+3H] ₃ +1617.6,[M+4H] ₄ +1213.4。

Example 15

Synthesis of polypeptide compound of SEQ ID NO 15

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The synthesis method is the same as that of example 11, 0.16g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4850.5.ESI-MS M/z calculated [ M+3H] ₃ +1617.8,[M+4H] ₄ +1213.6; observed value [ M+3H] ₃ +1617.6,[M+4H] ₄ +1213.4。

Test example:

the agonist activity of the polypeptide compounds prepared in the above examples on human GLP-1 receptor and GIP receptor was measured, and the specific test procedures and results were as follows:

(1) Polypeptide compound agonism assay

Agonism of the receptor by the polypeptide compounds is determined by a functional assay that measures cAMP response of HEK-293 cell lines stably expressing human GLP-1 or GIP receptors. The specific method comprises the following steps: cells stably expressing both receptors were split into T175 flasks and grown overnight in medium (DMEM/10% fbs) to near confluency, then the medium was removed, and the cells were washed with calcium and magnesium free PBS and then protease treated with Accutase enzyme. The detached cells were washed and resuspended in assay buffer (20mM HEPES,0.1%BSA,2mM IBMX,1 ×hbss) while cell density was determined and 25 μl aliquots were dispensed into wells of 96-well plates. For measurement, 25. Mu.L of the test polypeptide was subjected to polypeptideA solution of the compound in assay buffer was added to the wells and then incubated for 30 minutes at room temperature. The cAMP content of cells was determined based on Homogeneous Time Resolved Fluorescence (HTRF) using the Cisbio kit. After addition of HTRF reagents diluted in lysis buffer (kit components), the plates were incubated for 30 minutes and then the fluorescence ratio at 665/620nm was measured. By detecting the concentration that caused 50% of activation of the maximal response (EC ₅₀ ) To quantify the in vitro potency of the agonist.

The test data (nM) in embodiments of the invention is described in Table 1 below, and although the test data is stated in terms of a number of significant digits, it should not be considered to indicate that the data has been determined to be exactly a significant digit.

Table 1: agonistic activity of polypeptide compound on human GLP-1 receptor and GIP receptor

As shown in Table 1, the polypeptide compounds prepared in examples 1-15 of the present invention have agonistic activity at both GLP-1 and GIP receptors, indicating that these polypeptide compounds are compatible with the dual agonist characteristics described in this patent. Notably, some of the polypeptide compounds (e.g., SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO: 12) have stronger GLP-1 receptor agonistic activity than GLP-1 and Tirzepatide. The GIP receptor agonistic activity of the polypeptide compound of the invention is weaker than that of GIP and Tirzepatide, for example, the GIP receptor agonistic activity of SEQ ID NO:12 is about 102 times lower than that of natural GIP and about 84 times lower than that of Tirzepatide, which indicates that the agonistic activity of the polypeptide compound of the invention on the GIP receptor is weaker than that of GIP and Tirzepatide and also indicates that the ratio of the agonistic activity of the polypeptide compound of the invention on GLP-1 and GIP receptors is different from that of Tirzepatide.

(2) Stability of polypeptide Compounds against DPP-IV and NEP enzymes

The polypeptide compounds prepared in examples 1-15 were incubated with purified human DPP-IV or NEP enzyme at 37℃for 0,2,4,8 hours, the peak areas of residual samples in the solutions at each time point were measured by HPLC, and the half-lives of the samples were calculated, and the results are shown in Table 2.

Table 2: half-life (expressed as h) of polypeptide compounds in DPP-IV enzyme or NEP enzyme systems

As can be seen from Table 2, the polypeptide compounds prepared in examples 1-15 of the present invention have half-lives exceeding 8 hours in both DPP-IV enzyme-containing solution and NEP enzyme-containing solution systems, indicating that they are effective in tolerating degradation of DPP-IV and NEP enzymes.

(3) Effect of polypeptide Compounds on blood glucose in diabetes model mice (db/db mice)

Male db/db mice were randomly grouped, 6 per group. Normal saline (10 mg/kg) was subcutaneously administered in a blank group, and the administration composition was 4 groups, and the mice were free to eat and drink water during the experiment, and the mice were subcutaneously injected in a single injection of 25nmol/kg of liraglutide, semaglutinide, respectively, in a non-fasting state, as the polypeptide compounds prepared in examples 12-13. Blood glucose levels were measured in each group of mice at 0h prior to dosing, and at 2,6, 24, 34, and 48 hours post dosing.

