CN117624333A

CN117624333A - GLP-1 receptor, glucagon receptor and GIP receptor tri-excitation polypeptide compound and application thereof

Info

Publication number: CN117624333A
Application number: CN202311652574.2A
Authority: CN
Inventors: 韩京; 龙倩; 滕达
Original assignee: Jiangsu Normal University
Current assignee: Jiangsu Normal University
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-03-01

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

GLP-1 receptor, glucagon receptor and GIP receptor tri-excitation polypeptide compound and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a GLP-1 receptor, glucagon receptor and GIP receptor tri-excitation polypeptide compound and application thereof.

Background

Obesity and its related metabolic syndrome have become global public health problems, and the incidence and progression of many metabolic syndromes such as type 2 diabetes (T2 DM), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia are closely related to obesity. Studies have shown that clinically 80-90% of T2DM patients are associated with overweight or obesity. The current medicines for treating obesity have limited curative effects, and many medicines for treating obesity have obvious side effects.

Glucagon-like peptide-1 (GLP-1) is a glucose-dependent hypoglycemic polypeptide hormone secreted by small intestine L cells, and plays a hypoglycemic role after being specifically combined with 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, are currently 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 administration of GLP-1 drugs is easy to produce gastrointestinal side effects and 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.

Glucagon (glucon) is a hormone secreted by islet alpha cells. Acts on liver in stress state such as organism cold and hunger, and decomposes glycogen in liver to increase blood sugar. In addition to its glycemic effect, glucopon has effects of promoting lipolysis, fat oxidation, fever, etc. (diabetes, 2017,60,1851-1861), and long-term administration can exhibit weight-reducing effects by increasing energy metabolism, but the beneficial effects of glucopon on energy metabolism have not been utilized because of its inherent glycemic effect.

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.

GLP-1 receptors, glucopon receptors and GIP receptors corresponding to GLP-1, glucopon and GIP all belong to the GPCR receptor family, have similar protein structures and binding mechanisms, and make it possible to design tri-agonistic polypeptide compounds for these three receptors. The three-receptor tri-agonist polypeptide compound can simultaneously act on GLP-1 receptor, glucagon receptor and GIP receptor, and can simultaneously play the activities of GLP-1, glucagon and GIP. GLP-1 can reduce blood sugar and suppress appetite; glucopon can break down fat, reduce weight, raise blood sugar, but can be counteracted by the hypoglycemic activity of GLP-1; GIP mainly stimulates insulin secretion. The three activities of the three-agonism polypeptide compounds of the GLP-1 receptor, the glucogon receptor and the GIP receptor are mutually matched to form a feedback mechanism according to the blood sugar concentration, so that the blood sugar can be controlled, the fat can be decomposed, and the weight can be reduced. GLP-1 receptor, glucopon receptor and GIP receptor tri-agonistic polypeptide compounds have significant advantages over simple GLP-1 analogues for the treatment of metabolic diseases such as diabetes, obesity, etc.

Disclosure of Invention

The invention provides a GLP-1 receptor, a glucagon receptor and a GIP receptor tri-excitation polypeptide compound and application thereof, wherein the tri-excitation polypeptide compound can act on the GLP-1 receptor, the glucagon receptor and the GIP receptor simultaneously, and can exert the activities of GLP-1, glucagon and GIP simultaneously; not only has the therapeutic effect of GLP-1 on diabetes, but also has the beneficial effects of glucopon on weight, energy metabolism and lipid metabolism, and also has the beneficial effects of GIP on glucose, lipid metabolism and appetite suppression, thereby generating synergistic effect on glucose, lipid and energy metabolism; 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.

In order to achieve the above object, the present invention has the following technical scheme:

GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound, wherein the amino acid sequence general formula of the polypeptide compound is as follows:

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 ，

wherein: xaa ₁ Selected from Leu or αMeLeu; xaa ₂ Selected from Lys or Lys with modified side chain;

the side chain modified Lys is selected from

Wherein: n is a natural number, and n is more than or equal to 16 and less than or equal to 20.

Preferably, n is 16, 18 or 20.

Preferably, the sequence structure of the tri-agonistic 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 invention also provides pharmaceutically acceptable salts of the GLP-1 receptor, the glucagon receptor and the GIP receptor tri-excitation polypeptide compounds.

