CN115819551A

CN115819551A - GLP-1/glucagon/gastrin receptor triple agonist with site-specific modification and application thereof

Info

Publication number: CN115819551A
Application number: CN202211173424.9A
Authority: CN
Inventors: 耿月兵
Original assignee: Nanjing Sanuo Biopharmaceutical Co ltd
Current assignee: Nanjing Sanuo Biopharmaceutical Co ltd
Priority date: 2022-07-14
Filing date: 2022-09-26
Publication date: 2023-03-21

Abstract

The invention provides a GLP-1/glucagon/gastrin receptor triple agonist with fixed-point modification and application thereof. Compared with the prior patent, the GLP-1/glucagon/gastrin receptor triple agonist polypeptide compound has the advantages that Xaa4 and Xaa5 of the sequence are two OEG connecting arms, then Tyr-Gly-Trp-Leu-Asp-Phe-NH2 is the structure of gastrin-6, the sequence of gastrin-6 is continuously introduced into the C end on the basis of a GLP-1/GCGR double agonist, the agonistic activity of a CCK-2 receptor is endowed, and the hypoglycemic activity of the compound is further improved.

Description

GLP-1/glucagon/gastrin receptor triple agonist with site-specific modification and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a GLP-1/glucagon/gastrin receptor triple agonist modified at a fixed point and application thereof.

Background

Obesity and its associated metabolic syndrome have become global public health problems, and the incidence and course of many metabolic syndromes such as type 2 diabetes (T2 DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia are closely related to obesity. Studies have shown that clinically 80-90% of patients with T2DM are associated with overweight or obesity, and the use of weight loss therapy is beneficial in preventing and controlling disease conditions, including controlling blood glucose, reducing morbidity and disability (mortality). The ideal weight loss effect is generally difficult to achieve by only exercising and diet control to lose weight. The existing medicines for treating obesity have limited curative effect, and many medicines for treating obesity also have obvious side effects, such as mental symptoms caused by acting on central nerves, serious cardiovascular effects and the like. Among the drugs for treating T2DM, only glucagon-like peptide (GLP-1) receptor agonists and sodium-glucose cotransporter 2 (SGLT 2) inhibitors have a superior weight control effect (j.med.chem., 2018,61,5580-5593). Bariatric surgery has significant therapeutic effects on obesity, but patients suffer from a greater surgical risk and the long-term effects of surgery are still unclear. Therefore, there is still a great clinical need for a drug for weight control, and a drug that can safely and effectively control weight and has a therapeutic effect on primary diseases is an ideal choice.

The body's energy and blood glucose regulation signal system includes a number of different polypeptide endogenous gastrointestinal hormones, proglucagon (proglucagon), a precursor polypeptide of 160 amino acids that is cleaved in different tissues and converted into different products, such as GLP-1, glucagon-like peptide-2 (GLP-2), glucagon (Glucagon), and Oxyntomodulin (Oxyntomodulin, OXM). These endogenous gastrointestinal hormones are involved in the regulation of various physiological functions such as insulin secretion, food intake, gastric emptying and glucose homeostasis. Therefore, endogenous gastrointestinal hormone-based therapies have become a highly interesting research direction in the field of metabolic syndrome research.

OXM is an endogenous GLP-1 receptor and glucagon receptor dual agonist in humans, the agonistic activity of which at the GLP-1 receptor and the glucagon receptor is less potent than the natural ligand of each receptor (natural GLP-1 or glucagon). The acute physiological effects of OXM include inhibition of gastric emptying, ingestion, and exocrine secretion from the stomach and pancreas, elevation of resting energy expenditure, etc., which can produce weight loss effects. Experiments have shown that peripheral administration of OXM in animals and humans can reduce body weight and food consumption, and in obese subjects can increase metabolic rate and activity-related energy expenditure. In addition, large dose administration of OXM is not easy to cause nausea, vomiting and other common gastrointestinal side effects while reducing body weight in clinic. The above experiments demonstrate that therapies based on dual agonists of the OXM or GLP-1/glucagon receptor show potential utility in the treatment of metabolic syndrome.

The currently reported polypeptide GLP-1/glucagon receptor dual agonists can be classified into four categories based on glucagon, OXM, GLP-1 or Exendin-4 (exendin-4) according to sequence structures, and the published patent documents are as follows: CN201911103118.6, CN201780013643.1, CN201680021972.6, cn80030150. X, CN201380048137.8, WO2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO2011/075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO 2010/070253, WO2010/070255, WO 2011/160630, WO 2011/006497, WO 2011/087671, WO 2011/087672, WO2011/117415, WO 041/117416, WO 2012/177443, WO 20112012/1744, WO 20112012/150503, WO2013/004983, WO 201742703, WO 2014/041195, and WO 2014/375.

