CN117417431A - A class of polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors and their applications - Google Patents

Abstract

The invention discloses a type of polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors and its application. The polypeptide can act on the GLP-1 receptor, increase the secretion of insulin, and at the same time delay gastric emptying. Thereby lowering blood sugar; acting on glucagon receptors, producing beneficial effects on body weight, energy metabolism, and lipid metabolism; acting on GIP receptors, promoting insulin secretion, improving the sensitivity of pancreatic islet cells to glucose, and helping to reduce blood sugar. By simultaneously regulating these three receptors and affecting multiple physiological pathways, the peptide can improve metabolic function, provide better blood sugar control, weight loss and lipid-lowering effects, and is useful in the treatment of diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, It has greater potential in treating diseases such as steatohepatitis and dyslipidemia.

Description

A class of polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors and its application

Technical field

The present invention relates to the field of biomedicine technology, and in particular to a type of polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors and its application.

Background technique

Obesity has become a global epidemic problem, affecting human health and quality of life, and its prevalence has increased steadily since the mid-20th century. Obesity is an important risk factor for many chronic metabolic diseases, including type 2 diabetes (T2DM), hypertension, high cholesterol, cardiovascular disease, fatty liver, etc. Research shows that clinically 80-90% of T2DM patients are overweight or obese. Currently, there are few types of drugs for treating obesity, with limited efficacy and significant side effects.

Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by the intestines and mainly plays a role in regulating blood sugar. GLP-1 can protect pancreatic islet cells, reduce their damage and death, help maintain the function and number of islet cells, and can also stimulate islet cells to secrete insulin and reduce blood sugar levels. GLP-1 also inhibits the release of glucagon, thereby reducing the amount of glucose released by the liver. In addition, GLP-1 also has the effects of delaying gastric emptying, suppressing appetite, and has some weight loss effects. GLP-1 drugs increase the biological activity of GLP-1 through different mechanisms, thereby regulating blood sugar, improving insulin resistance and promoting weight loss. Drugs currently on the market include liraglutide, semaglutide, etc., which are widely used in the treatment of T2DM and have shown good efficacy and safety. However, GLP-1 drugs have short half-lives and are prone to gastrointestinal adverse reactions. Moreover, these drugs usually need to be administered by subcutaneous injection or continuous infusion, resulting in poor patient compliance. Therefore, there is a need to develop more convenient and safely tolerated therapeutic agents.

Glucagon is a hormone secreted by the alpha cells of the pancreas. In a state of hypoglycemia, glucagon secretion increases, prompting the liver to break down glycogen and release glucose into the blood, thereby restoring blood sugar levels and ensuring energy supply to the body's brain and other tissues. In addition, glucagon also has the effects of promoting lipolysis, fat oxidation, and fever in the body. Long-term administration can show weight loss effects by increasing energy metabolism, but it may also cause blood sugar to rise too fast or too high, causing hyperactivity. Blood sugar symptoms, so it cannot be used.

Glucose-dependent insulin peptide (GIP) is a peptide hormone secreted by small intestinal K cells. It is an incretin and has multiple functions in the human body, mainly involving blood sugar regulation, fat metabolism, and appetite regulation. GIP can stimulate pancreatic beta cells to secrete insulin, thereby lowering blood sugar and promoting glucose utilization. It can also protect pancreatic islet cells, reduce their damage and death, and help maintain the function and number of pancreatic islets. Moreover, GIP can promote fat metabolism, maintain the balance of fat metabolism, and can suppress appetite by acting on the central nervous system and affecting the appetite center. However, the biological activity of GIP is weak, and its blood sugar regulating effect is relatively mild.

GLP-1 receptor, glucagon receptor and GIP receptor all belong to the GPCR receptor family and have similar protein structures and binding mechanisms, making it possible to design multiple agonists targeting these three receptors. Multiple agonists of these three receptors can act on GLP-1 receptors, glucagon receptors and GIP receptors at the same time, and can exert the activities of GLP-1, glucagon and GIP at the same time. GLP-1 can lower blood sugar, delay gastric emptying, and suppress appetite; glucagon can promote lipolysis and increase blood sugar, but its blood sugar-raising effect can be offset by the hypoglycemic activity of GLP-1; GIP can protect pancreatic islet cells and stimulate insulin secretion , promote the utilization of glucose. The three activities of the GLP-1/glucagon/GIP receptor triple agonist cooperate with each other, which can not only strengthen the control of blood sugar, but also break down fat and reduce weight. For the treatment of T2DM, obesity and related metabolic syndrome, triple agonists targeting three receptors may provide better blood sugar control and weight loss effects, and have significant advantages over pure GLP-1 analogues.

