gIP AND GLP-1 DUAL AGONIST POLYPEPTIDE COMPOUNDS, PHARMACEUTICALLY ACCEPTABLE SALTS, AND USES - Patent application

Abstract

The GIP and GLP-1 dual agonist polypeptide compound belongs to the technical field of polypeptide pharmaceutical chemistry. The amino acid sequence of this GIP and GLP-1 dual agonist polypeptide compound is YXaa 1EGTFTSDYSIXaa 2LDKIAQXaa 3AFVQWLIAGGPSSGAPPPS, and the C-terminal amino acid of this polypeptide compound is amidated as a C-terminal primary amide. Also provided are a method for producing this polypeptide compound, pharmaceutically acceptable salts, pharmaceutical compositions, and pharmaceuticals. The polypeptide compound has the effects of lowering blood sugar and reducing weight, and can be used as an active ingredient in the manufacture of pharmaceuticals for treating or preventing diabetes or weight loss drugs.

Description

[Technical field]

[0001] The present invention relates to the field of polypeptide compounds, specifically to a polypeptide compound that is a dual incretin peptide analogue, which activates the receptors of human glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), and can be applied to the treatment of type 2 diabetes and obesity. The present invention also relates to a pharma- ceutically acceptable salt of the polypeptide compound, a polypeptide pharmaceutical composition, a pharmaceutical product, a method for preparation and its use. [Background technology]

[0002] The pathogenesis of metabolic syndrome is abnormal metabolism of various substances, such as proteins, fats, and carbohydrates. Overnutrition and decreased physical activity can lead to obesity and obesity-related diseases, such as diabetes. In recent years, the incidence of type 2 diabetes and dyslipidemia has increased. Type 2 diabetes is the most common form of diabetes, accounting for approximately 90% of all diabetes cases. Type 2 diabetes is characterized by high blood glucose levels caused by insulin resistance. The current standard treatment for type 2 diabetes includes diet, exercise, and treatment with oral and injectable hypoglycemic drugs. However, many patients with type 2 diabetes still have poorly controlled blood glucose levels.

[0003] GIP is a 42 amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose balance by stimulating pancreatic β cells to secrete insulin and protecting pancreatic β cells in the presence of glucose. GLP-1 is a 37 amino acid peptide that stimulates insulin secretion, protects pancreatic β cells, and reduces weight by inhibiting glucagon secretion, gastric emptying, and food intake. GIP and gLP-1 are called incretins. Incretin receptors signaling plays an important role in glucose balance. In normal physiology, GIP and GLP-1 are secreted from the intestine after a meal, and these incretins enhance physiological responses to food such as satiety, insulin secretion, and nutrient processing. The incretin response is reduced in type 2 diabetes patients. Currently available incretin analogs or dipeptidyl peptidase IV (DPP-IV) inhibitors control blood glucose by only a single mechanism of action. By using type 2 diabetes compounds with dual mechanisms of action, polypeptide compounds with superior hypoglycemic activity and weight loss effects can be obtained.

[0004] The administration of GLP-1 analogues is limited by side effects such as nausea and vomiting, and administration is often ineffective in improving glycemic control and weight loss. GIP alone has a very modest hypoglycemic potential in type 2 diabetes patients. Both native GIP and GLP-1 can only be used for short-term metabolic control, as they are rapidly inactivated by the ubiquitous protease DPP IV. DPP IV belongs to the epipeptidase class of protein hydrolases. The proteolytic stability of any peptide can be increased by introducing unnatural amino acids into the sequence. Thus, the use of unnatural amino acids can improve the stability of peptides against DPP IV protein degradation and other forms of degradation.

