CN117603364A

CN117603364A - GLP-1/glucon/Y 2 Receptor triple agonists and uses thereof

Info

Publication number: CN117603364A
Application number: CN202311601772.6A
Authority: CN
Inventors: 蒋能; 颜志明; 韩京; 汤春丽; 景林; 卢覃培
Original assignee: Guangxi Medical University Affiliated Tumour Hospital
Current assignee: Guangxi Medical University Affiliated Tumour Hospital
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2024-02-27
Also published as: CN115960258B; CN115960258A

Abstract

The invention provides a GLP-1/glucon/Y ₂ Receptor triple agonists and uses thereof, said triple agonists being for human GLP-1, glucagon and Y ₂ The receptor has triple agonistic activity. GLP-1/glucon/Y of the invention ₂ Receptor triple agonists have significant weight loss reduction and NAFLD treatment while effectively lowering blood glucoseHas beneficial effects. GLP-1/glucon/Y provided by the invention ₂ The receptor triple agonist has stable chemical property and pharmacokinetic characteristics supporting once-a-day administration, and is suitable for being used as an active ingredient of medicaments for treating metabolic diseases (such as diabetes, obesity, nonalcoholic fatty liver disease, dyslipidemia and the like) and the like.

Description

GLP-1/glucon/Y 2 Receptor triple agonists and uses thereof

Technical Field

The invention belongs to the technical field of biological medicine, in particular to a GLP-1/glucon/Y ₂ Receptor triple agonists and uses 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. GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by small intestine L cells, and the most main function of GLP-1 is to promote insulin secretion. Glucagon glycopeptide-1 (GLP-1) can suppress appetite and delay gastric emptying to achieve weight reduction. Although GLP-1 has excellent hypoglycemic effect and a certain weight-reducing effect, if better weight-reducing effect is required to be realized, the administration dosage is generally required to be increased, and large-dose administration of GLP-1 drugs is easy to generate gastrointestinal side effects and poor in 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 (glucagon) is a hormone produced in the α cells of the pancreas, and acts on the liver in a stress state such as cold or hunger of the body to decompose glycogen in the liver, thereby increasing blood sugar. In addition, glucopon has effects of promoting lipolysis, fat oxidation, fever, etc. (diabetes, 2017,60,1851-1861), and long-term administration can exhibit weight-loss efficacy by increasing energy metabolism, but the beneficial effects of glucopon on energy metabolism have not been used because of its inherent glycemic effect. GLP-1 and glucogen are given to animals and human bodies at inner and outer peripheries to achieve the effects of reducing blood sugar and reducing weight simultaneously, and various GLP-1/glucogen receptor dual agonists are in clinical research at present, but the GLP-1/glucogen receptor dual agonists have the defect of large gastrointestinal side effects.

Neuropeptide Y (NPY) is a neurotransmitter of 36 amino acid peptides, a member of the class of pancreatic polypeptides that has been shown to be neurotransmitter/neurohormone in both the peripheral and central nervous systems. NPY is one of the most effective appetizers known and has been shown to play an important role in regulating food intake in animals including humans. NPY receptors share 4 subtypes, Y ₁ 、Y ₂ 、Y ₄ And Y ₅ Wherein neuropeptide-2 (Y ₂ ) Receptors are widely distributed in the central nervous system of rodents and humans. In the hypothalamus, Y ₂ mRNA is localized in the arciform nucleus, the anterior nucleus and the dorsal nucleus. In the human brain, Y ₂ The receptor is the major subtype of NPY receptor. More than 80% of NPY neurons co-express Y in arcuately shaped nuclei ₂ Receptor mRNA. Selective activation Y ₂ The receptor can inhibit ingestion, and has weight reducing effect.

