CN117624333A - A kind of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds and their applications - Google Patents

Abstract

The invention discloses a GLP-1 receptor, glucagon receptor and GIP receptor three-excited polypeptide compound and application thereof, wherein the polypeptide compound can selectively act on the GLP-1 receptor, glucagon receptor and GIP receptor, can simultaneously exert the activities of GLP-1, glucagon and GIP, has a unique excited active proportion on the three receptors, and brings about better effects of reducing blood sugar, reducing weight and regulating lipid. The preparation method has great potential in preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, nonalcoholic fatty liver disease, nonalcoholic fatty hepatitis, dyslipidemia and the like.

Description

A class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds and its applications

Technical field

The present invention relates to the field of biomedicine technology, and in particular to a type of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds and their applications.

Background technique

Obesity and its related metabolic syndrome have become a global public health problem. Many metabolic syndromes such as type 2 diabetes (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and abnormal lipid metabolism The incidence and course of the disease are closely related to obesity. Research shows that clinically 80-90% of T2DM patients are overweight or obese. At present, the efficacy of drugs for treating obesity is relatively limited, and many drugs for treating obesity also have significant side effects.

Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic peptide hormone secreted by small intestinal L cells. It exerts hypoglycemic effects after specifically binding to the GLP-1 receptor. The main advantage of GLP-1 is that it has a blood glucose-dependent incretin secretion effect and avoids the risk of hypoglycemia that is often present in the treatment of diabetes. In addition to regulating blood sugar, GLP-1 can also prevent the degeneration of pancreatic beta cells, stimulate the proliferation and differentiation of beta cells, and improve the progression of diabetes from the source. In addition, GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, suppressing appetite, etc., and has a partial weight loss effect. There are currently a number of long-acting GLP-1 drugs on the market, such as liraglutide, semaglutide, etc. Although GLP-1 drugs have a safe hypoglycemic effect, if you want to achieve better weight loss, you generally need to increase the dosage. However, large doses of GLP-1 drugs are prone to gastrointestinal side effects and tolerance. Poor performance results in a narrow therapeutic window. Therefore, there remains a need for more safe and tolerable therapeutic agents that are effective for weight loss and glycemic control.

Glucagon is a hormone secreted by the alpha cells of the pancreas. It acts on the liver under stress conditions such as cold and hunger, decomposing glycogen in the liver and increasing blood sugar. In addition to its blood sugar-raising effect, glucagon also has the effects of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851–1861). Long-term administration can show weight loss effects by increasing energy metabolism. However, these beneficial effects of glucagon on energy metabolism cannot be applied due to its inherent blood sugar effect.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide. Both GLP-1 and GLP-1 are incretins, which play a key physiological role in the metabolism of blood sugar in the body. GIP exerts its physiological activity in the body by interacting with GIP receptors distributed in pancreatic beta cells, adipose tissue, and the central nervous system. Similar to GLP-1, GIP can stimulate pancreatic beta cells to secrete insulin to lower blood sugar, and can protect pancreatic beta cells to control glucose metabolism in the body. In addition, GIP can also stimulate GIP receptors in adipose tissue to promote fat metabolism, and GIP also has the effect of suppressing appetite.

The GLP-1 receptor, glucagon receptor and GIP receptor corresponding to GLP-1, glucagon and GIP all belong to the GPCR receptor family and have similar protein structures and binding mechanisms, making it possible to design triple agonists targeting these three receptors. Peptide compounds are possible. The tri-agonist polypeptide compounds of these three receptors can act on GLP-1 receptors, glucagon receptors and GIP receptors at the same time, and can simultaneously exert the activities of GLP-1, glucagon and GIP. GLP-1 can lower blood sugar and suppress appetite; glucagon can break down fat, reduce weight, and increase blood sugar, but it can be offset by the hypoglycemic activity of GLP-1; GIP mainly stimulates insulin secretion. The three activities of the tri-agonist polypeptide compound of GLP-1 receptor, glucagon receptor and GIP receptor cooperate with each other to form a feedback mechanism based on blood sugar concentration, which can not only control blood sugar, break down fat, but also reduce body weight. For the treatment of metabolic diseases such as diabetes and obesity, GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist peptide compounds have significant advantages over simple GLP-1 analogues.

