阅读说明：本技术 一类glp-1/胃泌素受体双重激动剂及其应用 (GLP-1/gastrin receptor dual agonist and application thereof ) 是由 韩京 孟庆华 周凤 杨启萌 于 2020-12-09 设计创作，主要内容包括：本发明提供了一类GLP-1/gastrin受体双重激动多肽化合物。本发明的GLP-1/gastrin受体双重激动多肽化合物在更为有效的降低血糖的同时具有更优异的促进胰腺组织的细胞增殖和胰岛再生作用,同时多肽化合物具有很好的减重和治疗糖尿病肾病的作用。本发明提供的多肽化合物化学性质稳定,具有低的免疫原性特性,适合作为治疗代谢性疾病,如糖尿病、肥胖、糖尿病肾病、NAFLD、NASH等药物的活性成分。(The invention provides a GLP-1/gastrin receptor dual-agonistic polypeptide compound. The GLP-1/gastrin receptor dual-agonist polypeptide compound has more excellent effects of promoting cell proliferation of pancreatic tissues and islet regeneration while effectively reducing blood sugar, and has good effects of reducing weight and treating diabetic nephropathy. The polypeptide compound provided by the invention has stable chemical properties and low immunogenicity, and 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.)

1. A GLP-1/gastrin receptor dual-agonistic polypeptide compound is characterized in that the amino acid sequence general formula of the polypeptide compound is as follows:

His-Xaa₁-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa₂-Ala-Ala-Xaa₃-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Xaa₄-Xaa₅-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

wherein:

Xaa₁taken from D-Ala, Gly or Aib;

Xaa₂from Glu, Lys or Lys modified in the side chain;

Xaa₃is taken from Lys or Lys modified in side chain;

Xaa₄taken from AEEA or absent;

Xaa₅taken from AEEA or absent;

wherein Lys having a modified side chain is selected from Lys (AEEA-AEEA-gamma-Glu-CO- (CH)₂)_n-COOH)

Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)_n-COOH) is represented by the formula:

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

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

3. The class of GLP-1/gastrin receptor dual agonist polypeptide compounds of claim 1, wherein the amino acid sequence of said polypeptide compounds is one of the following sequences:

(1)

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(2)

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(3)

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(4)

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(5)

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(6)

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

4. a pharmaceutically acceptable salt of a GLP-1/gastrin receptor dual agonist polypeptide compound of claim 1.

5. The salt of claim 4, wherein the salt is a salt of a GLP-1/gastrin receptor dual 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, malic acid, maleic acid, malic 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/gastrin receptor dual 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/gastrin receptor-like dual agonist polypeptide compound of claim 1 or a pharmaceutical composition of claim 6.

8. Use of a class of GLP-1/gastrin receptor dual agonist polypeptide compounds of claim 1, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, or pharmaceutical agents thereof for the manufacture of a medicament for the treatment of a metabolic disease or disorder.

Technical Field

The invention relates to biological medicines, in particular to a GLP-1/gastrin receptor dual agonist and application thereof.

Background

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, and is one of the leading chronic non-infectious diseases worldwide. Diabetes can be divided into insulin-dependent diabetes (type 1 diabetes, T1DM) and non-insulin-dependent diabetes (type 2 diabetes, T2DM), with T2DM accounting for over 85% of diabetics. The most effective method for treating T2DM is to inject insulin, but the risk of hypoglycemia occurs during the treatment. The severe hypoglycemic response may occur during insulin use, subject to factors such as dose size, individual variability, route of injection, site of injection, or lack of food intake after injection. Therefore, the search for safe and effective new hypoglycemic agents is an urgent task in the research of current diabetes treatment drugs.

GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells of the tail jejunum, ileum and colon, and has the function of reducing blood sugar after being specifically combined with GLP-1 receptors. The main advantage of GLP-1 is its blood sugar-dependent incretin secretion, avoiding the risk of hypoglycemia often present in diabetes treatment. In addition to regulating blood glucose, GLP-1 also prevents pancreatic beta cell degeneration, stimulates beta cell proliferation and differentiation, and can fundamentally improve the progression of diabetes. In addition, GLP-1 also has effects of inhibiting gastric acid secretion, delaying gastric emptying, and suppressing appetite, and has partial weight loss effect. However, the short half-life of native GLP-1 in vivo constitutes a significant pharmacological challenge in attempts to use GLP-1 as a drug. GLP-1 is rapidly degraded in vivo by dipeptidyl peptidase IV (DPP-IV), resulting in a half-life of only a few minutes.