Peptide YY _3-36 (PYY _3-36 ) Is a 34 amino acid linear peptide having Y ₂ Agonistic activity of the receptor, combined injection of GLP-1 and PYY in animals and humans _3-36 Has good hypoglycemic and weight-reducing effects, and shows that GLP-1 and Y are simultaneously excited ₂ The receptor can further utilize the excitation Y on the premise of maintaining the hypoglycemic effect of GLP-1 ₂ The appetite suppressing effect brought by the receptor produces better weight reducing effect. Novo Nordisk reported in 2021 that has GLP-1 and Y ₂ GLP-1 and short chain PYY with receptor dual agonistic activity _3-36 Hybrid peptides of the analogs. However, the compounds are not subjected to long-acting modification, the in vivo stability of the compounds is limited, and long-acting administration cannot be realized (Angew.chem).Int.ed.engl.,2021,6;60 (15):8268-8275). Brandon et al disclose a class of exendin-4 and short-chain PYY _3-36 Hybrid peptide of analogue with better GLP-1 and Y ₂ The receptor double-agonistic activity is similar to that reported by Novo Nordisk, and the compounds have no long-lasting modification and poor stability (J.Med. Chem.,202,28;64 (2): 1127-1138).

Currently, there is a lack of the ability to integrate GLP-1, glucon and PYY simultaneously _3-36 GLP-1/glucon/Y with beneficial effects on blood glucose, feeding and energy metabolism ₂ Receptor triple agonists, by which drugs against metabolic diseases having more excellent activity are developed.

Disclosure of Invention

The invention aims to provide a kind of compound with GLP-1/glucon/Y ₂ Receptor triple agonists and uses thereof, which agonists are useful for human GLP-1 receptor, glucagon and Y ₂ The receptor has triple agonistic activity and high stability, and has the pharmacokinetic characteristic of once-a-day administration; the preparation method has greater potential in the aspect of preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, nonalcoholic fatty liver disease, dyslipidemia and other diseases.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

GLP-1/glucon/Y ₂ Receptor triple agonists, GLP-1/glucon/Y ₂ The amino acid sequence of the receptor triple agonist is one of the following:

(1)SEQ ID NO:1

(2)SEQ ID NO:2

(3)SEQ ID NO:3

(4)SEQ ID NO:4

(5)SEQ ID NO:5

(6)SEQ ID NO:6

(7)SEQ ID NO:7

(8)SEQ ID NO:8

(9)SEQ ID NO:9

the invention also provides a GLP-1/glucon/Y ₂ A pharmaceutically acceptable salt of a receptor triple agonist.

Preferably, the salt is GLP-1/glucon/Y ₂ A salt of a receptor triple agonist with one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid, and succinic acid.

The invention also provides GLP-1/glucon/Y ₂ Prepared from receptor triple agonistsThe medicament is any one of tablets, capsules, inhalants, sprays, injections, films, patches, emulsions, suppositories or compound preparations which are pharmaceutically described and is prepared from GLP-1/glucon/Y ₂ The receptor triple agonist and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.

The invention also provides a liquid crystal display (GLP-1/glucon/Y) containing the same ₂ Pharmaceutical compositions of receptor triple agonists, which are based on GLP-1/glucon/Y as described above ₂ The receptor triple agonist is prepared from effective raw materials or pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers or diluents.

The invention also provides the GLP-1/glucon/Y of the invention ₂ Use of a receptor triplet agonist or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or a medicament thereof, in the manufacture of a medicament for the treatment of a metabolic disease or disorder. In a particular aspect, the metabolic disease or disorder is diabetes, obesity, NAFLD, dyslipidemia. In a particular aspect, the diabetes is T1DM, T2DM or gestational diabetes. In a particular aspect, the medicament is for the treatment of more than one metabolic disease or disorder, for example, diabetes and obesity; diabetes and NAFLD; diabetes and dyslipidemia; diabetes, dyslipidemia and obesity.

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

the invention provides a class of GLP-1 analogue-based analogues, glucagon and PYY _3-36 Sequence designed variants that retain the therapeutic effect of GLP-1 analogs on diabetes while having the beneficial effect of glucopon on metabolism and PYY _3-36 The beneficial effects on appetite suppression are achieved, so that synergistic effects on sugar, fat and energy metabolism are achieved, and the beneficial effects on reducing weight and treating NAFLD are achieved while blood sugar is effectively reduced. In addition, the invention provides GLP-1/glucon/Y ₂ The receptor triple agonists are chemically stable and have pharmacokinetic profiles that support once-a-day dosing. The triple agonist provided by the invention has better treatment effect on metabolic diseases such as T2DM, obesity, NAFLD, dyslipidemia and the like than the existing medicines on the market. Thus, the present inventionThe triple agonists provided are suitable as active ingredients of medicaments for the treatment of metabolic diseases (such as diabetes, obesity, NAFLD, dyslipidemia, etc.).