Contents of the invention

The invention provides a tri-agonist polypeptide compound of GLP-1 receptor, glucagon receptor and GIP receptor and its application. The tri-agonist polypeptide compound can act on GLP-1 receptor, glucagon receptor and GIP receptor at the same time. It can exert the activities of GLP-1, glucagon and GIP at the same time; it not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effects of glucagon on body weight, energy metabolism, and lipid metabolism, and also has the effect of GIP on sugar, lipid metabolism and appetite. Inhibit the beneficial effects, thereby having a synergistic effect on sugar, lipid, and energy metabolism; apply it to the preparation of drugs for the treatment of metabolic syndrome, such as diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia and other diseases has greater potential.

In order to achieve the above-mentioned object of the invention, the technical solutions of the present invention are as follows:

A type of tri-agonist polypeptide compound for GLP-1 receptor, glucagon receptor and GIP receptor. The general formula of the amino acid sequence of the polypeptide compound is:

Tyr-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa ₁ -Leu-Asp-Lys-Xaa ₂ -Ala-Gln-Aib-Ala-Phe-Ile-Glu- Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ ,

Among them: Xaa ₁ is selected from Leu or αMeLeu; Xaa ₂ is selected from Lys or Lys with modified side chain;

The Lys whose side chain is modified is selected from

Among them: n is a natural number, and 16≤n≤20.

Preferably, n is 16, 18 or 20.

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

SEQ ID NO:1

SEQ ID NO:2

The present invention also provides pharmaceutically acceptable salts of the above GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds.

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

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

The present invention also provides a pharmaceutical composition prepared from a type of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds. The pharmaceutical composition includes the type of GLP-1 receptor, glucagon receptor and GIP. Receptor triagonist polypeptide compounds, pharmaceutically acceptable carriers or diluents; or the pharmaceutical composition includes pharmaceutically acceptable GLP-1 receptor, glucagon receptor and GIP receptor triagonist polypeptide compounds. Salt, pharmaceutically acceptable carrier or diluent.

The present invention also provides the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound or the pharmaceuticals of the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound. The use of the above acceptable salts or the pharmaceutical agents or pharmaceutical compositions in the preparation of medicaments for treating metabolic diseases or disorders. The metabolic disease or condition includes diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.

The compound prepared by the present invention has strong agonistic activity on GLP-1 receptors, strong agonistic activity on GIP receptors, and weak agonistic activity on glucagon receptors, but achieves better blood sugar lowering and weight loss. It has lower gastrointestinal side effects and lower gastrointestinal side effects, providing a new idea for preparing such tri-agonist polypeptide compounds of GLP-1 receptor, glucagon receptor and GIP receptor.

Compared with the existing technology, the beneficial technical effects of the present invention are:

(1) The tri-agonist polypeptide compound of GLP-1 receptor, glucagon receptor and GIP receptor of the present invention can more effectively lower blood sugar while having significant weight loss and weight gain prevention effects, and better regulate lipid metabolism. ;

(2) The GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds of the present invention have a unique N-terminal 6-10 sequence structure (YTNDV), and a unique in vitro GLP-1 receptor, glucagon receptor The tri-agonist polypeptide compound of the present invention has a strong agonistic activity on GLP-1 receptors, a strong agonistic activity on GIP receptors, and a weak agonistic activity on glucagon receptors. It has significantly improved hypoglycemic, weight loss and lipid metabolism regulation effects, with unexpected beneficial effects;

(3) Compared with the reported GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds of the present invention have similar GLP-1 receptor agonist activity, close GIP receptor agonist activity, and significantly weaker glucagon receptor agonist activity, but the weight loss, lipid metabolism regulation and hypoglycemic activities of the tri-agonist polypeptide compound of the present invention are significantly improved. , has greater potential in the treatment of metabolic diseases, and provides new ideas for the drug development of such GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist peptide compounds;