Contents of the invention

The invention provides a type of polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors and its application. The polypeptide can act on the GLP-1 receptor, increase the secretion of insulin, and at the same time delay gastric emptying. Thereby lowering blood sugar; acting on glucagon receptors, producing beneficial effects on body weight, energy metabolism, and lipid metabolism; acting on GIP receptors, promoting insulin secretion, improving the sensitivity of pancreatic islet cells to glucose, and helping to reduce Blood sugar; by simultaneously regulating these three receptors, the present invention affects multiple physiological pathways. The polypeptide can improve metabolic function, provide better blood sugar control and weight loss effects, and is useful in the treatment of diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, and non-alcoholic fatty liver disease. Metabolic syndromes such as alcoholic steatohepatitis and dyslipidemia can show potential clinical application prospects.

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

A type of polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors. The general formula of the amino acid sequence of the polypeptide is:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Arg-Ala-Xaa ₁ -Glu-Xaa ₂ -Ile-Ala-Xaa ₃ -Arg-Le u-Phe-Val- Asp-Trp-Leu-Ile-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ , where: Xaa ₁ is selected from Leu or Met; Xaa ₂ is selected from Side The chain is chemically modified Lys-R1 or Lys-R2; Xaa ₃ is selected from Ala or Val;

The chemical structural formula of Lys-R1 is:

The chemical structure of Lys-R2 is:

Preferably, the sequence structure of the polypeptide is selected from any one of the amino acid sequences shown in SEQ ID NO: 1-4:

The present invention also provides pharmaceutically acceptable salts of the above-mentioned polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors.

Further, the pharmaceutically acceptable salt is a salt formed by a polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors 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, lauryl sulfate, citric acid, ascorbic acid, alcohol Stearic 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 polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors. The medicaments include any pharmaceutical tablets, capsules, syrups, Tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion, suppository or compound preparation.

The present invention also provides a pharmaceutical composition prepared from a polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors. A polypeptide with agonistic activity on GIP receptors, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition includes the polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors. Acceptable salts, pharmaceutically acceptable carriers or diluents.

The invention also provides the polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors or the polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors. The use of a pharmaceutically acceptable salt of a polypeptide or the medicament or the pharmaceutical composition in the preparation of a medicament for the treatment of metabolic diseases or disorders; the metabolic diseases or disorders include diabetes, obesity, non-alcoholic fatty liver disease, nonalcoholic steatohepatitis, or dyslipidemia.

The polypeptide compound prepared by the present invention has strong agonistic activity on GLP-1 receptors and good agonistic activity on glucagon receptors. However, its agonistic activity on GIP receptors is relatively weak, but it achieves better hypoglycemic and It has weight-reducing and lipid-regulating effects and has lower gastrointestinal side effects, providing a new idea for the preparation of such high-efficiency, low-toxicity multiple agonists.

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

(1) The polypeptide of the present invention can more effectively lower blood sugar and at the same time have significant weight loss and weight gain prevention effects, and better regulate lipid metabolism;

(2) The polypeptide of the present invention has a unique N-terminal 6-10 sequence structure (FTSDM) and a unique in vitro GLP-1 receptor, glucagon receptor and GIP receptor agonistic activity ratio, thus bringing about significantly improved Weight loss and lipid metabolism regulation, with lower gastrointestinal side effects, have unexpected beneficial effects;

(3) Compared with the reported GLP-1/glucagon/GIP receptor triple agonist, the polypeptide of the present invention has stronger GLP-1 receptor and glucagon receptor agonistic activity and weaker GIP receptor agonist activity. , but the weight loss, lipid metabolism regulation and hypoglycemic activity are significantly improved, gastrointestinal side effects are significantly reduced, and it has greater potential in the treatment of metabolic diseases, providing new ideas for the drug development of such multiple agonists;

(4) The polypeptides provided by the invention have stable chemical properties and have pharmacokinetic characteristics that support at least once-weekly administration; the polypeptides provided by the invention have better therapeutic effects on metabolic diseases such as T2DM, obesity, and dyslipidemia than existing ones. Marketed drugs. Therefore, the polypeptides provided by the present invention are suitable as active ingredients in drugs for treating metabolic diseases, such as diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and dyslipidemia.