Further, the pharmaceutically acceptable salt is a salt of a GLP-1 receptor, a glucagon receptor, and a GIP receptor tri-agonist polypeptide compound with 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, dodecylsulfuric acid, citric acid, ascorbic acid, wine stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid.

The invention also provides a medicament prepared from the GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound, wherein the medicament comprises any one of tablets, capsules, syrup, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound preparation.

The invention also provides a pharmaceutical composition prepared from the GLP-1 receptor, the glucagon receptor and the GIP receptor three-agonism polypeptide compound, and the pharmaceutical composition comprises the GLP-1 receptor, the glucagon receptor and the GIP receptor three-agonism polypeptide compound, a pharmaceutically acceptable carrier or a diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt, a pharmaceutically acceptable carrier or diluent of the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound.

The invention also provides application of the GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound or the pharmaceutically acceptable salt of the GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound or the medicament or the pharmaceutical composition in preparing medicaments for treating metabolic diseases or symptoms. The metabolic disease or disorder includes diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.

The compound prepared by the invention has strong agonistic activity to GLP-1 receptor, strong agonistic activity to GIP receptor and weak agonistic activity to glucagon receptor, but realizes better functions of reducing blood sugar, reducing weight and regulating lipid, has lower gastrointestinal side effect, and provides a new thought for preparing the GLP-1 receptor, glucagon receptor and GIP receptor three-agonistic polypeptide compound.

Compared with the prior art, the invention has the beneficial technical effects that:

(1) The GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound has remarkable weight reduction and weight increase prevention effects while reducing blood sugar more effectively, and can regulate lipid metabolism better;

(2) The GLP-1 receptor, the glucagon receptor and the GIP receptor three-agonism polypeptide compound provided by the invention has a unique N-terminal 6-10 bit sequence structure (YTNDV) and a unique in-vitro GLP-1 receptor, glucagon receptor and GIP receptor agonism activity proportion, and the three-agonism polypeptide compound provided by the invention has strong agonism activity on the GLP-1 receptor and strong agonism activity on the GIP receptor, and weak agonism activity on the glucagon receptor, but brings significantly improved effects of reducing blood sugar, reducing weight and regulating lipid metabolism, and has unexpected beneficial effects;

(3) Compared with reported GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compounds, the GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compounds have similar GLP-1 receptor agonism activity, approximate GIP receptor agonism activity and obviously weaker glucagon receptor agonism activity, but the three-agonism polypeptide compounds have significantly improved weight reduction, lipid metabolism regulation and glucose reduction activity, have higher potential in the aspect of treating metabolic diseases, and provide a new thought for drug development of the GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compounds;

(4) The GLP-1 receptor, glucagon receptor and GIP receptor tri-agonism polypeptide compound provided by the invention has stable chemical property and has pharmacokinetic characteristics for supporting at least once weekly administration; the GLP-1 receptor, glucagon receptor and GIP receptor three-excited polypeptide compound provided by the invention has better treatment effect on metabolic diseases such as T2DM, obesity, dyslipidemia and the like than the existing GLP-1 marketed drugs and the similar drugs under investigation. Therefore, the GLP-1 receptor, glucagon receptor and GIP receptor tri-excitation polypeptide compound provided by the invention is suitable for being used as an active ingredient of medicaments for treating metabolic diseases, such as diabetes, obesity, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, dyslipidemia and the like.

Drawings

FIG. 1 shows the percentage 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.

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

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

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 of SEQ ID NO. 1

(1) Swelling of the resin

0.382g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.262mmol/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 LDMF.

(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 20% piperidine/DMF (v/v) deprotection solvent, reacting with the resin for 15min, washing the resin for 4 times by 7mL of LDMF for 1.5min each time, and obtaining the Rink resin without Fmoc protecting groups.

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

Fmoc-Ser (tBu) -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 of LDMF, and the reaction coupling was checked for completion or not, if incomplete, 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. Wherein DIC/Oxyma (1.2 mmol/1.2 mmol) condensing agent is adopted when coupling the position Ile at 12, and the reaction is carried out by shaking at room temperature for 6 hours. The Lys site in which the side chain at position 17 was modified uses Fmoc-Lys (Dde) -OH protection strategy, while the His at the N-terminus was Boc-His (Boc) -OH.

(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 17 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-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

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.

