Long-acting GGG triple-target agonist

Abstract

The present invention discloses a long-acting GGG triple-target agonist. The compound of the present invention simultaneously achieves excellent agonist ability at the nM level for the three targets of GLP-1 / GIP / GCGR, and can fully excite the corresponding target organ tissue cells. The compound of the present invention can promote insulin secretion and lower blood sugar; it can also inhibit food intake, delay gastric emptying, increase energy consumption, and ultimately observe a weight loss effect. The compound of the present invention can reduce pancreatic β-cell apoptosis, increase the number of pancreatic β-cells, and improve pancreatic cell function. The compound of the present invention can also improve blood lipids, reduce liver fat accumulation, and inhibit the development of liver inflammation. The compound or composition of the present invention can be used to prevent and / or treat metabolic disorders and related complications. The compound or composition of the present invention can be used to treat bone diseases related to endocrine diseases, metabolic disorders, kidney disease, etc., such as osteoporosis and osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a long-acting GGG triple-target agonist. Background Art

[0002] GLP-1R (glucagon-like peptide-1 receptor) agonists have always been a hot research and development topic in the diabetes field, and have become the non-insulin diabetes drug with the highest global market share. Many star products such as dulaglutide and semaglutide have performed well in 2021. Therefore, the continued development of iterative products - multi-target agonists of GLP-1R - has also become a hot competitive direction.

[0003] Currently, the majority of multi-target agonists under development globally are focused on the GLP-1R / GIPR and GLP-1R / GCGR pathways. Eli Lilly's Tirzepatide leads the GLP-1R / GIPR field, having received FDA approval for marketing in the US. Among domestic companies, Hansoh Pharmaceuticals and Hongyun Huaning each have products in Phase I clinical trials, while Heze Pharmaceuticals and Borrui Biopharmaceuticals have also filed for INDs.

[0004] In contrast, competition in the GLP-1R / GCGR market is even more intense, with multinational companies such as AstraZeneca, Eli Lilly, and Boehringer Ingelheim all making moves. AstraZeneca is the first to initiate Phase III clinical trials. Innovent Biologics is the most advanced domestic company, with IBI362, jointly developed with Eli Lilly, in Phase II trials for the treatment of type 2 diabetes and obesity, respectively. Furthermore, Salubris, Page Pharmaceuticals, Tuwei Anchuang, and Hengrui Medicine each have products in Phase I clinical trials.

[0005] In addition to dual-targeting, triple-targeting GLP-1R / GIPR / GCGR agonists have the potential to demonstrate even stronger efficacy. Eli Lilly and Hanmi Pharmaceuticals have already advanced their respective products into Phase II clinical trials. Furthermore, there is the potential for GLP-1R to combine with GCGR, FGF21R, and GLP-2R to form single-molecule dual- and triple-receptor agonists.

[0006] Studies have shown that while mono-glucagon GCG receptor drugs can effectively lower blood glucose and glycosylated hemoglobin levels before and after meals in patients with type 2 diabetes, they are also associated with adverse reactions such as increased blood lipids and liver transaminases. However, the development and use of GLP-1R and GCGR as co-agonists not only lowers blood glucose but also effectively mitigates these adverse reactions. The successful development of various GLP-1 / GCG and GLP-1 / GIP dual receptor agonists has also triggered research on single-molecule agonists that simultaneously activate all three target receptors. Summary of the Invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0008] The present invention provides a GLP1R-GIPR-GCGR triple-target agonist peptide or a pharmaceutically acceptable salt thereof, wherein the agonist peptide or the pharmaceutically acceptable salt thereof comprises or consists of an amino acid sequence, and the amino acid sequence general formula I is shown below:

[0009] X1-Aib-QGTFTSDYSI-aMeL-LDK-X 17 -AQ-Aib-X 21 -FX 23 -EYLLE-XX1-R 1 ,

[0010] in,

[0011] X1 is H or Y;

[0012] X 17 is Ψ, Ψ is Lys with modified side chain;

[0013] X 21 D or A;

[0014] X 23 is V or I;

[0015] XX1 is GGPSSGAPPPSKVSRA (SEQ ID NO:1), GGGPSSGAPPPSKVSRA (SEQ ID NO:2), GGPSSGA d PPPSKVSRA(SEQ ID NO:3),GGGPSSGA d PPPSKVSRA (SEQ ID NO: 4), GGPSSGAPPPS (SEQ ID NO: 9), GGGPSSGAPPPS (SEQ ID NO: 10), GGPSSGA d PPPS (SEQ ID NO: 11), or GGGPSSGA d PPPS (SEQ ID NO: 12);

[0016] R1 is NH2 or OH, or a pharmaceutically acceptable salt and / or ester thereof.

[0017] Furthermore, the Ψ is a Lys with a side chain modified by a structure having the following general formula II, wherein the general formula II is: BZ; wherein B is (AEEA or Glu) a -(AEEA or Glu) b -(AEEA or Glu) c, wherein a, b, and c are each independently 0 or 1, and a, b, and c are not 0 at the same time; the carboxyl end of B is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m -R 2 , m is an integer between 6 and 24, R 2 Selected from -COOH.

[0018] Furthermore, the general formula II is AEEA-γG1u-CO(CH2) 18 COOH.

[0019] Furthermore, at least one site on the agonist peptide or a pharmaceutically acceptable salt thereof is chemically modified with a fatty acid side chain group.

[0020] Furthermore, the agonist peptide or a pharmaceutically acceptable salt thereof is selected from:

[0021] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPSKVSRA-NH2, designated as ZG1;

[0022] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPSKVSRA-NH2, designated as ZG2;

[0023] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPS-NH2, named ZG3;

[0024] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPS-NH2, named ZG4;

[0025] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPSKVSRA-NH2, named ZG5;

[0026] H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPSKVSRA-NH2, named ZG6;

[0027] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPS-NH2, named ZG7;

[0028] Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPS-NH2, named ZG8.

[0029] The amino acid sequence QGTFTSDYSI was named SEQ ID NO: 5; the amino acid sequence DFVEYLLE was named SEQ ID NO: 6; the amino acid sequence AFIEYLLE was named SEQ ID NO: 7; and the amino acid sequence LDKK was named SEQ ID NO: 8.

[0030] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 30% greater than that of native GLP-1 in agonist activity of cells transfected with the GLP-1 receptor. Furthermore, the activity is increased by at least 60%. Furthermore, the activity is increased by at least 80%. Furthermore, the activity is increased by at least 100%.

[0031] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 100% higher than that of native GIP in agonist ability of cells transfected with GIP receptors, and further, at least 150% higher.

[0032] Furthermore, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 10% higher than that of natural GCG in agonizing cells transfected with GCG receptors, and further, at least 30% higher.