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 DPP-IV, XenGLP-1 also shows a much greater stability to degradation by Neutral Endopeptidase (NEP) than GLP-1. XenGLP-1 is a potent agonist of the GLP-1 receptor, with many of the glucose-regulating effects observed with native GLP-1, and many preclinical studies have shown that XenGLP-1 has several beneficial anti-diabetic properties, including enhanced glucose-dependent insulin synthesis and secretion, slowed gastric emptying, decreased food intake and weight loss, and promotion of beta cell proliferation and restoration of islet function, among others (biochem. Pharmacol.,2017,142, 155-. These effects are beneficial not only for diabetics, but also for patients suffering from obesity.

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-. 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 mainly in three forms in human body, which are classified into Gastrin-34, Gastrin-17 and Gastrin-14 according to the number of amino acids, and a short peptide form, i.e., Gastrin-6, also 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" that also have beneficial effects in preventing and/or treating 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 demonstrate 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 type of treatment T2 DM. US10406207B2 discloses a truncated form of exendin-4 and a conjugated peptide of gastrin-6 (ZP3022), k. fosgerau et al disclose that ZP3022 exhibits improved therapeutic activity in diabetic model mice, and that such conjugated peptides have a short half-life due to the absence of long-acting modifications, and must be injected frequently (Diabetes Obes metabes, 2013,15, 62-71). Xinyu Chen et al disclose a class of conjugated peptides of XenGLP-1 and gastin-6, however, if the conjugated peptides adopt a modification means of once-a-week administration, the activity is remarkably reduced, so that only once-a-day administration can be realized, and once-a-week administration cannot be realized. And the required modification means involve various amino acid substitutions, resulting in significant immunogenicity problems for the compound (J.Med.chem.2020,63, 12595-12613).

Disclosure of Invention

The invention aims to provide a novel polypeptide compound with GLP-1/gastrin receptor dual-agonism, wherein the polypeptide is a variant designed based on a XenGLP-1 sequence, can excite a GLP-1 receptor and a gastrin receptor simultaneously, and further improves the treatment effect of the XenGLP-1 on diabetes. The polypeptide compound has stable property and low immunogenicity, and can be administrated once a week. Meanwhile, the polypeptide compound has good weight loss and diabetic nephropathy treatment effects, and has more potential than single receptor agonists and reported GLP-1/gastrin receptor dual agonists in the aspect of preparing medicines for treating metabolic syndrome, such as diabetes, obesity, NAFLD, NASH, diabetic nephropathy and the like.

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

a GLP-1/gastrin receptor dual-excitation polypeptide compound has an amino acid sequence general formula as follows:

His-Xaa₁-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Xaa₂-Ala-Ala-Xaa₃-Glu- Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Xaa₄-Xaa₅-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

wherein:

Xaa₁taken from D-Ala, Gly or Aib;

Xaa₂from Glu, Lys or Lys modified in the side chain;

Xaa₃is taken from Lys or Lys modified in side chain;

Xaa₄taken from AEEA or absent;

Xaa₅taken from AEEA or absent;

wherein Lys having a modified side chain is selected from Lys (AEEA-AEEA-gamma-Glu-CO- (CH)₂)_n-COOH)

Lys(AEEA-AEEA-γ-Glu-CO-(CH₂)_n-COOH) is represented by 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 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-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Gl u-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Tyr-Gly-Tr p-Leu-Asp-Phe-NH₂

(2)SEQ ID NO:2

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Gl u-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-Tyr- Gly-Trp-Leu-Asp-Phe-NH₂

(3)SEQ ID NO:3

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ-Gl u-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-AE EA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

(4)SEQ ID NO:4

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEE A-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Tyr-Gly-T rp-Leu-Asp-Phe-NH₂

(5)SEQ ID NO:5

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEE A-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-Tyr -Gly-Trp-Leu-Asp-Phe-NH₂

(6)SEQ ID NO:6

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AEE A-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-AE EA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

the invention also provides pharmaceutically acceptable salts of the GLP-1/gastrin receptor dual-agonist polypeptide compound.