[0005] As part of the present invention, unnatural amino acids have a positive effect on the balance of GIP and GLP-1 agonist activity. For example, fatty acids can improve the pharmacokinetics of peptides by extending half-life through albumin binding motifs. Although fatty acids can be used to improve the half-life of peptides, applicants have unexpectedly discovered that the length, composition, and position of the fatty acid chain and the linker between the peptide and the fatty acid chain can affect the balance of GIP and GLP-1 agonist activity and extend the half-life of the peptide as part of the present invention. Several GIP analogs have shown GIP and GLP-1 activity in patent documents such as WO2013 / 164483, WO2014 / 192284, and WO2011 / 119657. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a GIP and GLP-1 dual agonist polypeptide compound that utilizes a dual mechanism of action and has hypoglycemic activity and weight loss effect.

[0007] Another object of the present invention is to provide a method for preparing said GIP and GLP-1 dual agonist polypeptide compound.

[0008] Another object of the present invention is to provide pharma- ceutically acceptable salts of said GIP and GLP-1 dual agonist polypeptide compounds.

[0009] Another object of the present invention is to provide a pharmaceutical composition of said GIP and GLP-1 dual agonist polypeptide compound.

[0010] Another object of the present invention is to provide a pharmaceutical preparation of said GIP and GLP-1 dual agonist polypeptide compound.

[0011] Another object of the present invention is to provide uses of said GIP and GLP-1 dual agonist polypeptide compounds, their pharma- ceutically acceptable salts, their pharmaceutical compositions and pharmaceutical preparations. [Means for solving the problem]

[0012] The object of the present invention is achieved by the following technical means. The present invention discloses a GIP and GLP-1 dual agonist polypeptide compound, the amino acid sequence of which is: Y Xaa 1 EGTFTSDYSI Xaa 2 LDKIAQ Xaa 3 AFVQWLIAGGPSSGAPPPS, The C-terminal amino acid is amidated as a C-terminal primary amide, Xaa 1 teeth, [ka] is selected from Xaa 2 teeth, [ka] is selected from Xaa 1 and Xaa 2 The two may be the same or different. Xaa 3 teeth [ka] is selected from n is a natural number between 5 and 25.

[0013] In the polypeptide pharmaceutical composition according to the present invention, n is a natural number of 16 or 18.

[0014] In the polypeptide pharmaceutical composition according to the invention, the polypeptide amino acid sequence is preferably (1) SEQ ID NO:1 YVEGTFTSDYSIVLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (2) SEQ ID NO:2 YXEGTFTSDYSIXLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (3) SEQ ID NO:3 YX1EGTFTSDYSIX1LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (4) SEQ ID NO:4 YAEGTFTSDYSIALDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (5) SEQ ID NO:5 YAEGTFTSDYSIAibLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (6) SEQ ID NO:6 YAibEGTFTSDYSIALDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (7) SEQ ID NO:7 YXEGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSS GAPPPS-NH2 (8) SEQ ID NO:8 YXEGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16-CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (9)SEQ ID NO:9 YX1EGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (10)SEQ ID NO:10 YX2EGTFTSDYSIX1LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 (11)SEQ ID NO:11 YAibEGTFTSDYSIAibLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (12)SEQ ID NO:12 YVEGTFTSDYSIVLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (13)SEQ ID NO:13 YXEGTFTSDYSIXLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (14)SEQ ID NO:14 YX1EGTFTSDYSIX1LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (15)SEQ ID NO:15 YAEGTFTSDYSIALDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (16)SEQ ID NO:16 YAEGTFTSDYSIAibLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (17)SEQ ID NO:17 YAibEGTFTSDYSIALDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (18)SEQ ID NO:18 YXEGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (19)SEQ ID NO:19 YX2EGTFTSDYSIXLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (20) SEQ ID NO: 20 YX1EGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 (21)SEQ ID NO:21 YX2EGTFTSDYSIX1LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 and During the ceremony, [ka] It is.

[0015] The present invention further discloses a method for producing a GIP and GLP-1 dual agonist polypeptide compound, comprising the following steps (1) and (2): (1) After the resin is taken and activated, amino acids are linked one by one to obtain a peptide resin. (2) The peptide resin is taken, side chains are coupled, cleaved, and purified to give a side-chain protected polypeptide.