[0033] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist ability in cells transfected with the GLP-1 receptor, and the relative activity of the agonist peptide or its pharmaceutically acceptable salt on the GLP-1 receptor is increased by at least 30%-100% compared to natural GLP-1. Specifically, the relative activity of the agonist peptide or its pharmaceutically acceptable salt on the GLP-1 receptor is increased by at least 30%, 60%, 80%, or 100%.

[0034] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist activity on GIP receptor transfected cells that is at least 100%-150% higher than that of natural GIP.

[0035] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has an agonist activity on GCG receptor transfected cells that is at least 10%-30% higher than that of natural GCG.

[0036] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt increases the relative activity of the agonist peptide or its pharmaceutically acceptable salt on 3T3-L1 cells, representative of adipose tissue, by at least 60%-100%, compared to natural GIP.

[0037] In a specific embodiment of the present invention, the agonist peptide or its pharmaceutically acceptable salt has a relative activity of at least 60%-100% on human primary hepatocytes, a representative cell of liver tissue, compared to natural GCG.

[0038] In specific embodiments of the present invention, the peptide backbone of the agonist peptide or a pharmaceutically acceptable salt thereof is chemically modified with a fatty acid side chain group at at least one site. More specifically, the agonist peptide or a pharmaceutically acceptable salt thereof may have a stable peptide α-helical structure and enhanced albumin binding, thereby achieving improved stability of the peptide compound and prolonged duration of action.

[0039] The present invention provides a solid phase synthesis method for preparing the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, the method comprising the following steps:

[0040] 1) Resin pretreatment: Add the resin to the reactor, add DMF, stir and swell under nitrogen, filter and wash;

[0041] 2) Prepare amino acid solution: Dissolve Fmoc-Ser-tBu-OH and HOBt in DMF, then add DIC and mix thoroughly;

[0042] 3) Deprotection: Add 20% piperidine / DMF solvent to the reaction kettle, stir and wash with nitrogen;

[0043] 4) Coupling reaction: The amino acid solution prepared in step 2) is added to a reaction vessel, and the mixture is stirred under nitrogen gas at controlled temperature. After the reaction is completed, the resin is washed with DMF, and step 4) is repeated to sequentially couple the corresponding protected amino acids according to the aforementioned amino acid sequence of the agonist peptide or its pharmaceutically acceptable salt until the agonist peptide backbone is synthesized;

[0044] 5) Removal of the 17-position Lys side chain protecting group iLEde: Add 8% hydrazine hydrate / DMF solution to the reaction kettle, stir under nitrogen, and after the reaction is complete, filter to remove the solvent and wash;

[0045] 6) Lys side chain modification: Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and HOBt were dissolved in DMF, and DIC was added and mixed to prepare a side chain modification solution. The side chain modification solution was added to a reaction vessel, and the temperature was controlled and nitrogen was stirred. After the reaction was completed, the resin was washed with DMF, then with DCM, and then with methyl tert-butyl ether to obtain the peptide resin containing the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, which was then dried.

[0046] Furthermore, the swelling time in step 1) is 30 minutes.

[0047] Furthermore, in the step 2), the dissolution ratio of Fmoc-Ser-tBu-OH in DMF is 5.75 g / 75 mL.

[0048] Furthermore, in the step 2), the solubility ratio of HOBt in DMF is 2.02 g / 75 mL.

[0049] Furthermore, in the step 2), the dissolution ratio of DIC in DMF is 1.89 g / 75 mL.

[0050] Furthermore, the ratio of the amount of the 20% piperidine / DMF solvent added in step 3) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0051] Furthermore, the stirring time of nitrogen in step 3) is 35 minutes.

[0052] Furthermore, after washing in step 3), the resin is tested using a ninhydrin reagent to determine whether it is positive.

[0053] Furthermore, the ratio of the amount of the amino acid solution added in step 4) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0054] Furthermore, the temperature range of the temperature-controlled nitrogen stirring in step 4) is 25-35°C.

[0055] Furthermore, the completion of the reaction in step 4) is indicated by a negative result of the resin detected by the ninhydrin reagent.

[0056] Furthermore, the ratio of the amount of the 8% hydrazine hydrate / DMF solution in step 5) to the amount of the resin added in step 1) is 100 mL / 13.15 g.

[0057] Furthermore, the stirring time under nitrogen in step 5) is 1 h.

[0058] Furthermore, after washing in step 5), the resin is tested using a ninhydrin reagent to determine whether it is positive.

[0059] Furthermore, in the step 6), the dissolution ratio of Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH in DMF is 7.29 g / 150 mL.

[0060] Furthermore, in step 6), the solubility ratio of HOBt in DMF is 1.35 g / 150 mL.

[0061] Furthermore, in step 6), the dissolution ratio of DIC in DMF is 1.30 g / 150 mL.

[0062] Furthermore, the ratio of the amount of the side chain modification solution in step 6) to the amount of the resin in step 1) is 150 mL / 13.15 g.

[0063] Furthermore, the temperature range of the temperature-controlled nitrogen stirring in step 6) is 25-35°C.

[0064] Furthermore, the completion of the reaction in step 6) is indicated by a negative result of the resin detected by the ninhydrin reagent.

[0065] The present invention provides a post-treatment method for the solid-phase synthesis of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, the post-treatment method comprising the following steps:

[0066] 1) taking the peptide resin containing the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof finally synthesized by the method described above, adding a cutting solution, stirring the reaction, filtering, adding the filtrate to ice methyl tert-butyl ether for precipitation, filtering with suction, washing the filter cake with methyl tert-butyl ether, and vacuum drying to obtain a crude product;

[0067] 2) The crude product obtained in step 1) is passed through a refining purification system to obtain a purified product.

[0068] Furthermore, the volume ratio of the cutting fluid in step 1) is TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5.

[0069] Furthermore, the stirring reaction time in step 1) is 3 hours.

[0070] Furthermore, in step 1), the volume ratio of the filtrate to the glacial methyl tert-butyl ether is 1:5.

[0071] Furthermore, the refining and purification system in step 2) is a C18 reverse phase preparative chromatography system.

[0072] Furthermore, the purity of the purified product in step 2) is not less than 90%.

[0073] The present invention provides a product, which comprises the reagent used in the above-mentioned method and / or the reagent used in the above-mentioned post-treatment method.

[0074] Furthermore, the product includes a kit.

[0075] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount or a preventive effective amount of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0076] Furthermore, the pharmaceutical composition may further include other active pharmaceutical ingredients.

[0077] Furthermore, the pharmaceutical composition is an injection, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension, or syrup.

[0078] Furthermore, the pharmaceutical composition is administered orally, by inhalation or parenterally.

[0079] Furthermore, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection.

[0080] Furthermore, the therapeutically effective amount or preventively effective amount needs to be administered at a frequency of at least once a week or once every two weeks.