(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 is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-70% 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. Theory of relativityThe molecular mass is 4710.4.ESI-MS M/z calculated [ M+3H] ³⁺ 1517.1,[M+4H] ⁴⁺ 1178.6; observed value [ M+3H] ³⁺ 1517.0,[M+4H] ⁴⁺ 1178.4。

Example 2

Synthesis of polypeptide compound of SEQ ID No. 2

The synthesis was identical to that of example 1, except for the modification of the Lys side chain, and it was as follows: 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 17 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. And collecting target peaks, freeze-drying to obtain 0.21g of pure product, wherein the purity is more than 98%, and determining the molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 4855.6.ESI-MS M/z calculated [ M+3H] ³⁺ 1619.5,[M+4H] ⁴⁺ 1214.9; observed value [ M+3H] ³⁺ 1619.3,[M+4H] ⁴⁺ 1214.7。

Example 3

Determination of agonistic Activity of polypeptide Compounds on GLP-1 receptor, glucoago receptor and GIP receptor

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 receptor, glucoon receptor or GIP receptor. Cells stably expressing the three receptors were separately dispensed into T175 flasks and grown overnight in medium to near confluence, the medium was removed, the cells were washed with calcium and magnesium free PBS, and then Accutase was usedThe enzyme is subjected to protease treatment. The detached cells were washed and resuspended in assay buffer (20mM HEPES,0.1%BSA,2mM IBMX,1 ×hbss) and cell density was determined and 25 μl aliquots were dispensed into wells of 96-well plates. For measurement, 25 μl of a solution of the test polypeptide compound in the assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. 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 1 hour, 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 the examples of this patent application are shown 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: EC of polypeptide compounds to human GLP-1 receptor, glucagon receptor and GIP receptor ₅₀ Value (expressed in nM)

As shown in Table 1, both SEQ ID NO. 1 and SEQ ID NO. 2 have slightly more agonistic activity at the GLP-1 receptor than native GLP-1 (about 2-4 times stronger) and retatrutide (Cell metanolism, 2022,34,1234-1247, clinically developed GLP-1 receptor, glucogon receptor and GIP receptor tri-agonistic polypeptide compounds), about 2-4 times stronger); both SEQ ID NO. 1 and SEQ ID NO. 2 show significantly weaker agonistic activity at the glucagon receptor than native glucagon (about 259-282-fold weaker) and retatrutide (about 248-270-fold weaker); SEQ ID NO. 1 and SEQ ID NO. 2 show slightly weaker agonistic activity at the GIP receptor than native GIP (about 13-14 fold weaker) and retatrutide (about 7-8 fold weaker). The polypeptide compound disclosed by the invention has strong GLP-1 receptor agonism activity, weaker glucopon receptor agonism activity and better GIP receptor agonism activity, has a special agonism activity proportion to the GLP-1 receptor, the GIP receptor and the glucopon receptor, is obviously different from similar clinical research medicines in the agonism activity proportion to the GLP-1 receptor, the GIP receptor and the glucopon receptor, and also accords with the characteristics of the tri-agonism polypeptide compound disclosed by the patent.

Example 4

Pharmacokinetic properties of polypeptide Compounds in rats

Rats were given 50nmol/kg of subcutaneous (s.c.) injection and blood samples were collected 0.25h, 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, 36h and 48h after administration. After precipitation of the proteins using acetonitrile, plasma samples were analyzed by LC-MS. The pharmacokinetic parameters and half-life were calculated using WinnLin 5.2.1 (non-compartmental model) (Table 2).

Table 2: pharmacokinetic profile of polypeptide Compounds in rats

Sample of	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 the results in table 2 show, the in vivo half-life of the polypeptide compounds of the present invention is significantly prolonged, superior to once-a-week administration of semaglutinide and the clinically developed drug of the same type, retatrutinide (Cell metanolism, 2022,34,1234-1247), which demonstrate that the polypeptide compounds of the present invention have pharmacokinetic characteristics that support at least once-a-week administration.