(4) The GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds provided by the present invention have stable chemical properties and have pharmacokinetic characteristics that support at least once-weekly administration; the GLP- The therapeutic effect of tri-agonist polypeptide compounds on 1 receptor, glucagon receptor and GIP receptor on metabolic diseases such as T2DM, obesity, and dyslipidemia is better than existing GLP-1 marketed drugs and similar drugs under development. Therefore, the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds provided by the present invention are suitable for treating metabolic diseases, such as diabetes, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, and blood lipids. Active ingredient in drugs such as disorders.

Description of drawings

Figure 1 shows the body weight change percentage of each test substance of the present invention in DIO mice after long-term administration for 21 days.

Detailed ways

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

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

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

Further, the amino acids mentioned in the present invention can be abbreviated as follows according to the naming rules 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 stated, all amino acid residues in the polypeptide of the present invention are preferably in the L configuration.

Further, the " _-NH2 " portion on the C-terminus of the sequence indicates the amide group ( _-CONH2 ) on the C-terminus.

Furthermore, in addition to natural amino acids, unnatural amino acids, α-aminoisobutyric acid (Aib) and α-methylleucine (αMeLeu) are also used in the sequence of the present invention.

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

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

Example 1

Synthesis of polypeptide compound of SEQ ID NO:1

(1) Swelling of resin

Weigh 0.382g (0.1mmol equivalent) of RinkAmide MBHA resin with a loading capacity of 0.262mmol/g, put it into a 25mL reactor, wash the resin once with 7mL of DCM and methanol alternately, and wash the resin with 7mL of DCM twice. Then the resin was swollen with 7 mL of DCM for 1 h, and finally the resin was washed with 7 mL of DMF three times.

(2) Removal of resin Fmoc protective group

Transfer the swollen resin to the PSI-200 peptide synthesizer, add 7mL of 20% piperidine/DMF (v/v) and react at room temperature for 5 minutes. Filter out the deprotection solution, wash the resin once with 7mL of DMF, and then add 7mL of 20% piperidine/DMF. DMF (v/v) deprotection solvent reacted with the resin for 15 minutes, and finally washed the resin 4 times with 7 mL of DMF, 1.5 minutes each time, to obtain the Rink resin with the Fmoc protecting group removed.

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

Weigh Fmoc-Ser(tBu)-OH (0.4mmol), dissolve it with 3mL 10% DMF/DMSO (v/v), add 2mL DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activate for 30 minutes, and activate Add good amino acids into the reactor and react with shaking at room temperature for 2 hours. After filtering out the reaction solution, wash the resin 4 times with 7 mL DMF. Use Kaiser reagent to check whether the reaction coupling is complete. If it is incomplete, couple it twice.

(4) Extension of peptide chain

According to the sequence of the peptide chain, repeat the above deprotection and coupling steps to connect the corresponding amino acids in sequence until the synthesis of the peptide chain is completed. Among them, DIC/Oxyma (1.2mmol/1.2mmol) condensation agent was used when coupling 12-bit Ile, and the reaction was carried out with shaking at room temperature for 6 hours. The Lys site with modified side chain at position 17 adopts Fmoc-Lys(Dde)-OH protection strategy, while the N-terminal His uses Boc-His(Boc)-OH.

(5)Modification of Lys side chain

After the synthesis of the peptide chain is completed, add 7 mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove the Dde protecting group of Lys at position 17. After the Dde protecting group is removed, add 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44mmol HOBt, shaking condensation reaction for 2h. 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 condensation reaction was carried out with shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of eicosanedioic acid mono-tert-butyl ester, 0.4 mmol of DIC and 0.44 mmol of HOBt were added for condensation reaction for 2 hours. After the reaction was complete, the resin was washed 4 times with 7 mL of DMF.