[0081] Furthermore, the other active pharmaceutical ingredients include anti-diabetic active agents, GIP receptor agonists, GCG receptor agonists or antagonists, GLP-1 / GIP receptor agonists, GLP-1 / GCG receptor agonists, GIP / GCG receptor agonists, FGF-21 and its analogs, CCKB and its analogs, PYY and its analogs, leptin and its analogs, calcitonin and its analogs, lipid-regulating active drugs, PPAR-α, β, δ agonists or regulators, anti-platelet aggregation active agents, PCSK9 inhibitors, lipase inhibitors, anti-liver fibrosis or cirrhosis active agents, and anti-inflammatory active agents.

[0082] Furthermore, the diabetes active agents include insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, X-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1 / SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogs.

[0083] The present invention provides any of the following applications:

[0084] 1) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for promoting insulin secretion and lowering blood sugar.

[0085] 2) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for inhibiting food intake, delaying gastric emptying, increasing energy consumption, and reducing body weight.

[0086] 3) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for reducing pancreatic β-cell apoptosis, increasing pancreatic β-cell number, and improving pancreatic cell function.

[0087] 4) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a drug for improving blood lipids, reducing liver fat accumulation, and inhibiting the development of liver inflammation.

[0088] 5) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of drugs that promote brain neuron growth, eliminate neurotoxic substances, inhibit the development of inflammation, and exert neuroprotective effects.

[0089] 6) Use of the aforementioned agonist peptide or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating metabolic disorders.

[0090] Furthermore, the metabolic disorders include diabetes, obesity, non-alcoholic fatty liver disease, and dyslipidemia.

[0091] 7) Use of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of a medicament for treating neurodegenerative diseases.

[0092] Furthermore, the neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0093] 8) Use of the aforementioned agonist peptide or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of a medicament for treating bone diseases.

[0094] Furthermore, the bone diseases include bone diseases caused by endocrine diseases, metabolic disorders, and kidney diseases.

[0095] Furthermore, the bone diseases include osteoporosis and osteoarthritis.

[0096] In a specific embodiment of the present invention, the aforementioned agonist peptide or its pharmaceutically acceptable salt can be prepared by solid phase synthesis.

[0097] Beneficial effects of the present invention:

[0098] The compounds of the present invention simultaneously achieve excellent agonistic effects at the nM level on the three targets of GLP-1 / GIP / GCGR, and are able to fully excite the corresponding target organ tissue cells. The compounds of the present invention can promote insulin secretion and lower blood sugar; they can also inhibit food intake, delay gastric emptying, increase energy expenditure, and ultimately, a weight loss effect can be observed. The compounds of the present invention can reduce pancreatic β-cell apoptosis, increase the number of pancreatic β-cells, and improve pancreatic cell function. The compounds of the present invention can also improve blood lipids, reduce liver fat accumulation, and inhibit the development of liver inflammation. The compounds or compositions of the present invention can be used to prevent and / or treat metabolic disorders and their related complications. The compounds or compositions of the present invention can be used to treat bone diseases related to endocrine diseases, metabolic disorders, kidney disease, etc., such as osteoporosis and osteoarthritis.

[0099] Compared with existing compounds, the GLP-1 / GIP / GCGR triple agonist polypeptide molecules provided by the present invention have better GLP-1R, GIPRs, and GCGR agonist activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is the glucose consumption staining graph of each group of peptide compounds;

[0101] Figure 2 This is an Oil Red O staining image of mouse 3T3-L1;

[0102] Figure 3 It is a graph showing changes in blood sugar levels over time;

[0103] Figure 4 is a graph showing changes in mouse body weight over time;

[0104] Figure 5 is a graph of the CK content in serum of each group of peptide compounds;

[0105] Figure 6 is a graph of the ALT content in serum of each group of peptide compounds;

[0106] Figure 7 It is a statistical chart of the stability of each group of peptide compounds in serum. DETAILED DESCRIPTION

[0107] The amino acids in the compound sequences of the present invention are derived from natural amino acids or related amino acid variants and / or derivatives. The abbreviations and codes for the natural amino acids follow common rules well known to those in the industry. The chemical formula of Aib, αMel is as follows:

[0108]

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In the event of conflict, the present document, including definitions, will prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0110] The compounds of the present invention have agonist activity on GLP-1 receptor, GIP receptor and GCG receptor. The "agonist activity" mentioned above means that the compounds can stimulate specific receptor cells to produce cAMP. The cells used can be host cells overexpressing GLP-1 receptor, GIP receptor or GCG receptor constructed by those skilled in the art, or pancreatic tissue cells, adipocytes, hepatocytes, etc. The receptor agonist activity can be achieved by using ECs that stimulate receptor cells to produce cAMP. 50 The value is used as a measure. 50 The values are the drug concentrations required to achieve half of the maximal activity (50% activity) of the compound in a specific assay system.

[0111] As used herein, an "effective amount" refers to the amount, concentration, or dosage of one or more agonist peptides described herein, or a pharmaceutically acceptable salt thereof, that provides the desired effect in a subject being diagnosed or treated when administered in single or multiple doses to such subject in need thereof. An effective amount can be readily determined by one skilled in the art using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for a subject, a number of factors are considered, including, but not limited to, the species of mammal; its size, age, and general health; the specific disease or disorder involved; the extent of prevalence or severity of the disease or disorder; the response of the individual patient; the mode of administration; the bioavailability characteristics of the formulation to be administered; the dosing regimen selected; the use of concomitant medications; and other relevant circumstances.

[0112] For a human subject of about 50-70 kg, the unit dose of the pharmaceutical composition of the present invention or combination can contain about 1-1000 mg of active ingredient (one or more), or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredient. The therapeutically effective dose of a compound, pharmaceutical composition, or combination thereof depends on the type, body weight, age, and individual condition of the subject, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian of ordinary skill can easily determine the effective amount of each active ingredient necessary to prevent, treat, or inhibit a condition or disease progression.

[0113] Throughout this specification and subsequent claims, unless the context requires otherwise, variations such as words "comprise", "include" should be understood to imply the inclusion of the member, integer or step or the group of member, integer or step, but not excluding any other member, integer or step or the group of member, integer or step, but in some embodiments, such other member, integer or step or the group of member, integer or step can be excluded, i.e., the subject matter is to include the member, integer or step or the group of member, integer or step. Unless otherwise indicated herein or clearly contradicted by the context, the terms "one / a kind" and "described" used in the context of describing the present invention (especially in the context of the claims) should be interpreted as covering both the singular and the plural. The range of values listed herein is only intended to be used as a shorthand for individually mentioning each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if individually enumerated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not impose any limitation on the scope of the invention as originally claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0114] As used herein, the term "peptide" generally refers to an amino acid whose polymeric form is any length, which for example comprises about two or more, or about three or more, or about four or more, or about six or more, or about eight or more, or about nine or more, or about ten or more, or about 13 or more, or about 16 or more, or about 21 or more amino acids covalently linked by peptide bonds. A peptide can for example be made up of up to 100 amino acids. As used herein, the term "polypeptide" refers to a large peptide. In one embodiment, the term "polypeptide" refers to a peptide having more than about 100 amino acid residues. The terms "polypeptide" and "protein" are used interchangeably in this article.