[0016] The present invention further discloses a pharma- ceutically acceptable salt of a GIP and GLP-1 dual agonist polypeptide compound, which is any of the above-mentioned polypeptide compounds, and the C-terminal amino acid of the polypeptide compound is amidated as a C-terminal primary amide or a pharma- ceutically acceptable salt thereof.

[0017] In the pharma- ceutically acceptable salts of the GIP and GLP-1 dual agonist polypeptide compounds according to the present invention, the salts preferably comprise A GIP and GLP-1 dual agonist polypeptide compound, Hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectic acid, persulfuric acid, 3-phenylpropionic acid acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, fatty acids, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfate, and thiocyanate; It is a salt formed from

[0018] The present invention further discloses a pharmaceutical composition of GIP and GLP-1 dual agonist polypeptide compound. The pharmaceutical composition comprises any of the above-mentioned GIP and GLP-1 dual agonist polypeptide compounds as an active ingredient, or any of the above-mentioned GIP and GLP-1 dual agonist polypeptide compounds as an active ingredient, and further comprises a pharma- ceutically acceptable carrier or diluent.

[0019] The pharma- ceutically acceptable salts of the GIP and GLP-1 dual agonist polypeptide compounds according to the present invention are prepared from the GIP and GLP-1 dual agonist polypeptide compounds and the above-mentioned compounds according to conventional methods disclosed in the prior art.

[0020] The present invention further discloses a pharmaceutical product prepared by using any of the above-mentioned GIP and GLP-1 dual agonist polypeptide compounds.The pharmaceutical product is a tablet, capsule, elixir, syrup, lozenge, inhalant, spray, injection, membrane, patch, powder, granule, block, emulsion, suppository and combination.The pharmaceutical product is composed of a GIP and GLP-1 dual agonist polypeptide compound and a pharmaceutical acceptable pharmaceutical excipient, carrier or diluent.The pharmaceutical product according to the present invention can be prepared according to the conventional method in the prior art.

[0021] The use of the GIP and GLP-1 dual agonist polypeptide compound according to the present invention can be used as an active ingredient in the manufacture of medicines for treating or preventing diabetes or weight loss drugs.

[0022] A pharma- ceutically acceptable use of the GIP and GLP-1 dual agonist polypeptide according to the present invention is as an active ingredient in the manufacture of a medicament for treating or preventing diabetes or in the manufacture of a weight loss drug.

[0023] The pharmaceutical composition and medicine of the GIP and GLP-1 dual agonist polypeptide compound according to the present invention can be applied as a medicine for treating or preventing diabetes or as a weight loss drug.

[0024] The abbreviations used herein are listed below. <Abbreviation> <Meaning> DCM Dichloromethane DMF N-Methylpyrrolidone HBTU Benzotriazole-N,N,N',N'-tetramethyl benzotriazole Fluoronium Hexafluorophosphate HOBt 1-Hydroxybenzotriazole DIEA / DIPEA N,N'-Diisopropylethylamine Fmoc N-9-fluorenylmethyloxycarbonyl EDT Ethanedithiol HPLC High Performance Liquid Chromatography TFA Trifluoroacetic acid tBu tert-butyl DMSO Dimethyl sulfoxide Alloc allyloxycarbonyl Effect of the Invention

[0025] Compared with conventional polypeptide compounds and production techniques, the present invention has the following beneficial effects: The present invention discloses a novel GIP and GLP-1 dual agonist polypeptide compound. The polypeptide compound of the present invention has the effects of lowering blood sugar and reducing body weight, and can be used as an active ingredient in the manufacture of medicines or weight loss drugs for treating or preventing diabetes. According to animal energy consumption data, the polypeptide compound of the present invention has the effect of reducing the body weight of patients, and also has balanced co-agonist activity against GIP and GLP-1 receptors, selectivity against glucagon and GLP-2 receptors, low immunogenicity characteristics, and pharmacokinetic (PK) characteristics that allow it to be administered once a week, and can be used as an active ingredient in drugs for treating diabetes and obesity.