Example 5

Influence of polypeptide Compounds on blood lipid and body weight in diet-induced obese (DIO) mice

Male C57BL/6J mice, weighing about 22g, were kept on the D12492 high fat diet of Research Diets for 18 weeks to make DIO mouse models. Before the start of the administration, the DIO mice in each group were randomly grouped according to body weight, 6 in each group, respectively, physiological saline group (blank control group), positive control group (semaglutide, retatrutide (GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound in clinical stage; cell metaolism, 2022,34,1234-1247)), and test sample group (SEQ ID NO:1, SEQ ID NO: 2). Each group of mice was subcutaneously injected with normal saline (10 mg/kg), semaglutide (10 nmol/kg), retatrutide (10 nmol/kg), SEQ ID NO:1 (10 nmol/kg), SEQ ID NO:2 (10 nmol/kg) every two days for a 21 day dosing period. 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 collected and the serum glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), triglyceride (TG) and Total Cholesterol (TC) 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 (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 compared with the blank control group<0.001； ^### : comparison with semaglutide and retatrutide group P<0.001 (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 FIG. 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 by continuous administration in DIO mice for 3 weeks, and the effect of the polypeptide compounds of the present invention in reducing the body weight and body fat is significantly stronger than that of positive control drugs semaglutide and retatrutide. Notably, the GLP-1 receptor agonistic activity of retatrutide is similar to that of native GLP-1, the glucogen receptor agonistic activity is similar to that of native glucogen, and the GIP receptor agonistic activity is similar to that of native GIP. As can be seen, the GLP-1 receptor agonistic activity of retortide is similar to that of SEQ ID NO. 1 and SEQ ID NO. 2, the GIP receptor agonistic activity is only about 7-8 times stronger than that of SEQ ID NO. 1 and SEQ ID NO. 2, but the glucogen receptor agonistic activity of retortide is significantly stronger than that of SEQ ID NO. 1 and SEQ ID NO. 2, however, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention show significantly better weight and body fat reducing activity than retortide.

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)	9.3±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 ^***,###

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)	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 ^***,###

As shown in tables 4 and 5, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 prepared in the embodiment of the invention can be 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 compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the invention is obviously stronger than that of positive control drugs semaglutinide and retatrutinide, so that the polypeptide compounds of the invention have good prospects for treating non-alcoholic fatty liver diseases and non-alcoholic fatty liver diseases.

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

As shown by the results in Table 6, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention can significantly reduce serum triglyceride and total cholesterol contents of mice by continuously administering in DIO mice for 3 weeks, and the effect of the polypeptide compounds of the present invention for reducing serum lipid (triglyceride and cholesterol) contents is significantly stronger than that of positive control drugs semaglutide and retatrutide.

Example 6

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

Male db/db mice were randomly grouped, 6 per group. 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, 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), retatrutide (10 nmol/kg), SEQ ID NO:1 (10 nmol/kg), SEQ ID NO:2 (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)	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 ^***,###

As shown in the results of Table 7, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 of the present invention can significantly reduce HbA1c values of mice after 35 days of continuous administration in db/db mice, and HbA1c values of mice in the group of the polypeptide compounds of the present invention after treatment are significantly lower than those of positive controls semaglutide and retatrutide, indicating that the polypeptide compounds of the present invention have excellent glycemic control.

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

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％ ^***,###

As shown in the results of Table 8, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 prepared in the examples of the present invention can significantly reduce the fasting blood glucose value of db/db mice by continuous administration in db/db mice for 35 days, indicating that the polypeptide compounds of the present invention have excellent blood glucose control effect, and the blood glucose control effect of the polypeptide compounds of the present invention is significantly stronger than that of the positive control drugs semaglutinde and retatrutinde.

Example 7

Gastrointestinal side effects of polypeptide Compounds

Male SD rats (200-250 g) were randomly grouped and housed in a single cage, and each group of rats was given kaolin feed (Research Diets) in addition to normal feed 4 days prior to the experiment, and the kaolin feed was placed in separate compartments of a food funnel to allow the rats to become accustomed to the presence of kaolin feed in the cage. Rats were fasted for 12h prior to the experiment, and 10% DMSO/water (blank), 3mg/kg cisplatin (control group to determine whether the model was successful) and 10nmol/kg, 50nmol/kg and 100nmol/kg semaglutide, retatrutide, SEQ ID NO:1, SEQ ID NO:2 were intraperitoneally injected into each group of rats at 0 h. And then, quickly giving the common feed and the kaolin feed which are weighed in advance to each group of rats, recording the feeding amount of each group of rats in the common feed and the kaolin feed for 24 hours, and judging the strength of side effects caused by the compound according to the consumption amount of the common feed and the kaolin feed.