[0115] As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring amino acids, non-natural amino acids that function in a manner similar to the naturally occurring amino acids, amino acid analogs and / or amino acid mimetics, in their D and L stereoisomeric forms, if their structure permits such stereoisomeric forms. Amino acids are referred to herein by their name, their three letter symbols as known in the art, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0116] As used herein, the terms "naturally occurring" and "natural" when used in conjunction with biological materials (such as nucleic acid molecules, (poly)peptides, host cells, etc.) refer to materials that are found in nature and have not been manipulated by humans.

[0117] When used in conjunction with amino acids, the terms "naturally occurring" and "natural" refer to the 20 conventional amino acids (i.e., alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamate (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)), as well as selenocysteine, pyrrolysine (PYL), and pyrroline-carboxylysine (PCL).

[0118] As used herein, the term "non-natural amino acid" is intended to refer to an amino acid that is not naturally encoded or found in the genetic code of any organism. It can, for example, be a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelanediol, 2,3-diaminopropionic acid, N- Ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, D-alanine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0119] As used herein, the term "amino acid analogue" refers to a compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogues include natural amino acids and non-natural amino acids that are chemically blocked, reversibly or irreversibly, or chemically modified, for example, at one or any combination of their C-terminal carboxyl group, their N-terminal amino group, and / or their side chain functional groups. Such analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteine sulfone, aspartic acid-(β methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methylsulfonium.

[0120] The compounds of the present invention can form acid and / or base salts due to the presence of amino and / or carboxyl groups or similar groups. As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present invention. "Salt" particularly includes "pharmaceutically acceptable salts" and "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and are generally biologically or otherwise desirable. In particular, salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compounds provided by the present invention are acids, their corresponding salts can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include salts of aluminum, ammonium, calcium, copper (high and low valence), ferric iron, ferrous iron, lithium, magnesium, manganese (high and low valence), potassium, sodium, zinc, and the like. Salts of ammonium, calcium, magnesium, potassium, and sodium are particularly preferred. Pharmaceutically acceptable non-toxic organic bases capable of forming salts include primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines, such as naturally occurring and synthetic substituted amines. Other pharmaceutically acceptable non-toxic organic bases capable of forming salts include ion exchange resins, as well as arginine, betaine, caffeine, choline, N',N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, halamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0121] When compound provided by the invention is a base, it is possible to conveniently prepare its corresponding salt from pharmaceutically acceptable nontoxic acid, including inorganic acid and organic acid. Such acid includes inorganic acid and organic acid, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and p-toluenesulfonic acid etc. Preferably, the inorganic acid is hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid etc., and the organic acid is citric acid, lactic acid, malic acid, gluconic acid, tartaric acid, adipic acid, acetic acid, succinic acid, fumaric acid, septic acid, itaconic acid, methanesulfonic acid or benzenesulfonic acid etc. Since the compound represented by formula (I) is to be used as a medicine, it is preferably used with a certain purity, for example, at least 60% purity, more preferably at least 75% purity, and particularly preferably at least 98% purity (% is weight ratio).

[0122] Pharmaceutically acceptable salts of the present invention can be synthesized by alkaline or acidic moieties by conventional chemical methods. Usually, these salts can be prepared by reacting the free acid form of these compounds with the appropriate alkali (for example Na, Ca, Mg or K, hydroxide, carbonate, bicarbonate etc.) of stoichiometric amount, or by reacting the free base form of these compounds with the appropriate acid reaction of stoichiometric amount. Such reaction is usually carried out in water or in an organic solvent or in a mixture of the two. Usually, in the case of feasibility, it is relatively ideal to use non-aqueous media such as ether, ethyl acetate, tetrahydrofuran (THF), toluene, chloroform, methylene dichloride, methanol, ethanol, isopropyl alcohol or acetonitrile.

[0123] The term "administering" refers to administering a compound disclosed herein that can treat various diseases, or a compound that, although not explicitly disclosed, can be converted into a compound disclosed herein in vivo after administration to a subject. Conventional methods for selecting and preparing suitable prodrug derivatives are described in books such as "Design of Prodrugs" (ed. H. Bundgaard, Elsevier, 1985).

[0124] The compounds of the present invention may contain one or more asymmetric centers and may thus produce diastereomers and optical isomers. The present invention includes all possible diastereomers and racemic mixtures thereof, their substantially pure resolved enantiomers, all possible geometric isomers and pharmaceutically acceptable salts thereof.

[0125] In the present invention, the term "composition" refers to a product comprising a specified amount of each specified ingredient, as well as any product produced directly or indirectly by a combination of specified amounts of each specified ingredient. Therefore, pharmaceutical compositions containing a compound of the present invention as an active ingredient and methods for preparing the compound of the present invention are also part of the present invention. In addition, some crystalline forms of the compound may exist as polymorphs, and such polymorphs are included in the present invention. In addition, some compounds can form solvates with water (i.e., hydrates) or common organic solvents, and such solvates also fall within the scope of the present invention.

[0126] According to conventional drug mixing technology, the agonist peptide compound shown in formula I of the present invention, or a drug precursor, or a metabolite, or a pharmaceutically acceptable salt, can be used as an active ingredient and mixed with a drug carrier to form a pharmaceutical composition. The drug carrier can take a variety of forms, depending on the desired mode of administration, for example, oral or injection (including intravenous injection). Therefore, the pharmaceutical composition of the present invention can be in the form of a separate unit suitable for oral administration, such as a capsule, cachet or tablet containing a predetermined dose of the active ingredient. Further, the pharmaceutical composition of the present invention can be in the form of a powder, granules, solution, aqueous suspension, non-aqueous liquid, water-in-oil emulsion, or oil-in-water emulsion. In addition, in addition to the common dosage forms mentioned above, the compound shown in formula (I) or its pharmaceutically acceptable salt can also be administered by controlled release and / or a delivery device. The pharmaceutical composition of the present invention can be prepared by any pharmaceutical method. Generally, this method includes the step of associating the active ingredient with a carrier constituting one or more necessary ingredients. Generally, the pharmaceutical composition is prepared by uniformly and closely mixing the active ingredient with a liquid carrier or a finely divided solid carrier or a mixture of the two. In addition, the product can be easily prepared into a desired appearance.