As shown in the results of FIG. 1, the results of the hypoglycemic experiments in db/db mice indicate that the hypoglycemic effect of liraglutide can only be maintained for about 6 hours, and no hypoglycemic effect is obtained at all at 24 hours. semaglide showed a better hypoglycemic effect within 24 hours, but the hypoglycemic effect of semaglide was significantly reduced at 48 hours. The two polypeptide compounds of SEQ ID NO. 12 (example 12), SEQ ID NO. 13 (example 13) showed excellent hypoglycemic effect throughout the 48 hour experimental period, and the hypoglycemic effect did not decrease at 48 hours, indicating that they had a longer-acting hypoglycemic effect than semaglutide. Semaglutide is a GLP-1 receptor agonist that has been marketed for once-a-week administration, and SEQ ID NO:12 and SEQ ID NO:13 have longer-lasting hypoglycemic effects than Semaglutide, and also support their potential for development as once-a-week administration and even longer-period administration.

(4) Effects of polypeptide Compounds on diet-induced obesity (DIO) mice blood glucose and body weight

Male C57BL/6J mice, weighing about 22g, were kept in 36 model groups and were fed with D12492 high-fat feed from Research Diets for 18 weeks to make DIO mouse models. Before the start of the administration, DIO mice in each group were randomly grouped according to body weight, and 6 groups of 6 mice each were respectively physiological saline group (blank control group), positive control group (semaglutide and tirzepatide) and test sample group (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13). Each group of mice was subcutaneously injected with physiological saline (10)

mg/kg), semaglutide (10 nmol/kg), tirzepatide (10 nmol/kg), SEQ ID NO:11 (10 nmol/kg), SEQ ID NO:12 (10 nmol/kg), SEQ ID NO:13 (10 nmol/kg), and the administration period was 21 days. The body weight changes of mice were recorded daily and Nuclear Magnetic Resonance (NMR) was used to measure body fat mass before and at the end of the experiment. At the end of the experiment, each group of mice was sacrificed and liver tissues were taken to measure liver Triglyceride (TG) and Total Cholesterol (TC) content. Blood serum was also obtained and the serum glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), triglycerides and total cholesterol levels were measured.

Table 3: body weight and body fat changes in DIO mice over a 3 week dosing period

Sample (dose)	Overall weight change (%)	Body fat change (%)
			Blank control (give birth to)Brine group	-2.1±0.9	-0.8±0.2
Semaglutide(10nmol/kg)	-15.6±1.0 ^***	-20.3±3.4 ^***
			Tirzepatide(10nmol/kg)	-20.4±1.5 ^***	-29.3±3.1 ^***
SEQ ID NO:11(10nmol/kg)	-25.2±0.8 ^***,###	-35.9±4.8 ^***,###
			SEQ ID NO:12(10nmol/kg)	-38.2±2.0 ^***,###	-49.9±3.7 ^***,###
SEQ ID NO:13(10nmol/kg)	-34.6±1.7 ^***,###	-48.1±5.5 ^***,###

* **: p <0.001 compared to the placebo group; # # # #: the ratio P <0.001 (One-Way ANOVA, tukey post hoc test) to semaglutide and tirzepatide groups, expressed as mean.+ -. SD of 6 mice per group.

As shown in fig. 2 and table 3, the polypeptide compound of the present invention can significantly reduce the weight and body fat content of mice by continuously administering the polypeptide compound in DIO mice for 3 weeks, and the weight and body fat reducing effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs semaglutide and tirzepartial. Notably, the GIP receptor agonistic activity of SEQ ID NO. 11 was about 7 times stronger than that of SEQ ID NO. 12 and about 4.4 times stronger than that of SEQ ID NO. 13, but the weight loss effect of SEQ ID NO. 11 was significantly lower than that of SEQ ID NO. 12 and SEQ ID NO. 13.

Table 4: liver Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment

Sample (dose)	Total cholesterol (mg/g)	Triglyceride (mg/g)
			Blank control (normal saline group)	9.4±0.4	99.4±7.7
Semaglutide(10nmol/kg)	7.1±0.2 ^***	68.1±4.7 ^***
			Tirzepatide(10nmol/kg)	6.9±0.1 ^***	63.3±2.4 ^***
SEQ ID NO:11(10nmol/kg)	6.2±0.3 ^***,###	49.9±5.2 ^***,###
			SEQ ID NO:12(10nmol/kg)	5.2±0.4 ^***,###	40.2±2.8 ^***,###
SEQ ID NO:13(10nmol/kg)	5.8±0.4 ^***,###	45.2±2.2 ^***,###

Table 5: serum glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) levels after 3 weeks of DIO mice treatment