[0026] The GIP and GLP-1 dual agonist polypeptide compound produced by the production method of the present invention has high blood sugar lowering and weight gain suppressing activity, long duration of effect, high yield, short synthesis cycle, easy purification of the crude product, low production cost, and easy industrial automation production. [Brief description of the drawings]

[0027] [Figure 1] This figure shows the effect of a single administration of each test drug in the experiment on random blood glucose levels and AUC0-24hglu in ob / ob mice (▲X▼±s, n=7) (▲X▼ refers to the letter X with a - above it, the same applies below). [Diagram 2] FIG. 1 shows the effect of a single administration of each test drug in the experiment on random blood glucose levels and AUC0-24hglu in ob / ob mice (▲X▼±s, n=7). [Diagram 3] This is a diagram showing the effect of a single dose of each test drug on AUC0-24hglu in ob / ob mice (▲X▼±s, n=7). The data in the diagram are the rate of decrease in AUC0-24hglu.

[0028] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will be further described below with reference to examples, but these examples are not intended to limit the present invention.

[0030] Example 1: Synthesis of a polypeptide compound of SEQ ID NO: 1 YVEGTFTSDYSIVLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 1. Synthesis of peptide chain 1.1 Resin swelling 10 g of Fmoc-Rink Amide AM Resin (substitution degree 0.35 mmol / g) was weighed out and swollen in 100 mL of DCM for 30 min, then suction filtered to remove the DCM, swollen in 100 mL of DMF for 30 min, and washed with 100 mL of DMF and DCM, respectively.

[0031] 1.2 Synthesis of Fmoc-Ser(tBu)-Rink Amide AM Resin Fmoc-Ser(tBu)-OH (8 mmol), HOBT (16 mmol) and DIC (16 mmol) were dissolved in 100 mL of DMF, and the resulting solution was added to the resin obtained in the previous step and reacted for 2 hours. After the reaction was completed, the reaction solution was removed by filtration, and the resin was washed three times with 100 mL of DCM and 100 mL of DMF, respectively.

[0032] 1.3 Removal of Fmoc protecting group To the washed resin was added a 25% piperidine / DMF (V / V) solution containing 0.1 M HOBt to remove Fmoc, and after the reaction was completed, the resin was washed three times with 100 mL of DCM and 100 mL of DMF.

[0033] 1.4 Peptide chain elongation According to the sequence, the above deprotection and coupling steps were repeated to attach the corresponding amino acids in order until the synthesis of the peptide chain was completed and the peptide resin was obtained. Here, the 20th position K can adopt a protection strategy of Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(iVdde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(Boc)-OH. In this example, the protection strategy of Fmoc-Lys(Alloc)-OH was adopted.

[0034] 1.5 Side chain attachment The resulting peptide resin was placed in a reaction flask and the Alloc protecting group was removed using Pd(PPh3)4 with PhSiH3 as a scavenger. The Lys20 side chain was synthesized by Fmoc / t-Bu strategy or the Oct(OtBu)-γ-Glu(OtBu)-AEEA-AEEA-Osu fragment was directly coupled to obtain the fully protected peptide resin.

[0035] 1.6 Decomposition of peptide resin Trifluoroacetic acid was weighed and placed in a reactor, cooled to -10 to 0°C, triisopropylsilane, 1,2-ethanedithiol and purified water were added, and the mixture was stirred to mix uniformly. Peptide resin was slowly added, the temperature was raised to 20 to 30°C, and the cleavage reaction was carried out for 115 to 125 min. After the reaction was completed, the resin was removed by filtration, and the removed resin was washed with M×8×20% ml of TFA, and all the filtrate and washings were transferred to M×8×1.2×4 ml of ether, stirred for 5 to 10 min, and allowed to settle for 15 min or more. The precipitated suspension was placed in a centrifuge, centrifuged, and the solid was collected. The solid was washed with ether six times (5 L or more each time). The solid was vacuum-dried at 20 to 35°C for 6 to 10 h to obtain a crude peptide.