Description of the 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, the sequence of the present invention also uses unnatural amino acids, α-aminoisobutyric acid (Aib).

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 0.338g (0.1mmol equivalent) of RinkAmide MBHA resin with a loading capacity of 0.296mmol/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-MBHAResin

Weigh Fmoc-Ser(tBu)-OH (0.4mmol), add 2mL HBTU/HOBt (0.4mmol/0.44mmol) condensation agent and 1mL DIPEA (0.8mmoL) activation base. After pre-activation for 30 minutes, add the activated amino acids to the reactor. , shake the reaction in DMF for 2 hours at room temperature, filter out the reaction solution, and 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. The Lys at the Lys site whose side chain is modified can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH, etc. In this example, the Fmoc-Lys(Dde)-OH protection strategy is used, and the N-terminal His is 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 16. After the Dde protecting group is removed, add 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of HBTU and 0.44mmol HOBt were used for condensation reaction with shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of HBTU and 0.44 mmol of HOBt were added again, and the condensation reaction was carried out with shaking for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of HBTU 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, add 0.4 mmol of monotert-butyl octadecanedioate, 0.4 mmol of HBTU and 0.44 mmol of HOBt for condensation reaction for 2 hours. After the reaction is complete, the resin is washed 4 times with 7 mL of DMF.

(6) Cleavage of polypeptides

Transfer the polypeptide-connected resin obtained above to a round-bottom bottle, use 5 mL of cutting agent (triisopropylsilane/water/TFA, 2.5:2.5:95, V/V) to cut the resin, and keep it at a constant temperature of 30 in an oil bath. React at ℃ for 2 hours, pour the cutting solution into 40 mL of glacial ether, freeze and centrifuge, and wash the crude product three times with 15 mL of glacial ether, and finally blow dry with nitrogen to obtain the crude peptide.

(7) Purification of polypeptides

Dissolve the crude target polypeptide in water/methanol, 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×4.6mm, 5μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate is 0.8mL/min; The detection wavelength is 214nm. Use linear gradient (50% B ~ 90% B/15min) to elute, collect the target peak, remove methanol and freeze-dry to obtain 0.16g of pure product, with a purity greater than 99%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4874.6. ESI-MS m/z: calculated values [M+3H] ³⁺ 1625.9, [M+4H] ⁴⁺ 1219.7; observed values [M+3H] ³⁺ 1625.5, [M+4H] ⁴⁺ 1219.1.

Example 2

Synthesis of SEQ ID NO:2 polypeptide compounds

The synthesis method is the same as in Example 1. The target peak is collected and lyophilized to obtain 0.17g of pure product with a purity greater than 99%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 4828.5. ESI-MS m/z: calculated values [M+3H] ³⁺ 1610.5, [M+4H] ⁴⁺ 1208.1; observed values [M+3H] ³⁺ 1610.2, [M+4H] ⁴⁺ 1207.9.

Example 3

Synthesis of SEQ ID NO:3 polypeptide compounds

The synthesis method is the same as in Example 1, except that in the modification step of the Lys side chain, monotert-butyl octadecanedioic acid is replaced by monotert-butyl eicosanedioate. The target peak was collected and freeze-dried to obtain 0.18g of pure product, with a purity greater than 99%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4902.7. ESI-MS m/z: calculated values [M+3H] ³⁺ 1635.2, [M+4H] ⁴⁺ 1226.7; observed values [M+3H] ³⁺ 1634.8, [M+4H] ⁴⁺ 1226.4.