[0127] Specifically, the pharmaceutical carrier used in the present invention can be, for example, a solid carrier, a liquid carrier, or a gaseous carrier. Solid carriers include, but are not limited to, lactose, gypsum powder, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, and water. Gaseous carriers include, but are not limited to, carbon dioxide and nitrogen. When preparing oral pharmaceutical preparations, any pharmaceutically convenient medium can be used. For example, water, ethylene glycol, oils, alcohols, flavor enhancers, preservatives, colorants, etc. can be used for oral liquid preparations such as suspensions, elixirs, and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used for oral solid preparations such as powders, capsules, and tablets. Considering ease of administration, tablets and capsules are preferred for oral preparations, in which case solid pharmaceutical carriers are used. Alternatively, tablet coating can be performed using standard aqueous or non-aqueous formulation techniques.

[0128] The abbreviations used in the present invention have the following meanings:

[0129] Aib: a-amino isobutyric acid;

[0130] αMeL: α-methyl leucine;

[0131] AEEA: [2-(2-amino-ethoxy)-ethoxy]-acetyl;

[0132] cAMP: cyclic adenosine monophosphate;

[0133] Fmoc: fluorenylmethoxycarbonyl;

[0134] Boc: tert-butyloxycarbonyl;

[0135] DMF: dimethylformamide;

[0136] HOAt: N-hydroxy-7-azabenzotriazole;

[0137] Trt: trityl;

[0138] iLEde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl;

[0139] tBu: tert-butyl;

[0140] OtBu: tert-butyloxy;

[0141] TFA: trifluoroacetic acid;

[0142] TIS: triisopropylsilane;

[0143] DCM: dichloromethane;

[0144] DIPEA: N,N-diisopropylethylamine;

[0145] A d : D-alanine;

[0146] GLP-1: glucagon-like peptide-1;

[0147] GIP: glucose-dependent insulinotropic peptide;

[0148] GCG: glucagon;

[0149] GGG0: Eli Lilly's investigational product LY3437943.

[0150] Example 1 Synthesis of peptide compounds

[0151] The reagents and raw materials used in the present invention are readily available to those skilled in the art. Those skilled in the art can prepare the agonist peptides and pharmaceutically acceptable salts thereof according to the present invention using synthetic methods not limited to the examples herein. In particular, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0152] 1. Experimental Materials

[0153] The materials and reagents used in the present invention were purchased from commercial products. The protected amino acids used in the entire synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmo-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(iLEde)-OH, Fmoc-α-Me-Leu-OH, Fmoc-Ile-OH, Boc-Tyr(tBu)-OH, Eicosanedioic acid(mon-tBu)-γGlu(α-OtBu)-AEEA-OH.

[0154] 2. Taking ZG1 as an example, the synthesis and preparation method of the agonist peptide of the present invention is described (the preparation of other agonist peptides of the present invention only requires replacing the synthesis sequence of amino acid raw materials)

[0155] 1) Resin Pretreatment: Weigh 13.15 g of Rink Amide AM Resin into a reactor, add 100 mL of DMF, and allow to swell for 30 min under nitrogen. Once fully swollen, remove the solvent by filtration and wash the resin three times with 100 mL of DMF.

[0156] 2) Prepare amino acid solution: Dissolve 5.75 g of Fmoc-Ser(tBu)-OH and 2.02 g of HOBt in 75 mL of DMF, then add 1.89 g of DIC and mix thoroughly.

[0157] 3) Deprotection: Add 100 mL of 20% piperidine / DMF to the reaction vessel and stir under nitrogen for 35 minutes. After the reaction is complete, wash the resin six times with 100 mL of DMF each time. After washing, remove the resin and test it with ninhydrin, which should be positive.

[0158] 4) Coupling reaction: Add the prepared amino acid solution to the reactor, control the temperature at 25-35°C, and stir the reaction under nitrogen. Monitor the reaction progress with ninhydrin reagent, and the reaction is complete when the resin shows a negative reaction. After the reaction is complete, wash the resin with DMF three times, 100 mL each time. Repeat the above steps and sequentially couple the corresponding protected amino acids according to the ZG1 peptide until the peptide backbone is synthesized;

[0159] 5) Removal of the iLEde side chain protecting group at position 17 of Lys: 100 mL of 8% hydrazine hydrate / DMF solution was added to the reaction vessel and stirred under nitrogen for 1 h. When the reaction was complete, the solvent was removed by filtration. This process was repeated once. The resin was then washed 12 times with 100 mL of DMF each time. After washing, the resin was tested with ninhydrin, which was positive.

[0160] 6) Lys side chain modification: Weigh 7.29 g of Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and 1.35 g of HOBt, dissolve them in 150 mL of DMF, then add 1.30 g of DIC, mix well and add to the reactor, control the temperature at 25-35 ° C, and stir under nitrogen. The reaction progress was monitored with ninhydrin reagent until the resin showed a negative reaction, indicating that the reaction was complete. After the reaction was completed, the resin was washed with DMF 4 times, DCM 2 times, and methyl tert-butyl ether 3 times, each time 100 mL. The resin was dried and set aside for use;

[0161] 7) Peptide Resin Cleavage: Prepare a cleavage solution with TFA / TIS / DTT / H₂O = 90 / 2.5 / 2.5 / 5. Add the dried peptide resin to the cleavage solution and stir for 3 h. When the reaction is complete, filter the solution and add the filtrate to 5 volumes of glacial methyl tert-butyl ether for precipitation. Filter with suction, wash the filter cake three times with methyl tert-butyl ether, and vacuum dry to obtain the crude product.

[0162] 8) Purification: The crude product was purified by C18 reverse phase preparative chromatography to obtain a refined product of ZG1 with a purity of not less than 90%. The compound was analyzed by analytical HPLC and LC-MS.

[0163] 3. Preparation results

[0164] After the preparation was completed, the molecular weight test results of each agonist peptide compound were shown in Table 1, and the results showed that the prepared compounds were correct.

[0165] Table 1 List of synthetic agonist peptide compounds and molecular weight

[0166]

[0167] Example 2: Glucose consumption test of peptide compounds in human HepG2 cell insulin resistance model

[0168] 1. Experimental Materials

[0169] The materials and reagents used in this test were purchased from commercial products, including the basal culture medium for HepG2 cells (MEM+10% FBS+0.38 mg / ml G418+1% P / S).

[0170] 2. Experimental methods and results

[0171] The following is an example of a glucose consumption test using compounds ZG1 and ZG2, as follows:

[0172] HepG2 cells in the logarithmic growth phase were taken, the original culture medium was removed, serum-free culture medium was added, starved for 24 hours, and 10 - 6 mol / L insulin, cultured for 48 hours to establish an insulin resistance model. Discard the original culture medium, replace the culture medium (without FBS), add the peptide compound and incubate for 24 hours. Determine the glucose content of each group according to the instructions of the glucose staining kit. The peptide concentration used is 50nmol / L. The results are as follows Figure 1 Compared with the model group, the GGG0 group (50nmol / L), the ZG1 group (50nmol / L), and the ZG2 group (50nmol / L) all increased glucose consumption, indicating that ZG1 / 2 has significant in vitro hypoglycemic ability, and its hypoglycemic ability is stronger than that of GGG0.