(6) Cleavage of polypeptides

Transfer the polypeptide-connected resin obtained above to a round-bottomed bottle, use 5 mL of cutting agent Reagent R (TFA/anisole/phenol/EDT, 90:5:3:2, V/V) to cut the resin, and then The reaction was carried out in an oil bath at a constant temperature of 30°C for 2 hours. The cutting solution was poured into 40 mL of glacial ether. After refrigeration and centrifugation, the crude product was washed three times with 15 mL of glacial ether, and finally dried with nitrogen to obtain the crude peptide.

(7) Purification of polypeptides

Dissolve the crude target polypeptide in water, filter it with a 0.25 μm microporous membrane, and then purify it in a Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions are C18 reversed-phase preparative column (250mm×20mm, 12μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate is 8mL/min; detection wavelength is 214nm. Use linear gradient (20% B ~ 70% B/30min) to elute, collect the target peak, remove methanol and freeze-dry to obtain 0.20g of pure product, with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4710.4. ESI-MS m/z: calculated values [M+3H] ³⁺ 1517.1, [M+4H] ⁴⁺ 1178.6; observed values [M+3H] ³⁺ 1517.0, [M+4H] ⁴⁺ 1178.4.

Example 2

Synthesis of SEQ ID NO:2 polypeptide compounds

The synthesis method is the same as Example 1, except for the modification step of the Lys side chain, which is as follows: after the synthesis of the peptide chain is completed, add 7 mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove Lys at position 17 Dde protecting group, after removing the Dde protecting group, add 0.4mmol Fmoc-AEEA-OH, 0.4mmol DIC and 0.44mmol HOBt, and shake and condensate for 2 hours. 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 condensation 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 condensation reaction was carried out with shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of eicosanedioic acid mono-tert-butyl ester, 0.4 mmol of DIC and 0.44 mmol of HOBt were added for condensation reaction for 2 hours. After the reaction was complete, the resin was washed 4 times with 7 mL of DMF. The target peak was collected and freeze-dried to obtain 0.21g 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 4855.6. ESI-MS m/z: calculated values [M+3H] ³⁺ 1619.5, [M+4H] ⁴⁺ 1214.9; observed values [M+3H] ³⁺ 1619.3, [M+4H] ⁴⁺ 1214.7.

Example 3

Determination of the agonistic activity of polypeptide compounds on GLP-1 receptor, glucagon receptor and GIP receptor

Receptor agonism of polypeptide compounds is determined by functional assays that measure the cAMP response of HEK-293 cell lines stably expressing human GLP-1 receptor, glucagon receptor, or GIP receptor. Cells stably expressing the above three receptors were divided into T175 culture flasks and grown in the medium overnight to a nearly confluent state. The medium was then removed, and the cells were washed with calcium- and magnesium-free PBS, and then treated with protease using Accutase enzyme. . Wash the detached cells and resuspend them in assay buffer (20mM HEPES, 0.1% BSA, 2mM IBMX, 1×HBSS) and determine the cell density and aliquot 25 μL into 96-well plates. hole. For measurement, 25 μL of a solution of the test polypeptide compound in assay buffer is added to the wells and then incubated at room temperature for 30 min. Cisbio's kit was used to determine the cAMP content of cells based on homogeneous time-resolved fluorescence (HTRF). After adding HTRF reagent diluted in lysis buffer (kit component), the plate was incubated for 1 hour and the fluorescence ratio at 665/620 nm was measured. The in vitro potency of agonists was quantified by detecting the concentration that elicited 50% activation of the maximal response ( _EC50 ).

The detection data (nM) in the examples of this patent application are shown in Table 1 below. Although the detection data are stated with a certain number of significant figures, it should not be considered that the data has been determined to be an exact number of significant figures.