Example 4

Synthesis of polypeptide compounds of SEQ ID NO:4

The synthesis method is the same as in Example 1, except that in the modification step of the Lys side chain, monotert-butyl octadecanedioic acid is replaced by monotert-butyl eicosanedioate. The target peak was collected and freeze-dried to obtain 0.17g of pure product with a purity greater than 99%. The molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 4856.6. ESI-MS m/z: calculated values [M+3H] ³⁺ 1619.9, [M+4H] ⁴⁺ 1215.2; observed values [M+3H] ³⁺ 1619.5, [M+4H] ⁴⁺ 1214.6.

Example 5

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 activity of the polypeptide compounds of the present invention on GLP-1 receptors is stronger than that of natural GLP-1 (approximately 2.6-4.4 times stronger). The agonistic activity of the polypeptide compounds of the present invention on glucagon receptors is stronger than that of natural GLP-1. Glucagon is weak (about 1.4-8.7 times weaker), and the GIP receptor agonistic activity of the polypeptide compound of the present invention is weaker than that of natural GIP (about 25.6-38.4 times weaker), indicating that the polypeptide compound of the present invention has a strong GLP-1 receptor agonism. Body agonist activity, better glucagon receptor agonist activity and weaker GIP receptor agonist activity, with a special ratio of agonist activity for GLP-1 receptor, GIP receptor and glucagon receptor, and is also in line with the description of this patent Characteristics of triple agonists.

Example 6

Pharmacokinetic properties of peptide compounds in rats

SD 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 WinonLin 5.2.1 (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 9.4 498 SEQ ID NO:1 11.6 522 SEQ ID NO:2 13.1 519

As shown in the results in Table 2, the in vivo half-life of the polypeptide compound of the present invention is significantly prolonged, which is better than the marketed semaglutide administered once a week, indicating that the polypeptide compound of the present invention has pharmacokinetic characteristics that support at least once-weekly administration.

Example 7

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

Male C57BL/6J mice, weighing about 21g, were fed 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 was 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, tirzepatide and SAR441255 (GLP-1/glucagon/GIP recipient)). Triple agonist in vivo, Cell Metabolism, 2022, 34, 1–16)) and the test sample group (SEQ IDNO: 2). Mice in each group were subcutaneously injected with normal saline (10mg/kg), semaglutide (10nmol/kg), tirzepatide (10nmol/kg), SEQ ID NO: 2 (10nmol/kg) once every two days, or subcutaneously injected with SAR441255 twice a day. (10nmol/kg), the administration period 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) -1.2±3.3 -1.6±0.6 Semaglutide(10nmol/kg) -17.5±2.1 ^*** -23.8±3.8 ^*** Tirzepatide(10nmol/kg) -24.5±3.3 ^*** -36.9±2.7 ^*** SAR441255(10nmol/kg) -31.8±1.6 ^*** -40.5±4.6 ^*** SEQ ID NO:2(10nmol/kg) -39.2±2.8 ^***,### -54.6±5.0 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±SD.

As shown in Figure 1 and Table 3, the polypeptide compound 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 reduction of the polypeptide compound of the present invention can The effect of weight loss and body fat reduction is significantly stronger than that of the positive control drugs semaglutide, tirzepatide and SAR441255. It is worth noting that the GLP-1 receptor agonistic activity of SAR441255 is similar to natural GLP-1, the glucagon receptor agonistic activity is about 2 times lower than natural glucagon, and the GIP receptor agonistic activity is about 2 times lower than natural GIP (Cell Metabolism, 2022,34,1–16). It can be seen that the GLP-1 receptor agonistic activity and glucagon receptor agonistic activity of SAR441255 are similar to SEQ ID NO:2, but the GIP receptor agonistic activity of SAR441255 is significantly stronger than that of SEQ ID NO:2. However, this The invented polypeptide compound SEQ ID NO: 2 shows significantly better weight loss and body fat reduction activities than SAR441255.

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) 11.6±0.5 139.7±11.6 Semaglutide(10nmol/kg) 9.1±0.5 ^*** 78.9±6.2 ^*** Tirzepatide(10nmol/kg) 8.9±0.4 ^*** 74.6±5.9 ^*** SAR441255(10nmol/kg) 6.1±0.3 ^*** 56.6±3.6 ^*** SEQ ID NO:2(10nmol/kg) 4.1±0.1 ^***,### 35.4±2.1 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±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) 456±51 415±39 Semaglutide(10nmol/kg) 214±26 ^*** 266±31 ^*** Tirzepatide(10nmol/kg) 229±15 ^*** 278±24 ^*** SAR441255(10nmol/kg) 186±32 ^*** 196±18 ^*** SEQ ID NO:2(10nmol/kg) 116±12 ^***,### 96±9 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±SD.