Sample (dose)	Glutamic pyruvic transaminase (U/L)	Glutamic-oxaloacetic transaminase (U/L)
			Blank control (normal saline group)	665±152	632±104
Semaglutide(10nmol/kg)	297±13 ^***	250±62 ^***
			Tirzepatide(10nmol/kg)	210±22 ^***	219±30 ^***
SEQ ID NO:11(10nmol/kg)	175±39 ^***,###	169±28 ^***,###
			SEQ ID NO:12(10nmol/kg)	150±33 ^***,###	127±42 ^***,###
SEQ ID NO:13(10nmol/kg)	163±28 ^***,###	154±49 ^***,###

As shown in tables 4 and 5, the polypeptide compound prepared in the embodiment of the invention is continuously administered in DIO mice for 3 weeks, so that the liver triglyceride and total cholesterol content of the mice can be obviously reduced, the serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase content can be obviously reduced, and the effect of the polypeptide compound is obviously stronger than that of positive control medicines semaglutin and tirzepartial, which shows that the polypeptide compound has good prospect for treating non-alcoholic fatty liver disease and non-alcoholic fatty hepatitis.

Table 6: serum Triglyceride (TG) and Total Cholesterol (TC) levels 3 weeks after DIO mice treatment

Sample (dose)	Total cholesterol (mmol/L)	Triglyceride (mmol/L)
			Blank control (normal saline group)	9.7±1.8	1.6±0.1
Semaglutide(10nmol/kg)	7.8±0.3 ^***	1.1±0.2 ^***
			Tirzepatide(10nmol/kg)	6.7±0.6 ^***	1.0±0.1 ^***
SEQ ID NO:11(10nmol/kg)	5.6±0.3 ^**,###	0.8±0.2 ^***,###
			SEQ ID NO:12(10nmol/kg)	5.1±0.4 ^**,###	0.6±0.1 ^***,###
SEQ ID NO:13(10nmol/kg)	5.3±0.4 ^***,###	0.7±0.1 ^***,###

^*** : p compared with the blank control group<0.001； ^### : comparison with semaglutide and tirzepatide group P<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.

As the results in table 6 show, the polypeptide compound of the present invention can significantly reduce serum triglyceride and total cholesterol content of mice by continuously administering the polypeptide compound in DIO mice for 3 weeks, and the effect of reducing serum lipid (triglyceride and cholesterol) content of the polypeptide compound of the present invention is significantly stronger than that of the positive control agents semaglutide and tirzepatide.

(5) Effect of polypeptide Compounds on db/db mouse glycosylated hemoglobin (HbA 1 c) and fasting blood glucose

Male db/db mice were randomly grouped, 6 per group. Physiological saline (blank), positive control (semaglutide and tirzepatide) and test sample (SEQ ID NO:12, SEQ ID NO: 13), respectively. After one week of adaptive feeding, the tail bleed measures the initial HbA1c values and fasting blood glucose values before the start of the treatment. Each group of mice was subcutaneously injected with normal saline (10 mg/kg), semaglutide (10 nmol/kg), tirzepatide (10 nmol/kg), SEQ ID NO:12 (10 nmol/kg), SEQ ID NO:13 (10 nmol/kg) every two days with a dosing period of 35 days. The mice were fasted overnight after the end of treatment and blood was taken to measure the fasting blood glucose values and HbA1c (%).

Table 7: hbA1c (%) change in db/db mice over a 35 day dosing period

Sample (dose)	HbA1c% (before treatment)	HbA1c% (after treatment)
			Blank control (normal saline group)	5.6±0.3	7.1±0.6
Semaglutide(10nmol/kg)	5.8±0.4	6.1±0.3 ^***
			Tirzepatide(10nmol/kg)	5.5±0.2	6.0±0.3 ^***
SEQ ID NO:12(10nmol/kg)	5.6±0.2	5.1±0.4 ^*** ,##
			SEQ ID NO:13(10nmol/kg)	5.6±0.4	5.2±0.3 ^***,##

^*** : p compared with the blank control group<0.001； ^## : comparison with semaglutide and tirzepatide group P<0.01 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 mice per group.

As shown in the results of Table 7, the polypeptide compound of the present invention was administered continuously in db/db mice for 35 days, the increase in HbA1c value of the mice could be significantly suppressed, and the HbA1c value of the mice of the polypeptide compound group of the present invention after the treatment was significantly lower than that of the positive controls semaglutide and tirzepatide, indicating that the polypeptide compound of the present invention had a good glycemic control.

Table 8: fasting blood glucose changes in db/db mice over a 35 day dosing period

As shown in the results of Table 8, the polypeptide compound prepared in the embodiment of the invention can obviously reduce the fasting blood glucose value of db/db mice after being continuously administered in db/db mice for 35 days, which proves that the polypeptide compound has excellent blood glucose control effect and the blood glucose control effect of the polypeptide compound is obviously stronger than that of positive control drugs semaglutide and tirzepatide.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.