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

As shown in Table 1, the agonistic activities of SEQ ID NO:1 and SEQ ID NO:2 on GLP-1 receptors are slightly stronger than those of natural GLP-1 (approximately 2-4 times stronger) and retatrutide (Cell Metabolism, 2022, 34, 1234–1247, clinically developed tri-agonist polypeptide compounds for GLP-1 receptor, glucagon receptor and GIP receptor, approximately 2-4 times stronger); SEQ ID NO: 1 and SEQ ID NO: 2 have strong effects on glucagon receptors. The agonistic activities are significantly weaker than natural glucagon (approximately 259-282 times weaker) and retatrutide (approximately 248-270 times weaker); the agonistic activities of SEQ ID NO:1 and SEQ ID NO:2 on GIP receptors are slightly weaker than natural GIP (approximately 13-14 times weaker) and retatrutide (approximately 7-8 times weaker). It shows that the polypeptide compound of the present invention has strong GLP-1 receptor agonistic activity, weak glucagon receptor agonistic activity and good GIP receptor agonistic activity, and has strong effects on GLP-1 receptor, GIP receptor and glucagon receptor. It has a special agonistic activity ratio, and its agonistic activity ratio on GLP-1 receptor, GIP receptor and glucagon receptor is significantly different from similar clinical drugs under development, and is also in line with the characteristics of the three agonist polypeptide compounds described in this patent.

Example 4

Pharmacokinetic properties of peptide compounds in rats

Rats were given a subcutaneous (s.c.) injection of 50 nmol/kg, and blood samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h and 48h after administration. After protein precipitation using acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-life were calculated using WinonLin5.2.1 (non-compartmental model) (Table 2).

Table 2: Overview of pharmacokinetics of peptide compounds in rats

sample T _1/2 (h) C _max (ng/mL) Semaglutide 8.3 402 Retatrutide 8.1 411 SEQ ID NO:1 9.5 468 SEQ ID NO:2 10.9 471

As shown in the results in Table 2, the in vivo half-life of the polypeptide compound of the present invention is significantly extended, which is better than the marketed Semaglutide administered once a week and the same type of drug Retatrutide under clinical development (Cell Metabolism, 2022, 34, 1234-1247). It shows that the polypeptide compound of the present invention has pharmacokinetic characteristics that support at least once-weekly administration.

Example 5

Effects of polypeptide compounds on blood lipids and body weight in diet-induced obesity (DIO) mice

Male C57BL/6J mice, weighing about 22g, were fed with D12492 high-fat feed from Research Diets Company for 18 weeks to create a DIO mouse model. Before the start of drug administration, each group of DIO mice were randomly divided into groups according to body weight, with 6 mice in each group, namely the normal saline group (blank control group), the positive control group (semaglutide, retatrutide (clinical stage GLP-1 receptor, glucagon Receptor and GIP receptor tri-agonist peptide compounds; Cell Metabolism, 2022, 34, 1234-1247)) and test sample group (SEQ ID NO: 1, SEQ ID NO: 2). Mice in each group were subcutaneously injected with physiological saline (10 mg/kg), semaglutide (10 nmol/kg), retatrutide (10 nmol/kg), SEQ ID NO: 1 (10 nmol/kg), SEQ ID NO: 2 (10 nmol/kg) once every two days. kg), the dosing cycle is 21 days. The body weight changes of the mice were recorded every day, and body fat 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 tissues were taken to measure liver triglyceride (TG) and total cholesterol (TC) contents. At the same time, blood was taken to prepare serum, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total cholesterol (TC) were measured.

Table 3: Changes in body weight and body fat of DIO mice during the 3-week dosing period

Sample (dose) Overall weight change (%) Body fat change (%) Blank control (normal saline group) 2.9±3.7 3.8±0.8 Semaglutide(10nmol/kg) -15.4±2.1 ^*** -19.8±3.5 ^*** Retatrutide(10nmol/kg) -30.5±2.5 ^*** -35.9±2.1 ^*** SEQ ID NO:1(10nmol/kg) -44.5±3.0 ^***,### -54.3±5.8 ^***,### SEQ ID NO:2(10nmol/kg) -38.1±1.9 ^***,### -48.2±4.1 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