The GLP-1 effect in the body of the amphibian is similar to that of human GLP-1, so that the structure of the amphibian GLP-1 is modified, and a novel GLP-1 medicine with more efficient and long-acting hypoglycemic effect is expected to be found. The XenGLP-1 is GLP-1 analogues of animal sources found in Xenopus laevis bodies, and compared with natural GLP-1, the XenGLP-1 has better hypoglycemic activity and stability. Furthermore, in addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV), xenGLP-1 also appears to be much more stable to degradation by Neutral Endopeptidase (NEP) than GLP-1, OXM and glucagon. XenGLP-1 is a highly potent agonist of the GLP-1 receptor, however it does not activate the glucagon receptor. XenGLP-1 has many of the glucose-regulating effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial antidiabetic properties, including enhanced glucose-dependent insulin synthesis and secretion, slowed gastric emptying, reduced food intake and weight, and promotion of beta cell proliferation and restoration of pancreatic function, among others (biochem. Pharmacol.,2017, 142,155-167 fasebj., 2019,33,7113-7125. These effects are beneficial not only for diabetics, but also for patients suffering from obesity. Patients with obesity have an increased risk of hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular diseases.

Gastrin (gastrin) is secreted by gastric mucosal cells and duodenal G cells, and its main physiological effects in the human body are to stimulate gastric acid secretion and to assist gastric motility. Other effects of Gastrin include stimulation of cell growth, suggesting that Gastrin may play a role in islet neogenesis, i.e., stimulation of insulin-secreting beta cells in the islets (see Korc, m., j. Clin. Invest.,1993,92,1113-1114, rooman et al Diabetes,2002,51,686-690), thus contributing to the regulation of blood glucose. Gastrin and another gastrointestinal hormone Cholecystokinin (CCK) share receptors, which are divided into two classes, the CCK-1 receptor and the CCK-2 receptor (gastrostrin receptor), which have different affinities for gastrostrin and CCK analogs. The CCK-1 receptor acts primarily as a receptor for sulfated CCK, while the CCK-2 receptor has similar affinity for CCK and gastrin. Among them, the CCK-2 receptor is also known as a gastrin receptor because the level of gastrin in plasma is higher than that of CCK (Foucaud et al. Reg. Peptides,2008,145,17-23).

The CCK-2 receptor, when bound to a ligand, initiates a variety of intracellular pathways, one of the key pathways downstream of the CCK-2 receptor being the MAPK (mitogen activated protein kinase) or ERK (extracellular regulated kinase) pathways, which are also activated by several growth hormones, a key feature of gastrin in cell proliferation. Since the CCK-2 receptor is expressed in the pancreas, gastrin can promote cell proliferation and islet regeneration in pancreatic tissue. Gastrin exists in the human body in three forms, which are classified into, by the number of amino acids, gastrin-34, gastrin-17 and Gastrin-14, and, in addition, a short peptide form, gastrin-6, exists. The 6 amino acids of Gastrin-6 are key amino acids for binding of Gastrin to CCK-2 receptor, and the C-terminus is amidated.

WO2005/072045 discloses a combination of a "GLP-1 receptor agonist" and a "gastrin compound" having a beneficial effect in the prevention and/or treatment of conditions and/or diseases for which a "GLP-1 receptor agonist" or a "gastrin compound" has been demonstrated to have a therapeutic effect. WO2007/095737 discloses similar combinations of "Exendin-4" and "gastrin compounds" having beneficial effects as well in the prevention and/or treatment of conditions and/or diseases for which "Exendin-4 agonists" or "gastrin compounds" have been demonstrated to have therapeutic effects. It should be noted that neither WO2005/072045 nor WO2007/095737 provide any in vivo, in vitro or other data to confirm that a "GLP-1 receptor agonist"/"gastrin compound" or "Exendin-4"/"gastrin compound" combination, respectively, described and used therein may be beneficial, for example, in the type of treatment of T2DM. US10406207B2 discloses a truncated form of exendin-4 and a conjugated peptide of gastrin-6 (ZP 3022), k. Fosgerau et al disclose that ZP3022 exhibits improved therapeutic activity in diabetic model mice, which conjugated peptide has a short half-life due to the absence of long-lasting modifications and must be injected frequently (Diabetes Obes metab.,2013,15,62-71). In addition, the weight loss effect of ZP3022 is weak, and for patients with diabetes complicated with obesity, the medicament capable of remarkably reducing blood sugar and weight can achieve better treatment effect. Xinyu Chen et al disclose a class of conjugated peptides of xenoglp-1 and gastrin-6, however, the modification means required for such conjugated peptides involve various amino acid substitutions, resulting in significant problems with immunogenicity of the compounds (j.med. Chem.2020,63, 12595-12613). And the compound has similar weight loss problem with ZP 3022.

Disclosure of Invention

The invention aims to provide a GLP-1/glucagon/gastrin receptor triple agonist subjected to site-specific modification and application thereof, wherein the polypeptide is a variant designed based on a XenGLP-1 sequence, can stimulate a GLP-1 receptor, a glucagon receptor and a gastrin receptor simultaneously, and further improves the weight loss and the diabetes treatment effect of the XenGLP-1. The polypeptide compound has stable property and low immunogenicity, and can be administrated once a day or once a week. Meanwhile, the polypeptide compound has excellent weight-reducing effect and has more potential than a single receptor agonist and reported GLP-1/glucagon and GLP-1/gustin receptor double agonists in the aspect of preparing medicaments for treating metabolic syndrome, such as diabetes, obesity, NAFLD, NASH and other diseases.