[0173] Example 3: Functional activity test of peptide compounds on mouse 3T3-L1 adipocytes

[0174] 1. Experimental Materials

[0175] The materials and reagents used in this test were purchased from commercial products, including the basal culture medium for mouse 3T3-L1 cells (DMEM, 10% FBS, 1% P / S).

[0176] 2. Experimental methods and results

[0177] The lipid droplet formation inhibition test was carried out using compounds ZG1 and ZG2 as examples, as follows:

[0178] Mouse 3T3-L1 cells in the logarithmic growth phase were taken, and IBMX (0.5mM), dexamethasone (1μmol / L), and insulin (10μg / mL) were added to the basal culture medium to prepare an adipogenic induction medium. The medium was changed every 2 days, and the induction was continued for 8 days to establish an induced adipogenic model. The model cells were grouped into control group, GGG0 group (50nmol / L), ZG1 group (50nmol / L), and ZG2 group (50nmol / L). Then, peptide compounds were added to each group, and Oil Red O staining was performed after 24 hours. The peptide concentration used was 50nmol / L. The results are shown in Figure 2. Figure 2 Compared with the control group, the GGG0 group (50nmol / L), the ZG1 group (50nmol / L) and the ZG2 group (50nmol / L) could reduce the formation of lipid droplets, indicating that ZG1 / 2 has significant lipid-lowering ability in vitro.

[0179] Example 4: Hypoglycemic efficacy test of peptide compounds in mice

[0180] 1. Experimental Materials

[0181] 12-week-old wild-type C57 female mice.

[0182] 2. Experimental methods and results

[0183] The following is an in vivo hypoglycemic test using compounds ZG1 and ZG2 as examples, as follows:

[0184] Twelve-week-old C57 mice were divided into the vehicle group, the GGG0 group (15 nmol / kg), the ZG1 group (15 nmol / kg), and the ZG2 group (15 nmol / kg). Each peptide compound was subcutaneously injected once, followed by a subcutaneous injection of 2 g / kg glucose 12 hours later. Blood glucose levels were measured at the tail tip at 0, 15, 30, 60, and 120 minutes. N = 3. Results are shown in Figure 2. Figure 3 Compared with the vehicle group, the GGG0 group (15 nmol / kg), the ZG1 group (15 nmol / kg), and the ZG2 group (15 nmol / kg) all lowered blood glucose. The glucose-lowering ability of ZG1 / 2 in vivo was comparable to that of GGG0.

[0185] Example 5: Lipid-lowering efficacy test of peptide compounds in mice

[0186] 1. Experimental Materials

[0187] The animals were 16-week-old wild-type C57 female mice.

[0188] 2. Experimental methods and results

[0189] The following is an in vivo lipid-lowering test using compounds ZG1 and ZG2 as examples, as follows:

[0190] Establish high-fat induced obese mice. 16-week-old C57 female mice were divided into high-fat model combined with normal diet groups, and fed with high-fat feed and normal feed respectively for 8 consecutive weeks to establish high-fat model mice. Then, 24-week-old C57 female high-fat model mice were divided into Vehicle group, GGG0 group (15nmol / kg), ZG1 group (15nmol / kg) and ZG2 group (15nmol / kg). Each group received a subcutaneous injection of the peptide compound once every 3 days, and the body weight was measured every 3 days, N=3. The results are shown in Figure 3. Figure 4 Compared with the Vehicle group, the GGG0 group (15nmol / kg), the ZG1 group (15nmol / kg), and the ZG2 group (15nmol / kg) could all reduce the body weight of the model mice.

[0191] Example 6 Safety Evaluation of Peptide Compounds in Mice

[0192] 1. Experimental Materials

[0193] All materials and reagents used in this evaluation were purchased from commercial products. Wild-type C57 female mice were 12 weeks old.

[0194] 2. Experimental methods and results

[0195] The following is an example of compound ZG1 and ZG2 for safety evaluation in mice, as follows:

[0196] Twelve-week-old wild-type C57 female mice were divided into the Vehicle group, the GGG0 group (15 nmol / kg), the ZG1 group (15 nmol / kg), and the ZG2 group (15 nmol / kg). Each group received a subcutaneous injection of the peptide compound at 15 nmol / kg every three days. 21 days after the injection, tail tip blood was collected for serum creatine kinase (CK) and alanine aminotransferase (ALT) testing. N = 3. The results are shown in Figure 2. Figure 5 and 6 Compared with the Vehicle group, there was no significant difference in the levels of creatine kinase (CK) and alanine aminotransferase (ALT) in the serum of the GGG0 group (15nmol / kg), the ZG1 group (15nmol / kg) and the ZG2 group (15nmol / kg), indicating that ZG1 / 2 is safe in mice in vivo.

[0197] Example 7 Evaluation of Serum Stability of Peptide Compounds

[0198] 1. Experimental Materials

[0199] All materials and reagents used in this evaluation were purchased from commercial products. Wild-type C57 female mice were 12 weeks old.

[0200] 2. Experimental methods and results

[0201] The following is an example of compound ZG1 and ZG2 to evaluate the stability of mouse serum.

[0202] Six 12-week-old wild-type C57 female mice were enucleated and whole blood was collected. The blood was centrifuged at 1500 rpm for 10 minutes and the upper serum was collected. The mice were divided into 0-min, 15-min, 30-min, 45-min, and 60-min groups. 2.2 mmol / L of the peptide compound was added to each group at once, and after thorough mixing, samples were collected at the same time points as above. The content of the peptide compound was determined by HPLC. N = 3. Figure 7Compared with the 0-min group, the detection value of GGG0 decreased by 45.1% after 60-min incubation in serum, while the detection value of ZG1 decreased by 12.4% after 60-min incubation in serum, and the detection value of ZG2 decreased by 11.5% after 60-min incubation in serum, indicating that ZG1 / 2 has good stability in serum.

[0203] Example 8 EC50 test of peptide compounds

[0204] 1. Experimental Materials

[0205] The materials and reagents used in this test were purchased from commercial products.

[0206] 2. Experimental methods

[0207] The following is an example of compound ZG1, ZG2, ZG3 and ZG4 to conduct cell EC50 test, as follows:

[0208] 1) Construction of cell model

[0209] HEK293 cells that highly express human GLP1R, GIPR and GCGR were constructed (by transfecting human GLP1R, GIPR and GCGR).