[0036] 1.7 Purification of crude peptides The product was purified by preparative liquid chromatography. The chromatography conditions were: C18 column (100 mm x 250 mm, 10 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 20% to 60%, 60 min; flow rate: 200 mL / min, detection wavelength: 214 nm. Fractions with a purity of over 98.0% were collected, rotary evaporated, and then freeze-dried to obtain a 1.36 g sample.

[0037] Example 2: Synthesis of SEQ ID NO:2 Polypeptide Compound YXEGTFTSDYSIXLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.45 g of purified product.

[0038] Example 3: Synthesis of SEQ ID NO:3 Polypeptide Compound YXEGTFTSDYSIXLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.51 g of purified product.

[0039] Example 4: Synthesis of SEQ ID NO:4 Polypeptide Compound YAEGTFTSDYSIALDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.18 g of purified product.

[0040] Example 5: Synthesis of SEQ ID NO:5 Polypeptide Compound YAEGTFTSDYSIAibLDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.22 g of purified product.

[0041] Example 6: Synthesis of SEQ ID NO:6 Polypeptide Compound YAibEGTFTSDYSIALDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.97 g of the purified product.

[0042] Example 7: Synthesis of SEQ ID NO: 7 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 [ka] X2=Aib The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.55 g of purified product.

[0043] Example 8: Synthesis of SEQ ID NO:8 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 X1=Aib [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.01 g of purified product.

[0044] Example 9: Synthesis of SEQ ID NO:9 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(HOOC-(CH2) 16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 [ka] X2=Aib The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.64 g of the purified product.

[0045] Example 10: Synthesis of SEQ ID NO:10 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(HOOC-(CH2)16 -CO-γ-Glu-AEEA-AEEA)AFVQWLIAGGPSSGAPPPS-NH2 X1=Aib [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.81 g of purified product.

[0046] Example 11: Synthesis of SEQ ID NO:11 Polypeptide Compound YAibEGTFTSDYSIAibLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.84 g of the purified product.

[0047] Example 12: Synthesis of SEQ ID NO:12 Polypeptide Compound YVEGTFTSDYSIVLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.12 g of purified product.

[0048] Example 13: Synthesis of SEQ ID NO:13 Polypeptide Compound YXEGTFTSDYSIXLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.07 g of purified product.

[0049] Example 14: Synthesis of SEQ ID NO:14 Polypeptide Compound YXEGTFTSDYSIXLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.89 g of purified product.

[0050] Example 15: Synthesis of SEQ ID NO:15 Polypeptide Compound YAEGTFTSDYSIALDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.81 g of purified product.

[0051] Example 16: Synthesis of SEQ ID NO:16 Polypeptide Compound YAEGTFTSDYSIAibLDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.71 g of purified product.

[0052] Example 17: Synthesis of SEQ ID NO:17 Polypeptide Compound YAibEGTFTSDYSIALDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.53 g of the purified product.

[0053] Example 18: Synthesis of SEQ ID NO:18 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 [ka] X2=Aib The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.25 g of purified product.

[0054] Example 19: Synthesis of SEQ ID NO:19 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 X1=Aib [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 0.93 g of purified product.

[0055] Example 20: Synthesis of SEQ ID NO:20 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 [ka] X2=Aib The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 0.96 g of purified product.

[0056] Example 21: Synthesis of SEQ ID NO:15 Polypeptide Compound YX1EGTFTSDYSIX2LDKIAQK(γ-Glu-Palmitoyl)AFVQWLIAGGPSSGAPPPS-NH2 X1=Aib [ka] The synthesis method was the same as in Example 1. The collected solution was freeze-dried to obtain 1.47 g of purified product.