Drawings

FIG. 1 is a graph showing the results of feeding inhibition in ICR mice with a single administration of each test substance;

FIG. 2 is a graph showing the results of acute hypoglycemic events in ICR mice for a single administration of each test subject;

figure 3 shows a graphical representation of the percent change in body weight of each subject over 21 days of prolonged DIO mice administration.

Detailed Description

The invention is described in further detail below with reference to the drawings and the specific examples.

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

In addition, the amino acids to which the present invention relates are 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).

In addition, unless explicitly indicated, all amino acid residues in the polypeptide compounds of the invention are preferably in the L configuration.

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

In addition, in the sequences of the invention, non-natural amino acid dextrorotatory filaments are used in addition to natural amino acidsAmino acid% _d Ser， _d S)。

Example 1

Synthesis of polypeptide compound of SEQ ID No. 1

(1) Swelling of the resin

0.278g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.36mmol/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, and finally washing the resin for 4 times by 7mL of LDMF for 2min each time to obtain the Rink resin without Fmoc protecting groups.

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

Fmoc-Tyr (tBu) -OH (0.4 mmol) was weighed, dissolved in 2mL of DMF, 3mL of LDIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, the mixture was added to the reactor, the reaction was allowed to proceed with shaking at room temperature for 2h, the reaction solution was filtered off, the resin was washed 4 times with 7mL of DMF, and the Kaiser reagent was used to detect whether the coupling was complete or not, and if not, 2 times.

(4) Extension of the peptide chain of the PYY moiety (right-hand side)

According to the sequence of the PYY partial peptide chain (IKPEAPGEDA SPEELNRYYA SLRHY LNLVT RQRY-NH ₂ ) And repeating the deprotection and coupling steps to sequentially connect corresponding amino acids until the peptide chain synthesis is completed.

(5) Synthesis of linker arm

After the synthesis of the peptide chain of the PYY part is completed, continuing to synthesize a connecting arm part, adding 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt, and carrying out oscillation condensation reaction 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 Fmoc protecting groups were removed, 0.4mmol of 3-maleimidopropionic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added and the reaction was performed by shaking condensation for 2 hours.

(6) Cleavage of PYY moiety comprising a linker arm

The obtained polypeptide-linked resin was transferred to a round bottom bottle, the resin was cleaved with a cleavage agent Reagent R (TFA/phenylsulfide/phenol/EDT, 90:5:3:2, V/V) 5mL, reacted in an oil bath at a constant temperature of 30℃for 2 hours, the cleavage solution was poured into 40mL of glacial ethyl ether, the crude product was washed 3 times with 15mL of glacial ethyl ether after refrigerated centrifugation, and finally dried with nitrogen to obtain a crude peptide comprising the PYY moiety of the linker arm.

(7) Synthesis of GLP-1/glucon partial (left-hand part) sequence

0.278g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.36mmol/g was weighed into a 25mL reactor, the resin was alternately washed 1 time with 10mL of DCM and methanol, 2 times with 10mL of DCM, then the resin was swollen with 10mL of DCM for 1h, and finally the resin was washed 3 times with 10mL of LDMF. Transferring the swelled resin into a PSI-200 polypeptide synthesizer, adding 10mL of 20% piperidine/DMF (v/v) for reaction at room temperature for 5min, filtering off a deprotection solution, washing the resin once by 10mL of 20% piperidine/DMF (v/v) for reaction with the resin for 15min, and washing the resin for 4 times by 10mL of 20% piperidine/DMF (v/v) for 1.5min to obtain the Rink resin without Fmoc protecting groups. Fmoc-Cys (Trt) -OH (0.4 mmol) was weighed, dissolved in 2mL of 10% DMF/DMSO (v/v), 3mL of DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added, the mixture was added to the reactor, the reaction was stirred at room temperature for 2 hours, the reaction solution was filtered off, the resin was washed with 10mL of MF for 4 times, and the Kaiser reagent was used to detect whether the reaction coupling was complete or not, and if not, 2 times.