[0210] The specific steps are as follows:

[0211] A. Construction of plasmids overexpressing human GLP-1, GIP, and GCG receptors;

[0212] B. Transfection was performed using Lip3000 transfection reagent to obtain target cells that highly expressed GLP-1, GIP, and GCG receptors;

[0213] C. Perform cell verification and detect whether the transfection is successful by RT-qPCR experiment;

[0214] D. Extract total RNA from cells before and after transfection, perform RT-qPCR reaction, and analyze the results;

[0215] E. After successful cell transfection, drug (peptide compound) testing is performed.

[0216] 2) Peptide compound testing

[0217] A. HEK293 cell culture: HEK293 cells were cultured in DMEM + 10% FBS + 1% penicillin + 1% streptomycin at 37°C and 5% CO2.

[0218] B. When the cells are in the logarithmic growth phase, remove the culture medium and wash the cells with PBS without phosphorus and magnesium;

[0219] C. Protease treatment with Accutase;

[0220] D. Wash the separated cells and resuspend them in assay buffer to measure the cell density;

[0221] E. Cell suspension was adjusted to 4×10 5 Cells / ml were seeded in 96-well plates;

[0222] F, adding test peptide chemicals;

[0223] G. Incubate at room temperature for 30 minutes;

[0224] H. Use cAMP Gs kit for determination;

[0225] 1. Add lysis buffer, dilute HTRF reagent, and incubate for 1 hour;

[0226] J, measured at 665 / 620 nm;

[0227] K. Use the ELISA method to draw a standard curve and then calculate the EC50 value of the peptide.

[0228] 3. Experimental results

[0229] The experimental results are shown in Table 2. Regarding the agonist effects on the three targets of GLP1R, GIPR, and GCGR, the EC50 values of ZG1, ZG2, ZG3, and ZG4 were all better than those of GGG0.

[0230] Table 2 EC50 values of tested peptide compounds

[0231] GLP1R (nM) GIPR (nM) GCGR (nM) Testing GGG0 0.95±0.05 0.13±0.02 8.01±0.53 Testing ZG1 0.85±0.07 0.10±0.02 6.81±0.51 Testing ZG2 0.82±0.69 0.08±0.02 6.75±0.33 Testing ZG3 0.87±0.05 0.06±0.02 5.88±0.34 Testing ZG4 0.77±0.07 0.06±0.02 5.59±0.39

[0232] Example 9 PK test of peptide compounds

[0233] 1. Experimental Materials

[0234] The materials and reagents used in this test were purchased from commercial products.

[0235] 2. Experimental methods

[0236] The following is an in vivo PK test in rats using compounds ZG1, ZG2, ZG3 and ZG4 as examples, as follows.

[0237] The animals were randomly divided into two groups using a computer system, with 3 animals in each group, all female. The dosage and group information are shown in Table 3:

[0238] Table 3

[0239]

[0240] The specific contents are as follows:

[0241] 1) Route of administration: intravenous injection;

[0242] 2) Dosage frequency and duration: single dose;

[0243] 3) Administration: The test article should be administered via the tail vein of the animal at the appropriate dose, calculated based on the most recently obtained body weight. The injection should be completed within 1 minute.

[0244] 4) Blood sample collection: Whole blood (approximately 0.4 mL) was collected from the jugular vein of the animals for pharmacokinetic analysis;

[0245] 5) Sampling time: The blood samples of all animals were collected before each administration and at 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h after the end of administration;

[0246] 6) Sample Processing: Coat centrifuge tubes with K2EDTA, add 4 μL of aprotinin aqueous solution (100 μg / mL) and 4 μL of 100 mM PMSF ethanol solution to each tube, and store on ice. Centrifuge at 3000 g for 10 min at 4°C within 1 hour. Remove 160 μL of plasma from the centrifuged tubes and transfer them to new, labeled centrifuge tubes (pre-added with 2 μL of DMSO) and store below -60°C. After sample collection, Watson LIMS 7.5 was used for sample reception and management.

[0247] 7) Biological sample analysis: The feasibility-verified LC-MS / MS method was used to determine the content of Sell lipidated polypeptides in plasma samples. The analysis method is briefly described as follows: Verapamil was used as the internal standard, and the protein precipitation method was used in the treatment process. The specific method is as follows: 1) Take 50 μL of sample, add 5 μL of phosphoric acid-water solution (10:90, v / v), 5 μL of internal standard working solution (0.5 ng / mL) and 150 μL of acetonitrile, vortex to mix, and centrifuge for 10 minutes (4000g, 2-8°C). 2) Take all the supernatant and filter it through a phospholipid removal plate. The filtrate was diluted 2 times with ultrapure water and then injected for analysis. Use ACQUITY UPLC Protein BEH C4 ( Chromatographic separation was achieved using a 2.1 × 50 mm, 1.7 μm column, with a run time of 5 minutes per injection. The mobile phase consisted of water-formic acid (1000:2), and the organic phase consisted of acetonitrile-methanol-formic acid (800:200:2). The peptide linear range was 50–15,000 ng / mL, with a limit of quantification of 50 ng / mL.

[0248] The pharmacokinetic parameters of the drug-treated group were calculated using the non-compartmental model (NCA) method using WinNonlin 8.0 software, including AUC (area under the plasma concentration-time curve), C0, t1 / 2 (elimination half-life), Vd (apparent volume of distribution), Cl (clearance), and MRT (mean residence time).

[0249] 8) Data processing and statistical analysis: Microsoft EXCEL was used to describe the experimental data using mean (Mean), standard deviation (SD), and coefficient of variation (CV%).

[0250] 3. Experimental results

[0251] The results of metabolic kinetic parameters are shown in Table 4. The results show that the in vivo PK test results of ZG1, ZG2, ZG3 and ZG4 in rats, such as T1 / 2 (elimination half-life), Vd (apparent volume of distribution), Cl (clearance rate), and MRT (mean residence time), all performed excellently.