[0057] Example 22 The pharmacological experimental methods and results regarding the GIP and GLP-1 dual agonist polypeptide compound (abbreviated as "polypeptide compound") are shown below. 1. GLP-1 and GIP receptor agonist activity of polypeptide compounds HEK293 cells were co-transfected with cDNA encoding GLP-1R or GIPR. In the compound measurement test, the cells were seeded in a 96-well plate 2 hours before the test, and the polypeptide compounds were dissolved in DMSO, diluted in different folds in medium containing 0.1% bovine serum albumin, and added to the co-transfected cells. After incubating the cells for 20 min, the fluorescence intensity was measured using a microplate reader with a Cisbo ELISA kit, a standard curve was generated, and the fluorescence intensity was converted into the corresponding cAMP value, and the EC values ​​of the compounds were calculated by nonlinear regression using Graphpad Prism 7.0 software. 50 The numbers were calculated.

[0058] [Table 1] Peptides Semaglutide

[0059] As shown in Table 1, all of the polypeptide compounds retained high agonistic activity at the GLP-1R and reasonably high agonistic activity at the GIPR, and the modified unnatural amino acids and side chain groups did not significantly affect the agonistic activity.

[0060] 2. Blood sugar lowering experiment using polypeptide compounds After weaning, male ob / ob mice were raised in one cage and fed a high-fat diet. After 6-7 weeks of age, blood glucose levels were measured, and the mice were divided into a model control group, a polypeptide compound group (nine compounds were selected), a positive control liraglutide group, and a positive control semaglutide group according to random body weight, random blood glucose levels, and fasting blood glucose levels. Each test drug group and positive control group mice were subcutaneously injected with different doses of each test drug or positive drug liraglutide and semaglutide, and the model control group mice were subcutaneously injected with PBS buffer solution once. The random blood glucose levels of the mice in each group were measured before administration (0 hours) and 2, 4, 6, 10, and 24 hours after administration, and then the measurement time of the random blood glucose levels was adjusted according to the blood glucose status of the mice in each group and extended to 34, 48, 58, or 72 hours after administration. In addition, the random body weights of the mice in each group were measured before administration (0 hours) and 24 hours after administration, and the random body weights of the mice 48 or 72 hours after administration corresponding to the random blood glucose level measurement were measured. The blood glucose reduction rate and the area under the blood glucose curve within 24 hours of administration (AUC 0-24hglu ) and the rate of decline were calculated.

[0061] Blood glucose reduction rate = (blood glucose level of model control group - blood glucose level of treatment group) / blood glucose level of model control group x 100% AUC 0-24hglu (mmol / Lh·r)=(BG0+BG2)+(BG2+BG4)+(BG4+BG6)+(BG6+BG10)×2+(BG10+BG24)×7 BG0, BG2, BG4, BG6, BG10 and BG24 indicate blood glucose levels before administration (0 hour) and 2, 4, 6, 10 and 24 hours after administration, respectively. AUC 0-24hglu (mmol / Lh·r) Reduction rate = (Model control group AUC 0-24hglu -Treatment group AUG 0-24hglu ) / Model control group AUC 0-24hgln ×100% The experiment was conducted twice in total, with an interval of 8-10 days between each experiment. Each experiment was divided into 23 groups. The experimental groupings and dosage conditions are shown in Table 2.

[0062] [Table 2]

[0063] As shown in Figures 1-3, the results of the blood glucose lowering experiment showed that when the administration concentration of the polypeptide compound of the present invention was 100 μg / kg, the blood glucose lowering effect was almost equivalent to the blood glucose lowering effect of semaglutide and GIP, and in particular, the blood glucose lowering effect of sequence number 2 was almost identical to that of semaglutide.

[0064] 3. Effects of polypeptide compounds on random body weight in ob / ob mice As shown in the results of Table 3-5, single subcutaneous injection of 100g / kggIP, 100 and 300g / kggLP-1 had no obvious effect on the random body weight and its change in ob / ob mice. 24 and 48 hours after single subcutaneous injection of 100g / kg liraglutide and semaglutide groups, the random body weight and its change in ob / ob mice were significantly decreased, and 24, 48 and 72h after single subcutaneous injection of 300g / kg SEQ ID NO: 1-9, the random body weight and its change in ob / ob mice were significantly decreased. 24 hours after single subcutaneous injection of 100g / kg SEQ ID NO: 2, the change in random body weight of ob / ob mice was significantly decreased, and 24 and 48 hours after single subcutaneous injection of 300g / kg SEQ ID NO: 9, the change in random body weight of ob / ob mice was significantly decreased, and SEQ ID NO: 2 had almost the same effect as an equivalent dose of semaglutide. Therefore, a single subcutaneous injection of GIP and GLP-1 dual agonist compounds can significantly reduce random blood glucose levels in type 2 diabetic ob / ob mice, and SEQ ID NO: 2 is equivalent to the effect of equivalent doses of semaglutide. After long-term administration, all polypeptide compounds showed better body weight control effect.