As shown in Figure 1 and Table 3, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the body weight and body fat content of mice after continuous administration for 3 weeks in DIO mice, and the present invention The effect of weight loss and body fat reduction of the invented polypeptide compound is significantly stronger than that of the positive control drugs semaglutide and retatrutide. It is worth noting that the GLP-1 receptor agonistic activity of retatrutide is similar to natural GLP-1, the glucagon receptor agonistic activity is similar to natural glucagon, and the GIP receptor agonistic activity is similar to natural GIP. It can be seen that the GLP-1 receptor agonistic activity of retatrutide is similar to SEQ ID NO:1 and SEQ ID NO:2, and its GIP receptor agonistic activity is only about 7-7 stronger than SEQ ID NO:1 and SEQ ID NO:2. 8 times, but the glucagon receptor agonistic activity of retatrutide is significantly stronger than that of SEQ ID NO:1 and SEQ ID NO:2, but the polypeptide compounds SEQ ID NO:1 and SEQ ID NO:2 of the present invention show significantly better Greater weight loss and body fat reduction activity than retatrutide.

Table 4: Liver triglyceride (TG) and total cholesterol (TC) contents of DIO mice after 3 weeks of treatment

Sample (dose) Total cholesterol (mg/g) Triglycerides (mg/g) Blank control (normal saline group) 9.3±0.5 85.6±7.1 Semaglutide(10nmol/kg) 7.6±0.3 ^*** 68.1±5.6 ^*** Retatrutide(10nmol/kg) 6.5±0.3 ^*** 59.8±4.9 ^*** SEQ ID NO:1(10nmol/kg) 5.3±0.2 ^***,### 48.9±3.4 ^***,### SEQ ID NO:2(10nmol/kg) 5.5±0.4 ^***,### 50.1±4.3 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

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

Sample (dose) Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Blank control (normal saline group) 415±58 389±21 Semaglutide(10nmol/kg) 241±63 ^*** 211±33 ^*** Retatrutide(10nmol/kg) 234±51 ^*** 203±24 ^*** SEQ ID NO:1(10nmol/kg) 184±26 ^***,### 146±17 ^***,### SEQ ID NO:2(10nmol/kg) 199±37 ^***,### 161±12 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

As shown in Table 4 and Table 5, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the embodiments of the present invention can significantly reduce liver triglycerides and Total cholesterol content, and can significantly reduce serum alanine aminotransferase and aspartate aminotransferase contents, and the effects of the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention are significantly stronger than the positive control drugs semaglutide and retatrutide, indicating that the polypeptide compounds of the present invention Compounds hold good promise for the treatment of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Table 6: Serum triglyceride (TG) and total cholesterol (TC) contents of DIO mice after 3 weeks of treatment

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

As shown in Table 6, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the serum triglyceride and total cholesterol content of mice when administered continuously for 3 weeks in DIO mice, and the present invention The effect of the invented polypeptide compound on reducing serum lipid (triglyceride and cholesterol) content is significantly stronger than that of the positive control drugs semaglutide and retatrutide.

Example 6

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

Male db/db mice were randomly divided into groups, with 6 mice in each group. They are physiological saline group (blank control group), positive control group (semaglutide and retatrutide) and test sample group (SEQ ID NO: 1 and SEQ ID NO: 2) respectively. After one week of adaptive feeding, blood was taken from the tail to measure the initial HbA1c value and fasting blood glucose value before the start of treatment. Mice in each group were subcutaneously injected with physiological saline (10 mg/kg), semaglutide (10 nmol/kg), retatrutide (10 nmol/kg), SEQ ID NO: 1 (10 nmol/kg), SEQ ID NO: 2 (10 nmol/kg) once every two days. kg), the administration period is 35 days. After the treatment, the mice were fasted overnight and the fasting blood glucose value was measured, and blood was taken to measure the HbA1c (%) value.