As shown in Table 4 and Table 5, the polypeptide compound prepared in the embodiment of the present invention can significantly reduce the liver triglyceride and total cholesterol content of the mice when administered continuously in DIO mice for 3 weeks, and can significantly reduce serum glutamic acid. Transaminase and aspartate aminotransferase contents, and the effect of the polypeptide compound of the present invention is significantly stronger than the positive control drugs semaglutide, tirzepatide and SAR441255, indicating that the polypeptide compound of the present invention has good prospects for treating 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

Sample (dose) Total cholesterol (mmol/L) Triglycerides (mmol/L) Blank control (normal saline group) 13.6±2.2 2.0±0.2 Semaglutide(10nmol/kg) 9.5±0.6 ^*** 1.4±0.2 ^*** Tirzepatide(10nmol/kg) 9.4±0.3 ^*** 1.4±0.3 ^*** SAR441255(10nmol/kg) 6.0±0.4 ^*** 1.1±0.2 ^*** SEQ ID NO:2(10nmol/kg) 4.3±0.3 ^***,### 0.5±0.1 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±SD.

As shown in the results in Table 6, the polypeptide compound of the present invention can significantly reduce the serum triglyceride and total cholesterol content of the mice after continuous administration for 3 weeks in DIO mice, and the polypeptide compound of the present invention reduces serum lipids (glycerol). Triesters and cholesterol) content was significantly stronger than the positive control drugs semaglutide, tirzepatide and SAR441255.

Example 8

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 the normal saline group (blank control group), the positive control group (semaglutide, tirzepatide and SAR441255) and the test sample group (SEQ ID NO: 2). 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 normal saline (10mg/kg), semaglutide (10nmol/kg), tirzepatide (10nmol/kg), SEQ IDNO: 2 (10nmol/kg) once every two days, or subcutaneously injected with SAR441255 ( 10 nmol/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) 6.5±0.1 7.1±0.4 Semaglutide(10nmol/kg) 6.6±0.2 6.0±0.2 ^*** Tirzepatide(10nmol/kg) 6.7±0.1 5.9±0.2 ^*** SAR441255(10nmol/kg) 6.5±0.3 5.9±0.3 ^*** SEQ ID NO:2(10nmol/kg) 6.6±0.3 5.0±0.1 ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±SD.

As shown in Table 7, the polypeptide compound of the present invention can significantly reduce the HbA1c value of the mice after continuous administration for 35 days in db/db mice, and the HbA1c value of the mice in the polypeptide compound group of the present invention is significantly lower after treatment. Compared with the positive controls semaglutide, tirzepatide and SAR441255, it shows that the polypeptide compound of the present invention has 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) +4.6±0.7% Semaglutide(10nmol/kg) -3.5±0.4% ^*** Tirzepatide(10nmol/kg) -4.1±0.3% ^*** SAR441255(10nmol/kg) -4.6±0.3% ^*** SEQ ID NO:2(10nmol/kg) -11.6±0.4% ^***,###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 mice per group Mean±SD.

As shown in Table 8, the polypeptide compound prepared in the embodiment of the present invention can significantly reduce the fasting blood sugar of db/db mice after continuous administration for 35 days in db/db mice, indicating 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, tirzepatide and SAR441255.

Example 9

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 25 nmol/kg, 50 nmol/kg. and 100 nmol/kg of semaglutide, tirzepatide, SAR441255, 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 25.2±1.6g 0.2±0.1g Cisplatin 13.9±2.2g ^*** 3.3±0.5g ^*** Semaglutide(25nmol/kg) 20.2±1.3g ^*** 0.7±0.2g ^*** Tirzepatide(25nmol/kg) 18.3±2.0g ^*** 0.8±0.3g ^*** SAR441255(25nmol/kg) 18.1±1.2g ^*** 0.9±0.2g ^*** SEQ ID NO:2(25nmol/kg) 15.3±0.9g ^***,### 0.2±0.1g ^###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 rats per group Mean±SD.