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

the GLP-1/glucagon/gastin receptor triple-agonistic polypeptide compound has the amino acid sequence general formula as follows: his-Xaa1-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Xaa2-Tyr-Xaa 3-Asp-Ser-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Pro-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa 4-Xaa5-Tyr-Gly-Trp-Leu-Asp-Phe-NH2

Wherein:

xaa1 is taken from Aib, ser or D-Ser;

xaa2 is taken from Lys or Lys with modified side chain;

xaa3 is taken from Leu, lys or side chain modified Lys;

xaa4 is taken from AEEA or absent;

xaa5 is taken from AEEA or absent;

wherein Lys of which side chain is modified is Lys (gamma-Glu-CO- (CH 2) n-CH 3) or Lys (AEEA-AEEA-gamma-Glu-CO- (CH 2) n-COOH), and the structural formula of Lys (gamma-Glu-CO- (CH 2) n-CH 3) is shown as the following formula:

lys (AEEA-AEEA-gamma-Glu-CO- (CH 2) n-COOH) has the formula:

wherein n is a natural number, and n is more than or equal to 12 and less than or equal to 20.

Preferably, n is 14, 16, 18 or 20.

Preferably, the amino acid sequence of the polypeptide compound is one of the following sequences:

(1)SEQ ID NO:1

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Al a-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(2)SEQ ID NO:2

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys-Asp-Ser-Arg-Arg-Al a-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(3)SEQ ID NO:3

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH2)14-CH3)-T yr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-S er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(4)SEQ ID NO:4

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(C H2)16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-As n-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-As p-Phe-NH2；

(5)SEQ ID NO:5

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH2)14 -CH3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-S er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(6)SEQ ID NO:6

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Gl u-CO-(CH2)16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-As n-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-As p-Phe-NH2。

the invention also provides pharmaceutically acceptable salts of the GLP-1/glucagon/gastin receptor triple agonist polypeptide compounds.

Preferably, the salt is formed by the GLP-1/glucagon/gastin receptor triple agonist polypeptide compound and one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectinic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, eleostearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid, or sulfosalicylic acid.

The invention also provides a pharmaceutical composition of the GLP-1/glucagon/gustin receptor triple-agonist polypeptide compound, which comprises the following components in part by weight: any GLP-1/glucagon/gastin receptor triple agonist polypeptide compound or pharmaceutically acceptable salt thereof is used as an effective raw material, and a pharmaceutically acceptable carrier or diluent is added.

The invention also provides a medicament containing the GLP-1/glucagon/gastin receptor triple-agonistic polypeptide compound, wherein the medicament is any one of capsules, tablets, spraying agents, inhalants, injections, patches, emulsions, films, powders or compound preparations in pharmaceutics, and the medicament consists of the GLP-1/glucagon/gastin receptor triple-agonistic polypeptide compound and pharmaceutically acceptable pharmaceutic adjuvants, carriers or diluents.

The invention also provides an application of the GLP-1/glucagon/gastrin receptor triple agonist polypeptide compound, pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof or a medicament thereof in preparing a medicament for treating metabolic diseases or symptoms. In particular aspects, the metabolic disease or disorder is diabetes, diabetic nephropathy, NAFLD, NASH, or obesity. In a particular aspect, the diabetes is type 1 diabetes, T2DM, or gestational diabetes. In particular aspects, the medicament is for treating more than one metabolic disease or disorder, e.g., diabetes and NAFLD, NASH, or obesity; diabetic nephropathy and NAFLD, NASH or obesity; obesity and NASH or NAFLD; diabetes, NASH, and obesity; diabetes, NAFLD and obesity; or diabetes and obesity.

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

xaa4 and Xaa5 of the sequence are two OEG connecting arms, then Tyr-Gly-Trp-Leu-Asp-Phe-NH2 is the structure of gastrin-6, compared with the prior patent, the compound is characterized in that on the basis of GLP-1/GCGR double agonist, the sequence of gastrin-6 is continuously introduced into the C end, and the agonist activity of the CCK-2 receptor is endowed to the compound, thereby further improving the hypoglycemic activity of the compound.