Preferably, the salt is formed by the GLP-1/gastrin receptor dual 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, ethanolic acid, lithospermic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, malic 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/gastrin receptor dual agonist polypeptide compound, which comprises: any GLP-1/gastrin receptor dual-agonist polypeptide compound or pharmaceutically acceptable salt thereof is taken as an effective raw material, and a pharmaceutically acceptable carrier or diluent is added.

The invention also provides a medicament containing the GLP-1/gastrin receptor dual-agonist polypeptide compound, 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/gastrin receptor dual-agonist polypeptide compound and pharmaceutically acceptable pharmaceutic adjuvants, carriers or diluents.

The invention also provides application of the GLP-1/gastrin receptor dual-agonist polypeptide compound, the pharmaceutically acceptable salt thereof, the pharmaceutical composition thereof or the 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:

compared with the existing GLP-1 receptor agonist, the GLP-1/gastrin receptor dual-agonist polypeptide compound has more excellent effects of promoting cell proliferation and islet regeneration of pancreatic tissues while effectively reducing blood sugar, 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 receptors and gastrin receptors is higher than that of natural ligands of each receptor, the polypeptide compound provided by the invention has stable chemical property, is not easily degraded by DPP-IV and NEP in vivo, is not easily filtered by glomeruli, has remarkably improved stability, and has pharmacokinetic characteristic of supporting once-weekly administration. The polypeptide compound provided by the invention has improved biophysical properties, has higher solubility than natural GLP-1 and gastin-6 at neutral pH and pH 4.5, and has the property of being beneficial to preparation. Compared with the GLP-1 receptor agonist which is already on the market and the reported GLP-1/gastrin receptor dual agonist, the polypeptide compound provided by the invention has lower immunogenicity, more excellent hypoglycemic activity and weight loss effect, and better therapeutic effect on metabolic diseases such as T2DM, obesity, diabetic nephropathy, NAFLD, NASH and the like than the existing medicine which is on the market and the reported GLP-1/gastrin receptor dual agonist. 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.

Drawings

FIG. 1 shows the long-lasting hypoglycemic effect of a single administration of each test substance in the non-fasting state of db/db mice;

FIG. 2 shows the immunogenicity of each test subject in vitro.

Detailed Description

The following abbreviations are used throughout the specification:

english abbreviation Chinese

Gly glycine

Ser serine

Ala alanine

Thr threonine

Val valine

Ile isoleucine

Leu leucine

Tyr tyrosine

Phe phenylalanine

His histidine

Pro proline

Asp aspartic acid

Met methionine

Glu glutamic acid

Trp Tryptophan

Lys lysine

Arg arginine

Asn asparagine

Gln Glutamine

Cys cysteine

Aib alpha-aminoisobutyric acid

AEEA 8-amino-3, 6-dioxaoctanoic acid

DCM dichloromethane

DMF dimethyl formamide

Fmoc 9-fluorenylmethoxycarbonyl

Boc tert-butyloxycarbonyl group

DMSO dimethyl sulfoxide

DIC N, N' -diisopropylcarbodiimide

HOBT 1-hydroxy-benzotriazole

Alloc allyloxycarbonyl radical

Dde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -ethyl

Mtt 4-Methyltriphenylmethyl

ivDde 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) 3-methyl-butyl

TFA trifluoroacetic acid

EDT dimercaptoethane

HPLC high performance liquid chromatography

LC-MS liquid chromatography mass spectrometry

DMEM Dubecker's modified eagle's medium

FBS fetal bovine serum

PBS phosphate buffered saline

HEPES 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid

BSA bovine serum albumin

IBMX 3-isobutyl-1-methylxanthine

HBSS Hanks' balanced salt solution

AIMV serum-free cell culture medium

Example 1

Synthesis of polypeptide Compound of SEQ ID NO 1

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ- Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-Tyr-Gly- Trp-Leu-Asp-Phe-NH₂

(1) Swelling of the resin

0.262g (0.1mmol 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 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 (0.4mmol), dissolving with 3mL of 10% DMF/DMSO (v/v), adding 2mL of DIC/HOBt (0.4mmol/0.44mmol) condensing agent, pre-activating for 30min, adding the activated amino acid into a reactor, shaking for reaction at room temperature for 2h, filtering out the reaction solution, washing the resin with 7mL of DMF for 4 times, and detecting whether the reaction coupling is complete by using Kaiser reagent, if not, detecting the coupling for 2 times.