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

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 rats per group Mean±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 25.2±1.6g 0.2±0.1g Cisplatin 13.9±2.2g ^*** 3.3±0.5g ^*** Semaglutide(100nmol/kg) 18.1±1.9g ^*** 1.1±0.3g ^*** Tirzepatide(100nmol/kg) 16.8±1.4g ^*** 1.2±0.3g ^*** SAR441255(100nmol/kg) 16.2±1.6g ^*** 1.0±0.2g ^*** SEQ ID NO:2(100nmol/kg) 13.0±0.3g ^***,### 0.3±0.1g ^###

^*** : P<0.001 compared with the blank control group; ^### : P<0.001 compared with the semaglutide, tirzepatide and SAR441255 groups (One-Way ANOVA, Tukey post hoc test), the results are expressed as 6 rats per group Mean±SD.

As shown in the results in Tables 9 to 11, the polypeptide compounds prepared in the embodiments of the present invention have a good inhibitory effect on rat eating at doses of 25 nmol/kg, 50 nmol/kg, and 100 nmol/kg, which is better than the positive controls semaglutide, tirzepatide, and SAR441255. However, compared with the blank group, the polypeptide compounds prepared in the examples of the present invention did not cause obvious kaolin eating phenomena in rats at doses of 25 nmol/kg, 50 nmol/kg, and 100 nmol/kg. The kaolin intake of rats in the blank control group was significantly lower than the kaolin intake of rats in the positive control semaglutide, tirzepatide and SAR441255 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 significantly lower than the positive controls semaglutide, tirzepatide and SAR441255.

Claims (8)

1. A class of polypeptides with agonistic activity on GLP-1, glucagon and GIP receptors, characterized in that the general formula of the amino acid sequence of the polypeptide is: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Met-Ser-Arg-Ala-Xaa ₁ -Glu-Xaa ₂ -Ile-Ala-Xaa ₃ -Arg-Le u-Phe-Val- Asp-Trp-Leu-Ile-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ , in: Xaa ₁ is selected from Leu or Met; Xaa ₂ is selected from Lys-R1 or Lys-R2 with chemically modified side chains; Xaa ₃ is selected from Ala or Val; The chemical structure of Lys-R1 is: The chemical structure of Lys-R2 is: 2. A type of polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors according to claim 1, characterized in that the sequence structure of the polypeptide is selected from the group consisting of SEQ ID NO: 1-4 Any of the amino acid sequences shown: SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 3. A pharmaceutically acceptable salt of a polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors according to any one of claims 1-2. 4. A pharmaceutically acceptable salt of a polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors according to claim 3, characterized in that the pharmaceutically acceptable salt is Salts formed by polypeptides with agonistic activity of GLP-1, glucagon and GIP receptors and one of the following compounds; the following compounds include hydrochloric acid, formic acid, acetic acid, pyruvic acid, butyric acid, hexanoic acid, Benzoic 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 , malonic acid, maleic acid, fumaric acid, aspartic acid, sulfosalicylic acid. 5. A medicament prepared from a polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors according to any one of claims 1-2, characterized in that the medicament includes any pharmaceutical The above-mentioned tablets, capsules, syrups, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions, suppositories or compound preparations. 6. A pharmaceutical composition prepared from a polypeptide with agonistic activity on GLP-1, glucagon and GIP receptors, characterized in that the pharmaceutical composition includes a pharmaceutical composition according to any one of claims 1-2 A polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors, a pharmaceutically acceptable carrier or diluent; or the pharmaceutical composition includes a type of agonist according to any one of claims 3-4 Pharmaceutically acceptable salts, pharmaceutically acceptable carriers or diluents of polypeptides with agonistic activity for GLP-1, glucagon and GIP receptors. 7. A polypeptide having agonistic activity on GLP-1, glucagon and GIP receptors according to any one of claims 1-2 or a polypeptide having agonistic activity on GLP-1 according to any one of claims 3-4 , a pharmaceutically acceptable salt of a polypeptide with agonistic activity for glucagon and GIP receptors or a pharmaceutical agent according to claim 5 or a pharmaceutical composition according to claim 6 for use in the treatment of metabolic diseases or disorders uses in medicines. 8. The use according to claim 7, wherein the metabolic disease or condition is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or dyslipidemia.