Therefore, compared with the existing GLP-1 receptor agonist, the GLP-1/glucagon/gastrin receptor agonist polypeptide compound has more excellent functions of promoting cell proliferation of pancreatic tissues and islet regeneration while more effectively reducing blood sugar and body weight, can fundamentally treat diabetes, reverse insulin resistance and treat diabetic nephropathy complications, and has unexpected beneficial effects compared with the existing medicines. The agonistic activity of the polypeptide compound on GLP-1 receptor, glucagon receptor and gastrin receptor is higher than that of natural ligands of each receptor, the polypeptide compound provided by the invention has stable chemical property, is not easy to be degraded by DPP-IV and NEP in vivo, is not easy to be filtered by glomerulus, has obviously improved stability, and has the pharmacokinetic characteristic of supporting once-a-day and once-a-week administration. The polypeptide compound provided by the invention has improved biophysical characteristics, has higher solubility than natural GLP-1, glucagon and gastin-6 at neutral pH and pH 4.5, and has the characteristics of being beneficial to preparation. The polypeptide compound provided by the invention has low immunogenicity characteristic, more excellent hypoglycemic activity and weight-losing effect, and the therapeutic effect on metabolic diseases such as T2DM, obesity, diabetic nephropathy, NAFLD, NASH and the like is better than that of the existing marketed drugs. Therefore, the polypeptide compound provided by the invention is suitable for being used as an active ingredient of medicaments for treating metabolic diseases, such as diabetes, obesity, diabetic nephropathy, NAFLD, NASH and the like.

It should be understood that all combinations of the foregoing concepts and additional concepts described in greater detail below can be considered as part of the inventive subject matter of this disclosure unless such concepts are mutually inconsistent.

The foregoing and other aspects, embodiments and features of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings. Additional aspects of the present invention, such as features and/or advantages of exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of specific embodiments in accordance with the teachings of the present invention.

Drawings

FIG. 1 shows the immunogenicity of each test subject in vitro.

Detailed Description

In order to better understand the technical content of the present invention, specific embodiments are described below with reference to the accompanying drawings. In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. Embodiments of the present disclosure are not necessarily defined to include all aspects of the invention. It should be appreciated that the various concepts and embodiments described above, as well as those described in greater detail below, may be implemented in any of numerous ways, as the disclosed concepts and embodiments are not limited to any one implementation. In addition, some aspects of the present disclosure may be used alone, or in any suitable combination with other aspects of the present disclosure.

Example 1SEQ ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Ar g-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro -Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(1) Swelling of the resin

0.262g (0.1 mmol equiv.) of Rink Amide MBHA resin loaded at 0.382mmol/g was weighed into a 25mL reactor, the resin was washed 1 time with 7mL of DCM and methanol alternately, 2 times with 7mL of DCM at mL, then the resin was swollen with 7mL of DCM for 1h, and finally washed 3 times with 7mL of DMF.

(2) Removal of Fmoc protecting group of resin

Transferring the swelled resin into a PSI200 polypeptide synthesizer, adding 7mL of 20% piperidine/DMF (v/v) to react for 5min at room temperature, filtering out the deprotection solution, washing the resin once with 7mL of DMF, adding 7mL of 20% piperidine/DMF (v/v) deprotection solvent to react with the resin for 15min, and finally washing the resin 4 times with 7mL of DMF, wherein each time lasts for 1.5min, so that the Rink resin with the Fmoc protecting group removed is obtained.

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

Weighing Fmoc-Phe-OH (0.4 mmol), dissolving with 3mL of 10% DMF/DMSO (v/v), adding 2mL of DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent, pre-activating for 30min, adding activated amino acid into the reactor, shaking for reaction at room temperature for 2h, filtering off reaction solution, washing the resin with 7mL of DMF for 4 times, and detecting whether the reaction coupling is complete by Kaiser reagent, if not complete, 2 times coupling.

(4) Elongation of peptide chain

And repeating the deprotection and coupling steps according to the sequence of the peptide chain to connect corresponding amino acids in sequence until the synthesis of the peptide chain is finished.

(5) Cleavage of polypeptides

The polypeptide-linked resin obtained above was transferred to a round-bottom flask, and 5mL of the cleaved resin was reacted with a cleavage agent Reagent R (TFA/thioanisole/phenol/EDT, 90.

(6) Purification of polypeptides

And dissolving the crude target polypeptide in water, filtering by using a 0.25 mu m microporous filter membrane, and purifying by using an Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were C18 reverse phase preparative columns (250 mm. Times.20mm, 12 μm); mobile phase A:0.1% tfa/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-80% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.26g with purity higher than 98%, and determining the molecular weight of the target polypeptide by LC-MS.

Example 2SEQ ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys-Asp-Ser-Arg-Ar g-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro -Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

The synthesis method is the same as that of example 1, and the target peak is collected and lyophilized to obtain 0.23g of a pure product.

Example 3 synthesis of polypeptide compound of seq ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH2)14-C H3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2 ；

(1) Swelling of the resin

0.262g (0.1 mmol equiv.) of Rink Amide MBHA resin loaded at 0.382mmol/g was weighed into a 25mL reactor, the resin was washed 1 time with 7mL of DCM and methanol alternately, 2 times with 7mL of DCM, then the resin was swollen with 7mL of DCM for 1h, and finally washed 3 times with 7mL of DMF.