(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 for side chain-modified Lys, Fmoc-Lys (alloc) -OH, Fmoc-Lys (Dde) -OH, Fmoc-Lys (Mtt) -OH, or Fmoc-Lys (ivDde) -OH, etc. may be used. In this example, Fmoc-Lys (Dde) -OH protection strategy was used, while the N-terminal His was Boc-His (Boc) -OH.

(5) Modification of Lys side chain

After the peptide chain synthesis is finished, 7mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove the Dde protecting group of Lys, after the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH, 0.4mmol of DIC and 0.44mmol of HOBt are added, and the concussion condensation reaction is carried out 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

Transferring the obtained resin connected with the polypeptide into a round-bottom bottle, cutting the resin by using 5mL of cutting agent Reagent R (TFA/thioanisole/phenol/EDT, 90:5:3:2, V/V), reacting for 2h in an oil bath at constant temperature of 30 ℃, pouring the cutting liquid into 40mL of ethyl acetate, washing a crude product for 3 times by using 15mL of ethyl acetate after refrigerated centrifugation, and finally drying by using nitrogen to obtain the crude peptide.

(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.20 mm, 12 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); the flow rate is 8 mL/min; the detection wavelength was 214 nm. Eluting by linear gradient (20% B-80% B/30min), collecting target peak, removing methanol, lyophilizing to obtain pure product 0.20g with purity greater than 98%, and determining target polypeptide molecular weight by LC-MS.

Example 2

Synthesis of polypeptide Compound of SEQ ID NO 2

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ- Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-T yr-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 0.18g of a pure product.

Example 3

Synthesis of polypeptide Compound of SEQ ID NO 3

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Lys(AEEA-AEEA-γ- Glu-CO-(CH₂)₁₆-COOH)-Ala-Ala-Lys-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-A EEA-Tyr-Gly-Trp-Leu-Asp-Phe-NH₂

The synthesis method is the same as example 1, and the target peak is collected and lyophilized to obtain 0.19g of pure product.

Example 4

Synthesis of polypeptide Compound of SEQ ID NO. 4

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AE EA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-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 0.17g of a pure product.

Example 5

Synthesis of polypeptide Compound of SEQ ID NO 5

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AE EA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-T yr-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 0.16g of a pure product.

Example 6

Synthesis of polypeptide Compound of SEQ ID NO 6

His-Aib-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Thr-Glu-Tyr-Leu-Glu-Glu-Glu-Ala-Ala-Lys(AE EA-AEEA-γ-Glu-CO-(CH₂)₁₆-COOH)-Glu-Phe-Ile-Glu-Trp-Leu-Ile-Lys-AEEA-AEEA-AEEA-A EEA-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 0.17g of a pure product.

Example 7

Determination of agonistic Activity of polypeptide Compounds on GLP-1 receptor, GCG receptor and GIP receptor

Agonism of the polypeptide compound at the receptor is determined by a functional assay that measures the cAMP response of HEK-293 cell lines stably expressing the human GLP-1 receptor. Cells stably expressing the GLP-1 receptor were split into T175 flasks and grown overnight in media (DMEM/10% FBS) to near confluency, then the media was removed and the cells were washed with calcium and magnesium free PBS and then treated with Accutase enzyme for protease. Detached cells were washed and resuspended in assay buffer (20mM HEPES, 0.1% BSA,2mM IBMX,1 × HBSS) and cell density determined and 25 μ L aliquots 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. By detecting the maximumConcentration of 50% activation of response (EC)₅₀) To quantify the in vitro potency of agonists.

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). One day before the assay, cells were transferred to 384-well plates and the compound was dissolved in IP-One buffer (containing 10mmol/L HEPES,1mmol/L CaCl)₂4.2mmol/L KCl, 146mmol/L NaCl,5.5mmol/L glucose, 50mmol/L LiCl) and diluted 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 by detecting the concentration (EC) that caused 50% activation of the maximal response₅₀) To quantify the in vitro potency of agonists.