Claims

1. A GLP-1/GIP receptor dual agonism polypeptide compound characterized by having an amino acid sequence of formula:

wherein:

Xaa ₁ selected from Ala or Aib;

Xaa ₂ selected from Tyr or Phe;

Xaa ₃ selected from Tyr or Val;

Xaa ₄ selected from Tyr or Aib;

Xaa ₅ selected from Lys or Lys with modified side chain;

Xaa ₆ selected from Glu or Ala;

Xaa ₇ selected from Leu or Ile;

X _aa8 selected from any natural amino acid or peptide fragment consisting thereof, or is absent;

the side chain modified Lys is selected from

2. The GLP-1/GIP receptor dual agonist polypeptide compound of claim 1, wherein n is 14, 16, 18 or 20.

3. The GLP-1/GIP receptor double-agonism polypeptide compound according to claim 1, characterized in that the sequence of the GLP-1/GIP receptor double-agonism polypeptide compound is ordered from left to right, the amino acids at positions 6-10 being a combination of 5 amino acids, in particular Tyr-Thr-Asn-Asp-Val or Tyr-Thr-Asn-Asp-Tyr or Phe-Thr-Asn-Val or Phe-Thr-Asn-Asp-Tyr.

4. The GLP-1/GIP receptor double-agonism polypeptide compound of claim 1, wherein X _aa8 Is Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser.

5. The GLP-1/GIP receptor double agonism polypeptide compound of claim 1, wherein the sequence structure of the polypeptide compound is selected from any one of the amino acid sequences shown in SEQ ID NOs 1-15:

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:3

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

SEQ ID NO:7

SEQ ID NO:8

SEQ ID NO:9

SEQ ID NO:10

SEQ ID NO:11

SEQ ID NO:12

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SEQ ID NO:13

SEQ ID NO:14

SEQ ID NO:15

6. a derivative of a GLP-1/GIP receptor double-agonism polypeptide compound according to any one of claims 1 to 5, wherein the derivative is obtained by fusing the double-polypeptide compound according to any one of claims 1 to 5 with other polypeptide substances; such other polypeptide substances include, but are not limited to, glucagon, oxyntomodulin, tyrosin, fibroblast growth factor 21, or amylin.

7. A long-acting compound prepared from the GLP-1/GIP receptor double-agonist polypeptide compound according to any one of claims 1 to 5, wherein the long-acting compound is obtained by amino acid substitution or cyclization of the double-agonist polypeptide compound according to any one of claims 1 to 5, or further modification of polyethylene glycol, fusion of long-acting protein fragments, and modification of conjugate lipid acid chains; such long-acting protein fragments include, but are not limited to, bovine serum albumin, fc fusion proteins, human chorionic gonadotrophin, or unstructured biodegradable protein polymers.

8. A pharmaceutically acceptable salt of the GLP-1/GIP receptor dual agonist polypeptide compound of any one of claims 1-5.

9. The pharmaceutically acceptable salt of the GLP-1/GIP receptor dual agonist polypeptide compound of claim 8, wherein the pharmaceutically acceptable salt is a salt of the GLP-1/GIP receptor dual agonist polypeptide compound with one of the following compounds; the following compounds include hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectate acid, butyric acid, caproic 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, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, wine stearic acid, lithonic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid or sulfosalicylic acid.

10. A pharmaceutical formulation prepared from the GLP-1/GIP receptor double-agonism polypeptide compound of any one of claims 1 to 5, wherein the pharmaceutical formulation comprises any one of a tablet, capsule, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound formulation.

11. A pharmaceutical composition prepared from a GLP-1/GIP receptor dual agonist polypeptide compound, wherein the pharmaceutical composition comprises the GLP-1/GIP receptor dual agonist polypeptide compound of any one of claims 1-5, and further comprises a carrier or diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt of the GLP-1/GIP receptor dual agonist polypeptide compound of any one of claims 8-9, further comprising a carrier or diluent.

12. Use of the GLP-1/GIP receptor dual agonist polypeptide compound of any one of claims 1-5 or the GLP-1/GIP receptor dual agonist polypeptide compound of any one of claims 8-9, the pharmaceutically acceptable salt thereof or the medicament of claim 10 or the pharmaceutical composition of claim 11 in the manufacture of a medicament for the treatment of a metabolic disease or disorder; characterized in that the metabolic disease or disorder comprises diabetes, obesity, hypertension, nonalcoholic steatohepatitis, dyslipidemia or senile dementia.