(2) Removal of Fmoc protecting group of resin

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

(4) Elongation of peptide chain

And repeating the deprotection and coupling steps according to the sequence of the peptide chain to connect corresponding amino acids in sequence until the synthesis of the peptide chain is finished. As Lys at position 12, fmoc-Lys (Alloc) -OH, fmoc-Lys (Dde) -OH, fmoc-Lys (Mtt) -OH, fmoc-Lys (ivDde) -OH or the like can be used. In this example, fmoc-Lys (Dde) -OH protection strategy was used, while Boc-His (Boc) -OH was used for the N-terminal His.

(5) Modification of Lys side chain

After the peptide chain synthesis is finished, adding 7mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove the Dde protecting group of Lys at the 12 position, adding 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt after the Dde protecting group is removed, and carrying out oscillation reaction for 2 hours. After Fmoc protecting group was removed by the same method as above, 0.4mmol of palmitic acid, 0.4mmol of DIC and 0.44mmol of HOBt were added and the reaction was completed and the resin was washed 4 times with 7mL of DMF.

(6) Cleavage of polypeptides

(7) Purification of polypeptides

And dissolving the crude target polypeptide in water, filtering by using a 0.25 mu m microporous filter membrane, and purifying by using an Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were C18 reverse phase preparative columns (250 mm. Times.20mm, 12 μm); mobile phase A:0.1% tfa/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-70% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.29g with purity of more than 98%, and determining the molecular weight of the target polypeptide by LC-MS.

Example 4 synthesis of polypeptide compounds of seq ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-C O-(CH2)16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-L ys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-L eu-Asp-Phe-NH2；

(1) Swelling of the resin

(2) Removal of Fmoc protecting group of resin

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

(4) Elongation of peptide chain

(5) Modification of Lys side chain

After the peptide chain synthesis is finished, adding 7mL of 2% hydrazine hydrate/DMF (v/v) to selectively remove the Dde protecting group of Lys at the 12 position, adding 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt after the Dde protecting group is removed, and carrying out concussion condensation reaction for 2 hours. After Fmoc protecting group removal, 0.4mmol of Fmoc-AEEA-OH,0.4mmol of DIC and 0.44mmol of HOBt are added again, and the mixture is subjected to concussion condensation reaction for 2 hours. After removing the Fmoc protecting group, 0.4mmol of Fmoc-Glu-OtBu,0.4mmol of DIC and 0.44mmol of HOBt are added, and the mixture is subjected to concussion condensation reaction for 2 hours. After Fmoc protecting group removal, 0.4mmol of octadecanedioic acid mono-tert-butyl ester, 0.4mmol of DIC and 0.44mmol of HOBt were added for condensation reaction for 2 hours, and after the reaction was completed, the resin was washed 4 times with 7mL of DMF.

(6) Cleavage of polypeptides

(7) Purification of polypeptides

And dissolving the crude target polypeptide in water, filtering by using a 0.25 mu m microporous filter membrane, and purifying by using an Shimadzu preparative reversed-phase HPLC system. The chromatographic conditions were C18 reverse phase preparative columns (250 mm. Times.20mm, 12 μm); mobile phase A:0.1% tfa/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8mL/min; the detection wavelength was 214nm. Eluting with linear gradient (20-80% B/30 min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.27g with purity higher than 98%, and determining the molecular weight of the target polypeptide by LC-MS.

Example 5 synthesis of polypeptide compounds of seq ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH 2)14-CH3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-P ro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

The synthesis method is the same as that of example 3, and the target peak is collected and lyophilized to obtain 0.23g of a pure product.

Example 6 synthesis of polypeptide compound of seq ID no

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA- γ-Glu-CO-(CH2)16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-L ys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-L eu-Asp-Phe-NH2；

The synthesis method is the same as example 4, and the target peak is collected and lyophilized to obtain 0.26g of pure product.

EXAMPLE 7 determination of the agonistic Activity of polypeptide Compounds on GLP-1 receptor, glucagon receptor, GIP receptor, CCK-1 receptor and CCK-2 receptor

Agonism of the polypeptide compound at the receptor is determined by a functional assay that measures cAMP response in HEK-293 cell lines stably expressing the human GLP-1 receptor, glucogon receptor or GIP receptor. Cells stably expressing the above three receptors were divided into T175 flasks and grown overnight in medium (DMEM/10% FBS) to near confluency, then the medium was removed and the cells were washed with calcium and magnesium free PBS and then treated with Accutase enzyme. Detached cells were washed and resuspended in assay buffer (20mM HEPES,0.1% BSA,2mM IBMX,1 XHBSS), and the cell density was determined, and 25. Mu.L aliquots were dispensed into wells of a 96-well plate. For measurement, 25 μ Ι _ of a solution of the test polypeptide compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. Cell cAMP levels were determined based on Homogeneous Time Resolved Fluorescence (HTRF) using the Cisbio kit. After addition of HTRF reagent diluted in lysis buffer (kit components), the plates were incubated for 1 hour and then the fluorescence ratio at 665/620nm was measured. In vitro potency of agonists was quantified by measuring the concentration of 50% activation that elicits the maximal response (EC 50).