Table 7: Changes in HbA1c (%) in db/db mice during the 35-day dosing cycle

Sample (dose) HbA1c% (before treatment) HbA1c% (after treatment) Blank control (normal saline group) 6.9±0.3 7.6±0.4 Semaglutide(10nmol/kg) 6.8±0.2 6.3±0.2 ^*** Retatrutide(10nmol/kg) 7.0±0.4 6.2±0.3 ^*** SEQ ID NO:1(10nmol/kg) 7.1±0.4 5.3±0.2 ^***,### SEQ ID NO:2(10nmol/kg) 7.0±0.2 5.4±0.3 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

As shown in Table 7, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention can significantly reduce the HbA1c value of the mice after continuous administration for 35 days in db/db mice. The HbA1c value of the mice in the polypeptide compound group was significantly lower than the positive controls semaglutide and retatrutide, indicating that the polypeptide compound of the present invention has a good blood sugar control effect.

Table 8: Changes in fasting blood glucose in db/db mice during the 35-day dosing cycle

Sample (dose) Fasting blood glucose (%) Blank control (normal saline group) +4.1±0.3% Semaglutide(10nmol/kg) -3.6±0.2% ^*** Retatrutide(10nmol/kg) -3.5±0.4% ^*** SEQ ID NO:1(10nmol/kg) -5.9±0.3% ^***,### SEQ ID NO:2(10nmol/kg) -5.5±0.4% ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 mice in each group ±SD.

As shown in Table 8, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the embodiments of the present invention can significantly reduce the fasting blood glucose of db/db mice when administered continuously for 35 days. It shows that the polypeptide compound of the present invention has excellent blood sugar control effect, and the blood sugar control effect of the polypeptide compound of the present invention is significantly stronger than the positive control drugs semaglutide and retatrutide.

Example 7

Gastrointestinal side effects of peptide compounds

Male SD rats (200–250g) were randomly divided into groups and raised in single cages. Four days before the experiment, rats in each group were given additional kaolin diets (Research Diets) on the basis of regular diets. The kaolin diets were placed in a separate part of the food funnel. In the compartment, rats were allowed to become accustomed to the presence of kaolin chow in the cage. The rats were fasted for 12 hours before the experiment. At 0 h, the rats in each group were intraperitoneally injected with 10% DMSO/water (blank), 3 mg/kg cisplatin (the control group to determine whether the model was successful), and 10 nmol/kg and 50 nmol/kg. and 100 nmol/kg of semaglutide, retatrutide, SEQ ID NO: 1, SEQ ID NO: 2. Then the rats in each group were quickly given pre-weighed ordinary feed and kaolin clay feed, and the amount of ordinary feed and kaolin clay feed eaten by the rats in each group in 24 hours was recorded. Based on the consumption of ordinary feed and kaolin clay feed, the side effects caused by the compounds were judged. strength.

Table 9: Intake of ordinary feed and kaolin clay in SD rats in 24 hours

sample Ordinary feed intake (g) Kaolin intake (g) Blank control 23.2±2.5g 0.4±0.1g Cisplatin 14.9±0.8g ^*** 3.2±0.4g ^*** Semaglutide(10nmol/kg) 19.6±1.1g ^*** 0.5±0.1g Retatrutide(10nmol/kg) 16.8±1.2g ^*** 0.6±0.1g SEQ ID NO:1(10nmol/kg) 14.0±0.7g ^***,### 0.2±0.1g SEQ ID NO:2(10nmol/kg) 14.2±0.5g ^***,### 0.3±0.1g

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 rats in each group ±SD.

Table 10: Dietary intake of ordinary feed and kaolin clay for SD rats in 24 hours

sample Ordinary feed intake (g) Kaolin intake (g) Blank control 23.2±2.5g 0.4±0.1g Cisplatin 14.9±0.8g ^*** 3.2±0.4g ^*** Semaglutide(50nmol/kg) 18.2±1.3g ^*** 0.9±0.2g ^*** Retatrutide(50nmol/kg) 15.6±1.8g ^*** 1.0±0.3g ^*** SEQ ID NO:1(50nmol/kg) 12.1±0.9g ^***,### 0.5±0.1g ^### SEQ ID NO:2(50nmol/kg) 12.4±0.7g ^***,### 0.5±0.2g ^###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 rats in each group ±SD.