[0252] Table 4 Metabolic kinetic results

[0253] Group Dose (mg / kg) T1 / 2(h) Vd (mL / kg) Cl (mL / hr / kg) MRT0-48h(h) ZG1 12 9.56 137 6.8 10.2 ZG2 12 10.02 142 6.6 11.1 ZG3 12 9.98 133 6.5 10.4 ZG5 12 9.43 129 6.4 10.6

[0254] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A GLP1R-GIPR-GCGR triple-target agonist peptide or a pharmaceutically acceptable salt thereof, characterized in that: The agonist peptide or a pharmaceutically acceptable salt thereof is selected from: H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPSKVSRA-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPSKVSRA-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGAPPPS-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGAPPPS-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPSKVSRA-NH2; H-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPSKVSRA-NH2; Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-DFVEYLLE-GGPSSGA d PPPS-NH2: Y-Aib-QGTFTSDYSI-αMeL-LDK-K(AEEA-γGlu-CO(CH2) 18 COOH)-AQ-Aib-AFIEYLLE-GGGPSSGA d PPPS-NH2。 2. A solid phase synthesis method for preparing the agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The method comprises the following steps: 1) Resin pretreatment: Add the resin to the reactor, add DMF, stir and swell under nitrogen, filter and wash; 2) Prepare amino acid solution: Dissolve Fmoc-Ser-tBu-OH and HOBt in DMF, then add DIC and mix thoroughly. 3) Deprotection: Add 20% piperidine / DMF solvent to the reaction kettle, stir and wash with nitrogen; 4) Coupling reaction: Add the amino acid solution prepared in step 2) to the reactor, control the temperature and stir with nitrogen. After the reaction is complete, wash the resin with DMF and repeat step 4). The amino acids of the agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1 are sequentially coupled with corresponding protected amino acids until the main chain of the agonist peptide is synthesized; 5) Removal of the 17-position Lys side chain protecting group iLEde: Add 8% hydrazine hydrate / DMF solution to the reaction kettle, stir under nitrogen, and after the reaction is complete, filter to remove the solvent and wash; 6) Lys side chain modification: Eicosanedioic acid (mon-tBu) -γGlu (α-OtBu) -AEEA-OH and HOBt were dissolved in DMF, and DIC was added and mixed to prepare a side chain modification solution. The side chain modification solution was added to a reaction kettle, and the temperature was controlled and nitrogen was stirred. After the reaction was completed, the mixture was washed with DMF, then with DCM, and then with methyl tert-butyl ether to obtain a peptide resin containing the agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1, and then dried.

3. The method according to claim 2, characterized in that The swelling time in step 1) is 30 min.

4. The method according to claim 2, characterized in that In the step 2), the dissolution ratio of Fmoc-Ser-tBu-OH in DMF is 5.75 g / 75 mL.

5. The method according to claim 2, characterized in that In the step 2), the solubility ratio of HOBt in DMF is 2.02 g / 75 mL.

6. The method according to claim 2, characterized in that In the step 2), the dissolution ratio of DIC in DMF is 1.89 g / 75 mL.

7. The method according to claim 2, characterized in that The ratio of the amount of 20% piperidine / DMF solvent added in step 3) to the amount of resin added in step 1) is 100 mL / 13.15 g.

8. The method according to claim 2, characterized in that The stirring time during nitrogen flow in step 3) is 35 min.

9. The method according to claim 2, characterized in that After washing in step 3), the resin is tested using a ninhydrin reagent to determine whether it is positive.

10. The method according to claim 2, characterized in that The ratio of the amino acid solution added in step 4) to the resin added in step 1) is 100 mL / 13.15 g.

11. The method according to claim 2, characterized in that The temperature range of the temperature-controlled nitrogen stirring in step 4) is 25-35°C.

12. The method according to claim 2, characterized in that The completion of the reaction in step 4) is indicated by the resin showing a negative result when tested with a ninhydrin reagent.

13. The method according to claim 2, characterized in that The ratio of the 8% hydrazine hydrate / DMF solution in step 5) to the resin added in step 1) is 100 mL / 13.15 g.

14. The method according to claim 2, characterized in that The stirring time during nitrogen flow in step 5) is 1 h.

15. The method according to claim 2, characterized in that After washing in step 5), the resin is tested using a ninhydrin reagent to determine whether it is positive.

16. The method according to claim 2, characterized in that In the step 6), the dissolution ratio of Eicosanedioic acid(mon-tBu)-γGlu(α-OtBu)-AEEA-OH in DMF is 7.29 g / 150 mL.

17. The method according to claim 2, characterized in that In step 6), the dissolution ratio of HOBt in DMF is 1.35 g / 150 mL.

18. The method according to claim 2, characterized in that In step 6), the dissolution ratio of DIC in DMF is 1.30 g / 150 mL.

19. The method according to claim 2, characterized in that The ratio of the added amount of the side chain modification solution in step 6) to the added amount of the resin in step 1) is 150 mL / 13.15 g.

20. The method according to claim 2, characterized in that The temperature range of the temperature-controlled nitrogen stirring in step 6) is 25-35°C.

21. The method according to claim 2, characterized in that The completion of the reaction in step 6) is indicated by the resin showing a negative result when tested with a ninhydrin reagent.

22. A post-treatment method for the agonist peptide or pharmaceutically acceptable salt thereof after solid phase synthesis according to any one of claims 2 to 21, characterized in that: The post-processing method comprises the following steps: 1) adding a cutting solution to a peptide resin containing the agonist peptide or a pharmaceutically acceptable salt thereof as claimed in claim 1, which is synthesized by the method of any one of claims 2 to 21, stirring the reaction, filtering, adding the filtrate to ice-cold methyl tert-butyl ether to precipitate, filtering with suction, washing the filter cake with methyl tert-butyl ether, and vacuum drying to obtain a crude product; 2) The crude product obtained in step 1) is passed through a refining and purification system to obtain a purified product.

23. The post-processing method according to claim 22, characterized in that: The volume ratio of the cutting solution in step 1) is TFA / TIS / DTT / H2O=90 / 2.5 / 2.5 / 5.

24. The post-processing method according to claim 22, characterized in that: The stirring reaction time in step 1) is 3 hours.

25. The post-processing method according to claim 22, characterized in that: In step 1), the volume ratio of the filtrate to the glacial methyl tert-butyl ether is 1:

5.

26. The post-processing method according to claim 22, characterized in that: The refining and purification system in step 2) is a C18 reverse phase preparative chromatography system.

27. The post-processing method according to claim 22, characterized in that: The purity of the purified product in step 2) is not less than 90%.

28. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a therapeutically effective amount or a preventive effective amount of the agonist peptide or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition according to claim 28, characterized in that The pharmaceutical composition is an injection, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension, or syrup.

30. The pharmaceutical composition according to claim 28, characterized in that The pharmaceutical composition is administered orally, by inhalation or parenterally.

31. The pharmaceutical composition according to claim 30, characterized in that The parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration.

32. The pharmaceutical composition according to claim 28, characterized in that The therapeutically effective amount or preventively effective amount needs to be administered at a frequency of at least once a week or once every two weeks.

33. Use of the agonist peptide according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 28 to 32, in the preparation of a drug for promoting insulin secretion and lowering blood sugar.

34. Use of the agonist peptide or pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to any one of claims 28 to 32 in the preparation of a drug for inhibiting food intake, delaying gastric emptying, increasing energy expenditure, and reducing body weight.

35. Use of the agonist peptide according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 28 to 32, in the preparation of a medicament for preventing and / or treating metabolic disorders; The metabolic disorder is diabetes, obesity, non-alcoholic fatty liver disease or dyslipidemia.

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