1321-N1 cells stably expressing CCK-1 receptor or CCK-2 receptor were cultured with DMEM-31966 (containing 10% FBS,1% sodium pyruvate, 1% penicillin, 1% streptomycin). The day before the experiment, the cells were transferred to 384 well plates, and the compound was dissolved and diluted in IP-One buffer (containing 10mmol/L HEPES, 1mmol/L CaCl2,4.2mmol/L KCl,146mmol/L NaCl,5.5mmol/L glucose, 50 mmol/L LiCl) and added to 384 well plates. After 1 hour incubation at 37 ℃, intracellular inositol 1-phosphate concentrations were determined using the IP-One HTRF Assay kit, and the in vitro potency of the agonists was quantified by measuring the concentration that caused 50% activation of the maximal response (EC 50).

The test data (nM) in the examples of the present patent application are shown in table 1 below, and although the test data are stated in terms of a certain number of significant figures, it should not be considered as indicating that the data have been determined to be the number of significant figures exactly.

Table 1: EC50 values (in nM) of the polypeptide compounds for the GLP-1 receptor, the glucagon receptor, the GIP receptor CCK-1 receptor, and the CCK-2 receptor (gustin receptor)

As shown in Table 1, all the polypeptide compounds have higher agonistic activity to GLP-1 receptor than that of natural GLP-1 and ZP3022, part of the polypeptide compounds have higher agonistic activity to glucagon receptor than that of natural glucagon, all the polypeptide compounds have higher agonistic activity to CCK-2 receptor than that of gastrin-6 and ZP3022, all the polypeptide compounds show high agonistic selectivity to CCK-2 receptor, the selectivity is obviously better than that of gastrin-6 and ZP3022, and all the polypeptide compounds show weaker agonistic activity to GIP receptor.

Example 8 solubility and stability testing of polypeptide Compounds

Prior to testing the solubility and stability of a polypeptide compound, its purity is first determined using HPLC. Then, based on the determined% purity, 10mg of the polypeptide compound was dissolved in 1mL of solution in different buffer systems with gentle stirring for 2 hours. After centrifugation at 4500rpm for 20 minutes, the supernatant was analyzed by HPLC to determine peak area. And then comparing with the corresponding sample standard solution, and calculating to obtain the relative concentration of the tested sample solution. For stability testing, aliquots of the supernatants obtained from solubility were stored at 40 ℃ for 7 days, then the samples were centrifuged at 4500rpm for 20 minutes and the supernatants were analyzed by HPLC to determine peak areas. The "% remaining peptide" was obtained by comparing the peak area before the start of the stability experiment (t 0) and the peak area after 7 days of storage (t 7). Calculated according to the following formula: % remaining peptide = [ (peak area t 7) × 100 ]/peak area t0, stability is expressed as "% remaining peptide", the calculation results are shown in table 2 below.

Table 2: solubility and stability of polypeptide compounds

As shown in the results in Table 2, the polypeptide compound of the present invention has a greatly improved solubility under pH conditions acceptable for injection in a body, and has a property advantageous for formulation, as compared with native GLP-1, glucagon, gastin-6 and ZP 3022. The polypeptide compounds of the invention also have high solubility at pH 4.5, a property that may allow co-formulation for combination therapy with insulin or insulin derivatives. In addition, the polypeptide compound of the present invention has better stability under the conditions of pH 4.5 and neutral pH.

EXAMPLE 9 stability of polypeptide Compounds to DPP-IV and NEP enzymes

The test sample was incubated with purified human DPP-IV or NEP enzyme at 37 ℃ for 0,2,4,8 hours, and the peak area of the residual sample in the solution was measured at each time point by HPLC to calculate the half-life of the sample, and the results are shown in Table 3.

Table 3: half-life of polypeptide Compounds in DPP-IV enzyme or NEP enzyme System (denoted by h)

As shown in the results in Table 3, the polypeptide compound of the present invention has half-lives longer than 8 hours in both DPP-IV enzyme-containing solution and NEP enzyme-containing solution, and is superior to native GLP-1, glucagon and ZP3022, which indicates that the polypeptide compound can effectively resist the degradation of DPP-IV and NEP enzymes.

EXAMPLE 10 pharmacokinetic Properties of polypeptide Compounds in rats

Rats were given a subcutaneous (s.c.) injection of 50nmol/kg and blood samples were collected 0.25,0.5,1,2, 4,8, 16, 24, 36 and 48 hours post-administration. After precipitation of the protein using acetonitrile, plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-lives were calculated using WinonLin 5.2.1 (non-compartmental model) (table 4).

Table 4: pharmacokinetic profile of polypeptide Compounds in rats

Sample (I)	T1/2(h)	Cmax(ng/mL)
			ZP3022	1.1	435
Liraglutide	4.3	419
			Semaglutide	9.1	474
SEQ ID NO:5	5.8	529
			SEQ ID NO:6	12.2	592

As shown in the results of table 4, the polypeptide compounds of the present invention have significantly prolonged in vivo half-life, respectively superior to liraglutide administered once a day and semaglutide administered once a week, and significantly superior half-life to ZP3022, with pharmacokinetic profiles that support once daily and once weekly administrations.