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: EC of polypeptide compounds on GLP-1 receptor, CCK-1 receptor and CCK-2 receptor (gastrin receptor)₅₀Values (in nM)

Sample (I)	EC50(GLP-1 receptor)	EC50(CCK-1 receptor)	EC50(CCK-2 receptor)
				GLP-1	0.088	>100000	>100000
gastrin-6	>100000	1198.6	1.39
				ZP3022	0.099	1239.5	2.15
SEQ ID NO:1	0.035	>100000	1.25
				SEQ ID NO:2	0.026	>100000	0.98
SEQ ID NO:3	0.031	>100000	0.85
				SEQ ID NO:4	0.022	>100000	0.86
SEQ ID NO:5	0.033	>100000	1.11
				SEQ ID NO:6	0.038	>100000	1.30

As shown in Table 1, the agonistic activity of all the polypeptide compounds on GLP-1 receptor is higher than that of natural GLP-1 and ZP3022, the agonistic activity of all the polypeptide compounds on CCK-2 receptor is also higher than that of gastrin-6 and ZP3022, and all the polypeptide compounds show high selectivity of CCK-2 receptor agonism, and the selectivity is obviously better than that of gastrin-6 and ZP 3022.

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. By comparing the peak areas (t) before the start of the stability experiment₀) And peak area (t) after 7 days of storage₇) Get "% remaining peptide". Calculated according to the following formula: % remaining peptide ═ area of peak t₇)×100]Area per peak t₀Stability is expressed as "% remaining peptide", and the 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 compounds of the present invention have significantly improved solubility under pH conditions acceptable for injection in the body, and have advantageous properties for formulation, as compared to native GLP-1, gassin-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 compounds of the present invention also have better stability at 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, and 8 hours, and the peak area of the residual sample in the solution at each time point was measured 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)

Sample (I)	Half-life (DPP-IV middle)	Half-life (NEP middle)
			GLP-1	1.3	2.1
ZP3022	4.8	4.1
			SEQ ID NO:1	>8	>8
SEQ ID NO:2	>8	>8
			SEQ ID NO:3	>8	>8
SEQ ID NO:4	>8	>8
			SEQ ID NO:5	>8	>8
SEQ ID NO:6	>8	>8

As shown in the results in Table 3, the polypeptide compounds of the present invention have half-lives longer than 8 hours in both the DPP-IV enzyme-containing solution and NEP enzyme-containing solution systems, which are superior to native GLP-1 and ZP3022, and thus show effective resistance to degradation by DPP-IV and NEP enzymes.

Example 10

Pharmacokinetic Properties of polypeptide Compounds in rat

Rats were given a subcutaneous (s.c.) injection of 50nmol/kg and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36 and 48 hours post-dose. 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	1.0	425
Semaglutide	8.2	481
			SEQ ID NO:1	12.1	550
SEQ ID NO:6	12.8	581

As shown in the results of table 4, the polypeptide compound of the present invention has a significantly prolonged half-life in vivo, which is superior to semaglutide administered once a week, and a half-life which is significantly superior to ZP3022, and has pharmacokinetic characteristics supporting once a week administration.

Example 11

Effect of polypeptide Compounds on blood glucose in diabetes model mice (db/db mice)

Male db/db mice, randomly grouped, 6 per group. The blank group was given physiological saline (10mg/kg) by subcutaneous injection, the administration component was 6 groups, mice had free food and water during the experiment, and mice were given a single subcutaneous injection of 25nmol/kg ZP3022, semaglutide, SEQ ID NO:1, SEQ ID NO:6, respectively, in a non-fasting state. Blood glucose levels were measured with a glucometer 0h before dosing, and 4, 6, 24 and 48h after dosing for each group of mice.

As shown in the results of FIG. 1, the results of in vivo blood glucose reduction experiments in db/db mice show that the polypeptide compound of the present invention shows a long-lasting blood glucose reduction activity which is significantly superior to that of the positive control drugs semaglutide and ZP 3022.

Example 12

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 fed with D12492 high-fat diet from Research Diets for 18 weeks to make 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 normal saline group (control high fat diet group), a positive control group (ZP3022 and semaglutide), and a test sample group (SEQ ID NO:1, SEQ ID NO: 6). Control high fat diet group three times daily subcutaneous injection of normal saline (10mg/kg), ZP3022(25nmol/kg) three times daily subcutaneous injection, semaglutide, SEQ ID NO:1, SEQ ID NO:6 (all doses are 25nmol/kg) 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 (I)	Overall body weight change (%)	Body fat change (%)	Fasting blood glucose change (%)
				Control high fat diet	+1.2％(±0.6％)	+2.3％(±1.0％)	+2.8％(±1.1％)
ZP3022	-16.2％(±1.1％)***	-17.4％(±0.8％)***	-19.5％(±1.4％)***
				Semaglutide	-15.7％(±0.8％)***	-18.6％(±1.3％)***	-17.2％(±1.6％)***
SEQ ID NO:1	-29.5％(±1.6％)***,###	-35.6％(±2.5％)***,###	-28.5％(±1.9％)***,###
				SEQ ID NO:6	-27.3％(±1.2％)***,###	-37.4％(±3.2％)***,###	-29.6％(±2.2％)***,###