(8) Extension of GLP-1/glucon partial 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 Lys of the side chain modification site adopts Fmoc-Lys (Dde) -OH protection strategy, and Boc-His (Boc) -OH is used as His of the N terminal.

(9) Modification of GLP-1/Glucoagon partial Lys side chain

After the peptide chain synthesis is completed, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove the Dde protecting group of Lys, and after the Dde protecting group is removed, 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt are added to carry out oscillation reaction for 2h. After removal of Fmoc protecting groups, 0.4mmol Fmoc-Glu-OtBu,0.4mmol DIC and 0.44mmol HOBt were added again and the reaction was performed by shaking for 2h. After Fmoc protecting groups were removed, 0.4mmol of palmitic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2 hours, and after completion of the reaction, the resin was washed with 7 mM DMF for 4 times.

(10) Cleavage of GLP-1/glucon moiety

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 temperature is kept constant at 30 ℃ for 2 hours in an oil bath, 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 of GLP-1/glucopon part is obtained by drying by nitrogen.

(11) Synthesis of target polypeptide

0.05mmol of the crude peptide containing the PYY moiety of the linker arm and 0.05mmol of the crude peptide of the GLP-1/glucagon moiety were dissolved in 2mL of NMP, and after 20 minutes of reaction catalyzed by the addition of 0.005mmol of DIPEA, 5mL of 50% methanol/water (0.1% TFA) was added to stop the reaction, and the reaction solution was filtered with a 0.25 μm microporous filter membrane and purified by a Shimadzu preparative reverse 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 (20-90% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.012g, purity greater than 98%, and determining molecular weight of target polypeptide by MS. The theoretical relative molecular mass is 9242.3.ESI-MS M/z calculated [ M+8H] ⁸⁺ 1156.3,[M+9H] ⁹⁺ 1027.9; observed value [ M+8H] ⁸⁺ 1156.0,[M+9H] ⁹⁺ 1027.7。

Example 2

Synthesis of polypeptide compound of SEQ ID No. 2

The synthesis method was the same as that of example 1, and the target peak was collected and lyophilized to give 0.014g of pure product with a purity of more than 98%, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 9532.7.ESI-MS M/z calculated [ M+8H] ⁸⁺ 1192.6,[M+9H] ⁹⁺ 1060.2; observed value [ M+8H] ⁸⁺ 1192.3,[M+9H] ⁹⁺ 1059.9。

Example 3

Synthesis of polypeptide compound of SEQ ID No. 3

The synthesis method is the same as that of example 1, the target peak is collected and freeze-dried to obtain 0.016g of pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10113.2.ESI-MS M/z calculated [ M+9H] ⁹⁺ 1124.7,[M+10H] ¹⁰⁺ 1012.3; observed value [ M+9H] ⁹⁺ 1124.6,[M+10H] ¹⁰⁺ 1012.2。

Example 4

Synthesis of polypeptide compound of SEQ ID No. 4

The synthesis method is the same as that of example 1, 0.011g of a pure product is obtained by collecting a target peak and freeze-drying, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9413.4.ESI-MS M/z calculated [ M+7H] ⁷⁺ 1345.8,[M+8H] ⁸⁺ 1177.7; observed value [ M+7H] ⁷⁺ 1345.4,[M+8H] ⁸⁺ 1177.3。

Example 5

Synthesis of polypeptide compound of SEQ ID No. 5

The synthesis method is the same as that of example 1, 0.015g of a pure product is obtained by collecting a target peak and freeze-drying, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9703.8.ESI-MS M/z calculated [ M+10H ]] ¹⁰⁺ 971.4,[M+11H] ¹¹⁺ 883.2; observed value [ M+10H] ¹⁰⁺ 971.2,[M+11H] ¹¹⁺ 882.9。

Example 6

Synthesis of polypeptide compound of SEQ ID No. 6

The synthesis method is the same as that of example 1, 0.013g of the target peak is collected and freeze-dried to obtain a pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10284.4.ESI-MS M/z calculated [ M+9H] ⁹⁺ 1143.7,[M+11H] ¹¹⁺ 935.9; observed value [ M+9H] ⁹⁺ 1143.5,[M+11H] ¹¹⁺ 935.6。