Table 11: Dietary intake of ordinary feed and kaolin clay for SD rats in 24 hours

sample Ordinary feed intake (g) Kaolin intake (g) Blank control 23.2±2.5g 0.4±0.1g Cisplatin 14.9±0.8g ^*** 3.2±0.4g ^*** Semaglutide(100nmol/kg) 17.1±1.8g ^*** 1.2±0.4g ^*** Retatrutide(100nmol/kg) 14.3±1.4g ^*** 1.4±0.2g ^*** SEQ ID NO:1(100nmol/kg) 10.2±0.7g ^***,### 0.5±0.2g ^### SEQ ID NO:2(100nmol/kg) 10.6±0.6g ^***,### 0.6±0.1g ^###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide and retatrutide groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as the average of 6 rats in each group ±SD.

As shown in Table 9 to Table 11, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the embodiments of the present invention have good inhibitory effects on rat eating at doses of 10 nmol/kg, 50 nmol/kg, and 100 nmol/kg. The effect is significantly better than the positive controls semaglutide and retatrutide. However, the polypeptide compounds SEQ ID NO: 1 and SEQ ID NO: 2 prepared in the examples of the present invention did not cause kaolin eating in rats at doses of 10 nmol/kg, 50 nmol/kg, and 100 nmol/kg. The kaolin intake of the polypeptide compound group was similar to that of the blank group. At doses of 50 nmol/kg and 100 nmol/kg, the kaolin intake of rats in the SEQ ID NO: 1 and SEQ ID NO: 2 groups was significantly lower than the kaolin intake of rats in the positive control semaglutide and retatrutide groups. This shows that the polypeptide compounds prepared in the examples of the present invention did not cause gastrointestinal side effects in rats, and the gastrointestinal side effects were lower than the positive controls semaglutide and retatrutide.

Claims (9)

1. A class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, characterized in that the general formula of the amino acid sequence of the polypeptide compound is: Tyr-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa ₁ -Leu-Asp-Lys-Xaa ₂ -Ala-Gln-Aib-Ala-Phe-Ile-Glu- Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ , in: Xaa ₁ is selected from Leu or αMeLeu; Xaa ₂ is selected from Lys or Lys with modified side chain; The Lys whose side chain is modified is selected from Among them: n is a natural number, and 16≤n≤20. 2. A kind of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to claim 1, characterized in that, the n is 16, 18 or 20. 3. A kind of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to claim 1, characterized in that the sequence structure of the polypeptide compound is selected from the group consisting of SEQ ID NO: 1-2 Any of the amino acid sequences shown: SEQ ID NO:1 SEQ ID NO:2 . 4. A pharmaceutically acceptable salt of the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to any one of claims 1 to 3. 5. The pharmaceutically acceptable salt of a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to claim 4, characterized in that the pharmaceutically acceptable salt is GLP- Salts formed by 1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds and one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, caproic acid, benzenesulfone Acid, pamoic acid, benzoic acid, salicylic acid, lauric acid, cinnamic acid, propionic acid, dodecyl sulfate, citric acid, ascorbic acid, stearic acid, stearic acid, oxalic acid, lactic acid, succinic acid, propionic acid Diacid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid. 6. A medicament prepared from the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to any one of claims 1 to 3, characterized in that the medicament includes any pharmaceutical The tablets, capsules, syrups, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions, suppositories or compound preparations. 7. A pharmaceutical composition prepared from a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound, characterized in that the pharmaceutical composition includes a GLP according to any one of claims 1-3 -1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds, pharmaceutically acceptable carriers or diluents; or the pharmaceutical composition includes a type of GLP-1 according to any one of claims 4-5 Receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds pharmaceutically acceptable salts, pharmaceutically acceptable carriers or diluents. 8. A kind of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to any one of claims 1-3 or a kind of GLP-1 receptor according to any one of claims 4-5 , glucagon receptor and GIP receptor tri-agonist polypeptide compounds pharmaceutically acceptable salts or a pharmaceutical agent according to claim 6 or a pharmaceutical composition according to claim 7 in the preparation of drugs for the treatment of metabolic diseases or disorders use in. 9. Use according to claim 8, characterized in that the metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.