^***: p compared to control high fat diet group<0.001；^###: ratio P to ZP3022 and semaglutide<0.001

As shown in table 5, the polypeptide compound of the present invention can significantly reduce the 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 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	1.26±0.07	2.3±0.2
ZP3022	1.02±0.08***	1.7±0.1***
			Semaglutide	1.03±0.06***	1.8±0.2***
SEQ ID NO:1	0.80±0.04***,##	1.1±0.1***,##
			SEQ ID NO:6	0.78±0.03***,##	1.0±0.1***,##

^***: p compared to control high fat diet group<0.001；^##: ratio P to ZP3022 and semaglutide<0.01

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

Example 13

Effect of polypeptide Compounds on glycated hemoglobin (HbA1c), fasting plasma glucose and islet area in db/db mice

Male db/db mice, randomly grouped, 6 per group. One week after acclimatization, tail bleeds were performed to measure initial HbA1c (%) and fasting plasma glucose values prior to initiation of treatment. The blank group was given physiological saline (10mg/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 a day), semaglutide (every two days), SEQ ID NO:1 (every two days), and SEQ ID NO:6 (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 (%)

Sample (dosage)	HbA1c Change (%)	Fasting blood glucose change (%)
			Physiological saline	+2.87％(±1.18％)	+9.27％(±0.90％)
ZP3022	-4.16％(±0.83％)***	-6.07％(±0.97％)***
			Semaglutide	-4.27％(±0.80％)***	-5.39％(±0.70％)***
SEQ ID NO:1	-8.39％(±0.62％)***,##	-13.62％(±1.22％)***,##
			SEQ ID NO:6	-8.11％(±0.99％)***,##	-12.39％(±1.27％)***,##

^***: p compared to control high fat diet group<0.001；^##: ratio P to ZP3022 and semaglutide<0.01

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

Table 8: islet area (in μm) of db/db mice after 5 weeks of dosing²Is shown)

Sample (dosage)	Area of islet
		Physiological saline	21296±1847
ZP3022	29698±1530***
		Semaglutide	28156±1357***
SEQ ID NO:1	38956±1436***,###
		SEQ ID NO:6	37156±1356***,###

^***: comparison with physiological saline group P<0.001；^###: ratio of Liraglutide to semaglutide P<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 and semaglutide.

Example 14

Immunogenicity of polypeptide compounds

Immunogenicity experiments to induce T cell proliferation were performed using Peripheral Blood Mononuclear Cells (PBMCs) from 50 chinese donors. PBMC were cultured in AIMV medium and added to 24-well plates (2mL) to reach final concentrations-3X 10⁶cells/mL, and then PBMC stimulated by addition of ZP3022, semaglutide, 6a (selected from J.Med. chem.2020,63,12595-12613), SEQ ID NO:1, SEQ ID NO:6 to AIMV medium. 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. By [3H ]]Thymidine cultures were treated and incubated for a further 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 course of time (5-8 days) as a percentage of the total number of donors tested.

As shown in the results of fig. 2, 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.