EXAMPLE 11 Effect of polypeptide Compounds on blood glucose, body weight and blood lipid in diet-induced obese (DIO) mice

Male C57BL/6J mice, weighing about 22g, were bred to 42 mice in the model group, and were bred on D12492 high-fat diet of Research Diets for 18 weeks to produce DIO mouse models. Before the start of administration, groups of DIO mice were randomly divided by body weight into 5 groups of 6 mice each, namely, a saline group (control high fat diet group), a positive control group (ZP 3022, liraglutide and semagllutide), and a test sample group (SEQ ID NO:5, SEQ ID NO. Control high fat diet group three times daily subcutaneous saline (10 mg/kg), ZP3022 (25 nmol/kg) three times daily subcutaneous injection, SEQ ID NO:5 and liraglutide (both at 25 nmol/kg) twice daily subcutaneous injection, semaglutide, SEQ ID NO:6 (both at 25 nmol/kg) once every two days, administration cycle 21 days. Changes in body weight of the mice were recorded daily, and body fat mass was measured before and at the end of the experiment using Nuclear Magnetic Resonance (NMR), and fasting plasma glucose was measured using a glucometer. After the experiment was completed, each group of mice was bled and the triglyceride and cholesterol contents in the serum were measured.

Table 5: body weight, body fat and fasting blood glucose changes (%) -in DIO mice over a 3-week dosing period

Sample(s)	Overall body weight change (%)	Body fat change (%)	Fasting blood glucose change (%)
				Control high fat diet	+1.6％(±0.5％)	+2.8％(±1.2％)	+2.9％(±1.0％)
ZP3022	-15.1％(±2.3％)	-15.4％(±1.7％)	-17.9％(±2.9％)
				Liraglutide	-16.2％(±3.1％)	-19.9％(±3.6％)	-15.8％(±2.1％)
Semaglutide	-16.8％(±0.9％)	-20.5％(±1.7％)	-17.9％(±3.7％)
				SEQ ID NO:5	-28.9％(±1.9％),	-34.9％(±2.9％),	-28.9％(±1.5％),
SEQ ID NO:6	-27.1％(±1.4％),	-33.2％(±3.5％),	-27.8％(±2.7％),

: p <0.001 compared to control high fat diet group; : the ratio P of ZP3022 to liraglutide to semaglutide is less than 0.001

As shown in table 5, the polypeptide compound of the present invention can significantly reduce body weight and body fat content of mice and reduce fasting blood glucose value after continuous administration in DIO mice for 3 weeks, and the effect of the polypeptide compound of the present invention is significantly stronger than that of positive control drugs ZP3022, liraglutide and semaglutide.

Table 6: blood lipid data (in mmol/L) of DIO mice after a 3-week dosing period

Sample (I)	Triglycerides	Cholesterol
			Control high fat diet	1.31±0.09	2.9±0.4
ZP3022	1.00±0.13	1.6±0.3
			Liraglutide	1.05±0.07	1.7±0.2
Semaglutide	1.04±0.08	1.6±0.3
			SEQ ID NO:5	0.77±0.10,	1.0±0.1,
SEQ ID NO:6	0.79±0.09,	1.1±0.3,

: p <0.001 compared to control high fat diet group; : the ratio P of ZP3022 to liraglutide to semaglutide is less than 0.01

As shown in table 6, the polypeptide compound of the present invention can significantly reduce triglyceride and cholesterol levels in DIO mice when administered continuously for 3 weeks in vivo, and the effect of the polypeptide compound of the present invention is significantly stronger than that of positive control drugs ZP3022, liraglutide and semaglutide.

Example 12 Effect of polypeptide Compounds on glycated hemoglobin (HbA 1 c), fasting plasma glucose and islet area in db/db mice

Male db/db mice, randomly grouped, 6 per group. One week after acclimatization, tail blood was taken to measure initial HbA1c (%) and fasting plasma glucose values before treatment began. The blank group was given physiological saline (10 mg/kg) by subcutaneous injection three times a day, and the administration groups were 4 groups, each of which was given by subcutaneous injection of 25nmol/kg of ZP3022 (three times per day), semaglutide (once every two days), liraglutide (twice per day), SEQ ID NO:5 (twice per day), and SEQ ID NO:6 (once every two days). The treatment period was 5 weeks, and fasting blood glucose values were measured after the mice were fasted overnight after the end of the treatment, while HbA1c (%) values were measured from blood. Finally, the mice were sacrificed, pancreas was sectioned, and islet area was measured for each group of mice under a 10-fold microscope after HE staining.

Table 7: hbA1c and fasting plasma glucose changes (%)

As shown in the results in Table 7, the polypeptide compound of the present invention, when continuously administered in db/db mice for 5 weeks, can reduce HbA1c and fasting plasma glucose, which are significantly superior to positive control drugs ZP3022, liraglutide and semaglutide, indicating that it has a good blood glucose control effect.