[0065] [Table 3]

[0066]

Table 4

[0067]

Table 5

Claims

1. The amino acid sequence is (2) SEQ ID NO: 2 (CH ２ ) １６ -G--γ-G+-AEEA-AEEA)AFVQ=LIAGGEIGAAMI-E ２ (3) SEQ ID NO:3 YE １ EGTFTSDYSIE １ LDKIAQK(HOOC-(CH ２ ) １６ -G--γ-G+-AEEA-AEEA)AFVQ=LIAGGEIGAAMI-E ２ is selected from During the ceremony, 【Chemistry 4】 2. A GIP and GLP-1 dual agonist polypeptide compound comprising:

2. A method for producing the GIP and GLP-1 dual agonist polypeptide compound according to claim 1, comprising the steps of: (1) After the resin is taken and activated, amino acids are linked one by one to obtain a peptide resin; (2) A production method comprising the steps of: taking a peptide resin, coupling a side chain to it, decomposing it, and purifying it to obtain a side-chain-protected polypeptide.

3. The GIP and GLP-1 dual agonist polypeptide compound is a polypeptide compound according to claim 1, characterized in that the C-terminal amino acid of the polypeptide compound is amidated as a C-terminal primary amide or a pharma-ceutically acceptable salt thereof.

4. The salt is a GIP and GLP-1 dual agonist polypeptide compound; Hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectic acid, persulfuric acid, 3-phenylpropionic acid with one of the following: carboxylic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, fatty acids, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfate, and thiocyanate; 4. A pharma- ceutically acceptable salt of the GIP and GLP-1 dual agonist polypeptide compound according to claim 3, characterized in that it is a salt formed from:

5. A pharmaceutical product produced using the GIP and GLP-1 dual agonist polypeptide compound of claim 1, The pharmaceutical contains a GLP-1 agonist polypeptide compound as an active ingredient, and is in the form of any one of tablets, capsules, elixirs, syrups, lozenges, inhalants, sprays, injections, membranes, patches, powders, granules, blocks, emulsions, suppositories, and combination drugs.

6. 1. A pharmaceutical composition of a GIP and GLP-1 dual agonist polypeptide compound, comprising: The pharmaceutical composition comprises the GIP and GLP-1 dual agonist polypeptide compound according to claim 1 as an active ingredient, or a pharma- ceutical acceptable salt of the GIP and GLP-1 dual agonist polypeptide compound according to claim 3 or 4 as an active ingredient, and further comprises a pharma- ceutical acceptable carrier or diluent.

7. Use of the GIP and GLP-1 dual agonist polypeptide compound according to claim 1 or the GIP and GLP-1 dual agonist polypeptide compound produced by the method according to claim 2, The use is characterized in that the use is a GIP and GLP-1 dual agonist polypeptide compound as an active ingredient in the manufacture of a medicine for treating or preventing diabetes or in the manufacture of a weight loss drug.

8. Use of a pharma- ceutical acceptable salt of the GIP and GLP-1 dual agonist polypeptide compound according to claim 3 or 4, a pharmaceutical product according to claim 5, or a pharmaceutical composition according to claim 6, comprising The use is characterized in that it is use of a pharma- ceutically acceptable salt, medicine or pharmaceutical composition of a polypeptide compound as an active ingredient in the manufacture of a medicine for treating or preventing diabetes or in the manufacture of a weight loss drug.

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