Example 15

Activity of polypeptide Compounds in diabetic nephropathy mice

SD male rats were adaptively bred for one week, and then 6 rats were randomly selected as normal control groups, and the remaining groups were divided into 5 groups of 8 rats each for animal model creation using a high fat and high sugar diet plus low dose STZ (30mg/kg) molding method, and fed with high fat feed for 1 month (high fat feed composition: 66.5% conventional feed, 20.0% sucrose, 10.0% lard, 20.0% sucrose, 2.5% cholesterol, 1.0% cholate). After feeding the rats with the high-fat and high-sugar feed for four weeks, the rats in the model group were intraperitoneally injected with a low dose of STZ (30mg/kg), and the rats in the normal group were intraperitoneally injected with a citric acid buffer (pH 4.4). After one week, tail vein blood is taken, blood sugar of the rat is measured by a glucometer and test paper, if the blood sugar of the model building group is less than or equal to 16.7mmol/L, the model building group can be injected once again, the model building group is measured again after four weeks, the mark that the model building success is marked by the blood sugar of more than or equal to 16.7mmol/L, and all rats do not use insulin in the model building process. The rats successfully molded were randomly distributed into normal saline group, ZP3022 group, semaglutide group, SEQ ID NO:1 group, and SEQ ID NO:6 group in equal amount according to the random number table method. Normal saline was given as an intraperitoneal injection (three times a day), in the model group, saline was given as an intraperitoneal injection three times a day, ZP3022 was given as an intraperitoneal injection three times a day (25nmol/kg), semaglutide was given as an intraperitoneal injection every two days (25nmol/kg), SEQ ID NO:1 was given as an intraperitoneal injection every two days (25nmol/kg), SEQ ID NO:6 was given as an intraperitoneal injection every two days (25nmol/kg), and the administration was continued for 3 weeks. After the 3-week administration, the urine of rats was collected in a metabolic cage for 24 hours, the rats were fasted without water supply during the collection of the urine in the metabolic cage, and after the collection of the urine of the rats, the urine volume of each group of rats was recorded, and then the rats were subjected to low-speed centrifugation at 3000 rpm for 10 minutes to absorb the upper layer of urine, and the collected urine was measured for 24 hours of urine microalbumin (ELISA kit assay). After the experiment, the rats were sacrificed and blood serum was collected and measured for the values of blood creatinine (Scr) and Blood Urea Nitrogen (BUN).

Table 9: comparison of urinary microalbumin excretion (UAER) rates at 24h over the end of 3 weeks for various groups of rats

Sample (dosage)	UAER(μg/24h)
		Normal control group	75.6±12.8
Physiological saline group	1658.5±126.9
		Group ZP3022	1413.4±156.5
Semaglutide group	1368.5±198.6
		Group 1 of SEQ ID NO	189.1±29.4***,###
Group 6 of SEQ ID NO	254.3±45.8***,###

^***: comparison with physiological saline group P<0.001；^###: ratio P to ZP3022 and semaglutide<0.001

At the end of 3 weeks of the experiment, the excretion rate of the microalbumin in 24h urine of the normal saline group of the model group is obviously increased compared with that of the normal control group. The polypeptide compound can effectively reduce UAER, and the effect of the polypeptide compound is obviously stronger than that of positive control drugs ZP3022 and semaglutide, while the effect of ZP3022 and semaglutide on UAER is not obviously reduced.

Table 10: comparison of blood creatinine (Scr) and Blood Urea Nitrogen (BUN) at 3 weekend in rats of each group

^***: comparison with physiological saline group P<0.001；^###: ratio P to ZP3022 and semaglutide<0.001

At the end of experiment 3 weeks, the blood creatinine and blood urea nitrogen of the model group normal saline group are obviously increased compared with the normal control group. The polypeptide compound can obviously reduce the values of blood creatinine and blood urea nitrogen, and the effect of the polypeptide compound is obviously stronger than that of positive control medicaments ZP3022 and semaglutide. In conclusion, the polypeptide compound can obviously reduce blood urea nitrogen, reduce blood creatinine and proteinuria, improve renal function, delay and treat the disease progression of diabetic nephropathy rats, and has excellent effect of treating diabetic nephropathy.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

<110> university of Jiangsu profession

<120> GLP-1-gastrin receptor dual agonists and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Tyr Gly

20 25 30

Trp Leu Asp Phe

35

<210> 2

<211> 37

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Xaa Tyr

20 25 30

Gly Trp Leu Asp Phe

35

<210> 3

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Lys Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Xaa Xaa

20 25 30

Tyr Gly Trp Leu Asp Phe

35

<210> 4

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Tyr Gly

20 25 30

Trp Leu Asp Phe

35

<210> 5

<211> 37

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Xaa Tyr

20 25 30

Gly Trp Leu Asp Phe

35

<210> 6

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

His Xaa Glu Gly Thr Tyr Thr Asn Asp Val Thr Glu Tyr Leu Glu Glu

1 5 10 15

Glu Ala Ala Lys Glu Phe Ile Glu Trp Leu Ile Lys Xaa Xaa Xaa Xaa

20 25 30

Tyr Gly Trp Leu Asp Phe

35