Table 9: normal diet and kaolin food intake by SD rats at 24 hours

Sample of	Feed intake (g) of common feed	Kaolin food intake (g)
			Blank control	23.2±2.5g	0.4±0.1g
Cisplatin (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 compared with the blank control group<0.001； ^### : comparison with semaglutide and retatrutide group P<0.001 (One-Way ANOVA, tukey post hoc test) the results are expressed as mean ± SD of 6 rats per group.

Table 10: normal diet and kaolin food intake by SD rats at 24 hours

Sample of	Feed intake (g) of common feed	Kaolin food intake (g)
			Blank control	23.2±2.5g	0.4±0.1g
Cisplatin (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 ^###

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

Table 11: normal diet and kaolin food intake by SD rats at 24 hours

Sample of	Feed intake (g) of common feed	Kaolin food intake (g)
			Blank control	23.2±2.5g	0.4±0.1g
Cisplatin (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 ^###

As shown in the results of tables 9-11, the polypeptide compounds SEQ ID NO. 1 and SEQ ID NO. 2 prepared in the examples of the invention have good rat feeding inhibition effect at the dosages of 10nmol/kg, 50nmol/kg and 100nmol/kg, and are obviously superior to positive controls semaglutide and retatrutide. However, neither the polypeptide compounds SEQ ID NO. 1 nor SEQ ID NO. 2 prepared in the examples of the present invention resulted in kaolin feeding in rats at doses of 10nmol/kg, 50nmol/kg, 100nmol/kg, and the kaolin feeding in the group of polypeptide compounds prepared in the examples of the present invention was similar to that in the blank group. At doses of 50nmol/kg and 100nmol/kg, the kaolin feeding of rats in the groups SEQ ID NO. 1 and SEQ ID NO. 2 was significantly lower than that of rats in the positive control semaglutide and retatrutide groups. This demonstrates that the polypeptide compounds prepared in the examples of the present invention do not cause gastrointestinal side effects in rats, which are lower than those of the positive controls semaglutinide and retatrutinide.

Claims

1. A GLP-1 receptor, glucagon receptor and GIP receptor three-agonism polypeptide compound is characterized in that the amino acid sequence of the polypeptide compound has a general formula:

wherein:

Xaa ₁ selected from Leu or αMeLeu;

Xaa ₂ selected from Lys or Lys with modified side chain;

the side chain modified Lys is selected from

2. The GLP-1 receptor, glucopon receptor and GIP receptor tri-agonism polypeptide compound according to claim 1, wherein n is 16, 18 or 20.

3. The GLP-1 receptor, glucopon receptor and GIP receptor tri-agonism polypeptide compound according to claim 1, wherein the sequence structure of the polypeptide compound is selected from any one of the amino acid sequences as shown in SEQ ID NOs 1-2:

SEQ ID NO:1

SEQ ID NO:2

。

4. a pharmaceutically acceptable salt of the GLP-1 receptor, the glucagon receptor and the GIP receptor tri-agonist polypeptide compound of any one of claims 1-3.

5. The pharmaceutically acceptable salt of a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to claim 4, wherein the pharmaceutically acceptable salt is a salt of the GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound with 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, dodecylsulfuric acid, citric acid, ascorbic acid, wine stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid.

6. A pharmaceutical formulation comprising a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to any one of claims 1 to 3, 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.

7. A pharmaceutical composition prepared from a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound, wherein the pharmaceutical composition comprises a GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compound according to any one of claims 1-3, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition comprises a pharmaceutically acceptable salt, a pharmaceutically acceptable carrier or diluent of a GLP-1 receptor, a glucogon receptor and a GIP receptor tri-agonist polypeptide compound of any one of claims 4-5.

8. Use of a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to any one of claims 1 to 3 or a class of GLP-1 receptor, glucagon receptor and GIP receptor tri-agonist polypeptide compounds according to any one of claims 4 to 5, a pharmaceutically acceptable salt thereof or a class of agents according to claim 6 or a pharmaceutical composition according to claim 7 for the manufacture of a medicament for the treatment of a metabolic disease or disorder.

9. The use according to claim 8, wherein the metabolic disease or disorder is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.