Example 7

Synthesis of polypeptide compound of SEQ ID No. 7

The synthesis method is the same as that of example 1, the target peak is collected and freeze-dried to obtain 0.010g of pure product, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9329.3.ESI-MS M/z calculated [ M+9H] ⁹⁺ 1037.6,[M+10H] ¹⁰⁺ 933.9; observed value [ M+9H] ⁹⁺ 1038.9,[M+10H] ¹⁰⁺ 933.7。

Example 8

Synthesis of polypeptide compound of SEQ ID No. 8

The synthesis method is the same as that of example 1, and the target peak is collected and freeze-dried to obtain pure0.014g of the product with a purity of more than 98%, and the molecular weight of the target polypeptide was confirmed by MS. The theoretical relative molecular mass is 9619.7.ESI-MS M/z calculated [ M+9H] ⁹⁺ 1069.9,[M+10H] ¹⁰⁺ 963.0; observed value [ M+9H] ⁹⁺ 1069.8,[M+10H] ¹⁰⁺ 962.8。

Example 9

Synthesis of polypeptide compound of SEQ ID No. 9

The synthesis method is the same as that of example 1, 0.015g of a pure product is obtained by collecting a target peak and freeze-drying, the purity is more than 98%, and the molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10200.3.ESI-MS M/z calculated [ M+9H] ⁹⁺ 1134.4,[M+10H] ¹⁰⁺ 1021.0; observed value [ M+9H] ⁹⁺ 1133.9,[M+10H] ¹⁰⁺ 1020.7。

Example 10

Polypeptide compounds for human GLP-1 receptor, glucagon and Y ₂ Determination of agonistic Activity of receptors

Agonism of the receptor by the polypeptide compounds was determined by functional assays, GLP-1 receptor and glucopon receptor agonism activity was measured by measuring cAMP responses of HEK-293 cell lines stably expressing human GLP-1 or glucopon receptors. Cells stably expressing the GLP-1 receptor or the glucoon receptor were split into T175 flasks and grown overnight in medium (DMEM/10% FBS) to near confluence, then the medium was removed, and the cells were washed with calcium and magnesium free PBS and then protease treated with Actuase enzyme. 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 30min and then assayedFluorescence ratio at 665/620 nm. By detecting the concentration that caused 50% of activation of the maximal response (EC ₅₀ ) To quantify the in vitro potency of the agonist.

Use of stable expression of human Y ₂ HEK-293 cell assay of receptor and cAMP sensitive calcium ion channels compounds against Y ₂ Agonism of the receptor. First, cells were cultured in a medium (DMEM, 10% fbs, geneticium, geneticin, penicillium/streptomycin), and then 20 μl of cell suspension per well was added to 384-well plates (20000 cells/well). Then using a calcium sensitive dye at 37 ℃ and 5% CO ₂ The cells were pre-treated for 50 minutes under conditions followed by 10 minutes at 25 ℃. Test compounds were serially diluted 10 times in 4-fold amounts and 750nL of test compound was transferred to 384 well plates. The 384 well plates were then removed from the incubator and placed into FLIPR Tetra System, fluorescent signals were measured (excitation 494 nm/emission 516 nm), and the concentration of 50% activation that caused the greatest response was measured (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: polypeptide compound for human GLP-1 receptor, glucago receptor and Y ₂ Agonistic activity of the receptor

As shown in Table 1, all polypeptide compounds showed activity against GLP-1 receptor, glucopon receptor and Y ₂ The triple agonistic activity of the receptor indicates that the polypeptide compounds have the characteristics of triple agonists. At the same time, some of the polypeptide compounds showed a complex with GLP-1, glucagon and PYY _3-36 Proximity or proximity toBetter GLP-1 receptor, glucon receptor and Y ₂ Agonistic activity of the receptor.

Example 11

Effect of polypeptide Compounds on ICR mouse feeding

Male ICR mice were randomized into 5 groups of 6 mice each. Mice were fasted for 12h before the experiment, normal saline (10 mg/kg) was subcutaneously administered in a blank group, the administration composition was 4 groups, and liraglutide, GEP44 (GLP-1/Y) was subcutaneously administered in a single injection of 30nmol/kg in a non-fasting state, respectively ₂ Receptor dual agonists, J.Med.chem.2021,64,2,1127-1138), xGLP/GCG-15 (GLP-1/glucon receptor dual agonists, european Journal ofMedicinal Chemistry,2021,212,113118) and SEQ ID NO:6. Immediately after this, the mice were given pre-weighed feed and the feed was weighed again at 2h,4h,6h,12h and 24h, and the feeding rate of the mice was calculated at different time points.