Table 8: islet area (expressed as μm 2) in db/db mice after 5 weeks of dosing

Sample (dosage)	Area of islet
		Physiological saline	21125±1726
ZP3022	28614±1618
		Liraglutide	28918±1348
Semaglutide	29158±1541
		SEQ ID NO:1	37986±2158,
SEQ ID NO:6	36715±1948,

P <0.001 compared to saline group; : the ratio P to ZP3022, liraglutide and semaglutide is less than 0.001;

as shown in the results of Table 8, the polypeptide compound of the present invention administered continuously in db/db mice for 5 weeks significantly increased the islet area in db/db mice, indicating that it has a high effect of promoting cell proliferation and islet regeneration in pancreatic tissues, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs ZP3022, liraglutide and semaglutide.

EXAMPLE 13 immunogenicity of polypeptide Compounds

Immunogenicity experiments to induce T cell proliferation were performed using Peripheral Blood Mononuclear Cells (PBMCs) from 50 chinese donors. PBMCs were cultured in AIMV medium and added to 24-well plates (2 mL) to reach final concentrations of-3 × 106cells/mL, then PBMCs were stimulated by addition of ZP3022, semaglutide,6a (selected from j.med.chem.2020,63, 12595-12613), SEQ ID NO:5, SEQ ID NO. The 24-well plates were incubated at 37 ℃ in a carbon dioxide incubator (5%) for 8 days. On

days

5,6, 7 and 8, cells from each well of the culture plate were transferred to a 96-well plate. Cultures were treated with [3H ] -thymidine and incubated for an additional 18 hours and counts per minute (cpm) were determined for each well. Stimulation Index (SI) was calculated by dividing the proliferative response (cpm) of the test wells of each donor by the proliferative response of the medium treatment (cpm), SI greater than 2.0 was considered positive. The percent response of the donors was calculated by taking the number of donors with positive reactions over the entire time course (5-8 days) as a percentage of the total number of donors tested.

As shown in the results of fig. 1, the donor response ratio of the polypeptide compound of the present invention is significantly lower than that of ZP3022, 6a and semaglutide, indicating that the polypeptide compound of the present invention has lower immunogenicity.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A GLP-1/glucagon/gustin receptor triple-agonistic polypeptide compound is characterized in that the amino acid sequence of the polypeptide compound has a general formula as follows:

His-Xaa1-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Xaa2-Tyr-Xaa3-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa4-Xaa5-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

wherein:

xaa1 is taken from Aib, ser or D-Ser;

xaa2 is taken from Lys or Lys with modified side chain;

xaa3 is taken from Leu, lys or side chain modified Lys;

xaa4 is taken from AEEA or absent;

xaa5 is taken from AEEA or absent;

wherein Lys of which the side chain is modified is Lys (gamma-Glu-CO- (CH 2) n-CH 3) or Lys (AEEA-AEEA-gamma-Glu-CO- (CH 2) n-COOH), and the structural formula of Lys (gamma-Glu-CO- (CH 2) n-CH 3) is shown as the following formula:

lys (AEEA-AEEA-gamma-Glu-CO- (CH 2) n-COOH) has the formula:

2. The class of GLP-1/glucagon/gastrin receptor triple agonist polypeptide compounds of claim 1, wherein n is 14, 16, 18, or 20.

3. The GLP-1/glucagon/gastin receptor triple agonist polypeptide compound of claim 1, wherein the amino acid sequence of the polypeptide compound is one of the following sequences:

(1)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(2)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(3)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH2)14-CH3)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(4)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH2)16-COOH)-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(5)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH2)14-CH3)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2；

(6)

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH2)16-COOH)-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH2。

4. a pharmaceutically acceptable salt of a GLP-1/glucagon/gastin receptor triple agonist polypeptide compound of the class described in claim 1.

5. The salt of claim 4, wherein the salt is a salt of a GLP-1/glucagon/gastin receptor triple agonist polypeptide compound with one of the following compounds: hydrobromic acid, hydrochloric acid, methanesulfonic acid, phosphoric acid, ethanesulfonic acid, formic acid, p-toluenesulfonic acid, acetic acid, acetoacetic acid, pyruvic acid, pectinic acid, butyric acid, caproic acid, benzenesulfonic acid, heptanoic acid, undecanoic acid, benzoic acid, salicylic acid, lauric acid, 2- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, camphoric acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, camphorsulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, sulfamic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, ethanolic acid, lithospermic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptonic acid, aspartic acid, thiocyanic acid, or sulfosalicylic acid.

6. A pharmaceutical composition comprising a GLP-1/glucagon/gastin receptor triple agonist polypeptide compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

7. An agent comprising a GLP-1/glucagon/gastin receptor triple agonist polypeptide-like compound of claim 1 or a pharmaceutical composition of claim 6.

8. Use of a GLP-1/glucagon/gastrin receptor triple agonist polypeptide compound of the class described in claim 1, a pharmaceutically acceptable salt thereof of claim 4, a pharmaceutical composition thereof of claim 6, or an agent thereof of claim 7 in the manufacture of a medicament for the treatment of a metabolic disease or disorder.