As shown in the results of FIG. 1, the results of the feeding experiment in ICR mice show that the polypeptide compound of SEQ ID NO:6 significantly reduces the feeding rate of the mice in 24 hours, and the feeding inhibition effect of the polypeptide compound is significantly better than liraglutide, GEP and xGLP/GCG-15, which indicates that the polypeptide compound of the invention has excellent feeding inhibition effect.

Example 12

Pharmacokinetic properties of polypeptide Compounds in rats

SD rats were given 50nmol/kg of liraglutide and SEQ ID NO:6 subcutaneously (s.c.) and blood samples were collected 0.25h,0.5h,1h,2h,4h,8h,16h and 24h after dosing. 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)
			Liraglutide	2.3	489
SEQ ID NO:6	4.6	398

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 liraglutide, with pharmacokinetic profiles supporting once-a-day dosing.

Example 13

Polypeptide compound acute hypoglycemic activity in mice

Male ICR mice, randomly grouped, 6 per group. Only drinking water, fasted overnight. Saline (10 mg/kg) was administered by intraperitoneal injection in a blank group, the administration composition was 4 groups, and liraglutide, GEP (GLP-1/Y) was administered by intraperitoneal single injection of 30nmol/kg in the non-fasting state to mice, respectively ₂ Receptor dual agonists, J.Med.chem.2021,64,2,1127-1138), xGLP/GCG-15 (GLP-1/glucon receptor dual agonists, european Journal ofMedicinal Chemistry,2021,212,113118) and SEQ ID NO:6. After 30 minutes, each group of mice was given 3g/kg of glucose solution intraperitoneally. Blood glucose levels were measured with a glucometer at-30 min,0min,15min,30min,60min, and 120min

As shown in the results of FIG. 2, the acute hypoglycemic experiment in ICR mice shows that the polypeptide compound of SEQ ID NO:6 significantly improves the glucose tolerance level of the mice, has excellent hypoglycemic effect, and the hypoglycemic effect is better than that of liraglutide and GEP 44.

Example 14

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

Male C57BL/6J mice, weighing about 22g, were 18 in model groups, and were fed with D12492 high-fat feed from Research Diets for 18 weeks to make DIO mouse models. Before the start of the administration, the DIO mice in each group were randomly grouped according to body weight, and 6 mice in each group were divided into 5 groups, namely, a normal saline group (blank control group), a positive control group (liraglutide, GEP, xGLP/GCG-15) and a test sample group (SEQ ID NO: 6). Each group of mice was subcutaneously injected twice daily with physiological saline (10 mg/kg), liraglutide (30 nmol/kg), xGLP/GCG-15 (30 nmol/kg), SEQ ID NO:6 (10 nmol/kg), and GEP44 (30 nmol/kg) three times daily for 21 days of the administration period. Mice body weight changes were recorded daily. At the end of the experiment, each group of mice was sacrificed, blood serum was taken, liver homogenates were taken, and Triglyceride (TG) and Total Cholesterol (TC) contents of the liver and serum were measured, respectively.

As shown in the results of FIG. 3, the polypeptide compound SEQ ID NO 6 of the present invention can reduce the body weight of 36.2% of mice by continuous administration in DIO mice for 3 weeks at a dose of 10 nmol/kg. While three positive control compounds, at a dose of three times SEQ ID NO. 6 (30 nmol/kg), liraglutide reduced only 15.1% of the mouse body weight, GEP44 reduced only 24.9% of the mouse body weight, and xGLP/GCG-15 reduced only 27.0% of the mouse body weight. The results show that the SEQ ID NO. 6 can realize the weight reduction effect which is obviously higher than those of liraglutide, GEP and xGLP/GCG-15 at low dosage, which shows that the SEQ ID NO. 6 has excellent weight reduction effect.

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

^*** : p compared with the blank control group<0.001； ^### : group ratio P to liraglutide, GEP and xGLP/GCG-15<0.001(One-WayANOVA, tukey post hoc test), the results are expressed as mean ± SD of 6 mice per group.

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

Sample (dose)	Total cholesterol (mg/g)	Triglyceride (mg/g)
			Blank control (normal saline group)	14.26±1.59	94.28±6.71
Liraglutide(30nmol/kg)	12.11±1.25 ^*	73.07±4.78 ^***
			GEP44(30nmol/kg)	11.65±0.86 ^**	77.72±6.62 ^***
xGLP/GCG-15(30nmol/kg)	10.15±0.75 ^***	61.65±6.98 ^***
			SEQ ID NO:6(10nmol/kg)	7.15±0.50 ^***,###	40.93±4.39 ^***,###

^* : p compared with the blank control group<0.05； ^** : p compared with the blank control group<0.01； ^*** : p compared with the blank control group<0.001； ^### : group ratio P to liraglutide, GEP and xGLP/GCG-15<0.001 (One-WayANOVA, tukey post hoc test) and the results are expressed as mean.+ -. SD of 6 mice per group.

As shown in the results of tables 3 and 4, the polypeptide compounds SEQ ID NO:6 of the present invention can significantly reduce the Triglyceride (TG) and Total Cholesterol (TC) contents of serum and liver of mice by continuous administration in DIO mice at a dose of 10nmol/kg for 3 weeks, and the serum and liver lipid lowering effect of SEQ ID NO:6 is significantly stronger than that of positive control drugs liraglutide (30 nmol/kg), GEP44 (30 nmol/kg) and xGLP/GCG-15 (30 nmol/kg) at high doses. The results show that the SEQ ID NO. 6 at low dosage can realize the effects of reducing weight and reducing liver and serum blood fat which are obviously better than those of liraglutide, GEP and xGLP/GCG-15, and the polypeptide compound has the effects of reducing weight, regulating fat and treating NAFLD.

Claims

1. GLP-1/glucon/Y ₂ A receptor triple agonist characterized in that the GLP-1/glucon/Y ₂ The amino acid sequence of the receptor triple agonist is one of the following:

(1)SEQ ID NO:1

(2)SEQ ID NO:2

(3)SEQ ID NO:3

(7)SEQ ID NO:7

(8)SEQ ID NO:8

(9)SEQ ID NO:9

。

2. GLP-1/glucon/Y ₂ A pharmaceutically acceptable salt of a receptor triple agonist, characterized by: the GLP-1/glucon/Y ₂ The amino acid sequence of the receptor triple agonist is one of the amino acid sequences described in claim 1.

3. A GLP-1/glucon/Y type according to claim 2 ₂ A pharmaceutically acceptable salt of a receptor triple agonist, which is characterized in that the salt is GLP-1/glucon/Y ₂ A salt of a receptor triple agonist with one of the following compounds: hydrochloric acid, acetic acid, salicylic acid, lauric acid, cinnamic acid, citric acid, oxalic acid, lactic acid, and succinic acid.

4. A GLP-1/glucon/Y set forth in claim 1 ₂ The preparation of the receptor triple agonist is characterized in that the preparation is any one of tablets, capsules, inhalants, sprays, injections, films, patches, emulsions, suppositories or compound preparations which are described in pharmaceutics, and the preparation is prepared from GLP-1/glucagon/Y ₂ Receptor triple agonists and pharmaceutical uses thereofThe pharmaceutical excipients, carriers or diluents are accepted.

5. Comprises GLP-1/glucon/Y ₂ A pharmaceutical composition of a receptor triple agonist, characterized in that the pharmaceutical composition is a GLP-1/glucon/Y composition according to claim 1 ₂ Receptor triple agonists as effective raw materials or as GLP-1/glucon/Y as claimed in claim 2 or 3 ₂ The receptor triple agonist is prepared from pharmaceutically acceptable salts serving as effective raw materials and pharmaceutically acceptable carriers or diluents.

6. GLP-1/glucon/Y according to any of the claims 1-5 ₂ Use of a receptor triplet agonist or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or a medicament thereof, in the manufacture of a medicament for the treatment of a metabolic disease or disorder.

7. The use according to claim 6, wherein the metabolic disease or disorder is diabetes, obesity, non-alcoholic fatty liver disease and dyslipidemia.