CN114349828B - GLP-1/glucagon receptor dual agonist and application thereof - Google Patents

Abstract

The GLP-1/GCG receptor double-excited polypeptide compound has the effects of promoting weight reduction and weight gain prevention, reversing insulin resistance and regulating lipid metabolism while reducing blood sugar more effectively. The polypeptide compound of the present application has higher agonistic activity to GLP-1 receptor and GCG receptor than natural ligand of each receptor, and has lower agonistic activity to GIP receptor. The polypeptide compound provided by the application has stable chemical property and low immunogenicity, and is suitable for being used as an active ingredient of medicines for treating metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH and the like.

Description

GLP-1/glucagon receptor dual agonists and their applications

This application is a divisional application of the original application. The filing date of the original application is November 27, 2020, the application number is 2020113561546, and the name of the invention is a class of GLP-1/glucagon receptor dual agonists and their applications.

Technical Field

The present invention relates to biomedicine, and in particular to a class of GLP-1/glucagon receptor dual agonists and applications thereof.

Background Art

Obesity and its related metabolic syndrome have become global public health issues. The incidence and course of many metabolic syndromes, such as type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and dyslipidemia, are closely related to obesity. Studies have shown that 80-90% of T2DM patients are overweight or obese. The use of weight loss therapy is beneficial for the prevention and control of the disease, including controlling blood sugar, reducing morbidity and disability (mortality), etc. It is generally difficult to achieve the ideal weight loss effect by relying solely on exercise and diet control to lose weight. The efficacy of current drugs for the treatment of obesity is relatively limited, and many drugs for the treatment of obesity also have significant side effects, such as psychiatric symptoms and serious cardiovascular effects caused by acting on the central nervous system. Currently, only a few drugs can achieve a 5-10% weight loss when used alone. Among the drugs for the treatment of T2DM, only glucagon-like peptide (GLP-1) receptor agonists and sodium-glucose co-transporter 2 (SGLT2) inhibitors have a good weight control effect (J. Med. Chem., 2018, 61, 5580-5593). Bariatric surgery has a significant therapeutic effect on obesity, but the surgical risks suffered by patients are relatively high, and the long-term effects of the surgery are still unclear. Therefore, there is still a huge clinical demand for drugs for weight control. Drugs that can safely and effectively control weight and have the effect of treating primary diseases are ideal choices.

The body's energy and blood sugar regulation signaling system includes a variety of different polypeptide endogenous gastrointestinal hormones. Proglucagon is a 160-amino acid precursor polypeptide that is cleaved in different tissues and converted into different products, such as GLP-1, glucagon-like peptide-2 (GLP-2), glucagon (GCG) and oxyntomodulin (OXM). These endogenous gastrointestinal hormones are involved in the regulation of multiple physiological functions such as insulin secretion, food intake, gastric emptying, and glucose homeostasis. Therefore, therapies based on endogenous gastrointestinal hormones have become a research direction that has attracted much attention in the field of metabolic syndrome research.

GLP-1 is a glucose-dependent hypoglycemic polypeptide hormone secreted by L cells in the terminal jejunum, ileum and colon. It exerts a hypoglycemic effect after specifically binding to the GLP-1 receptor. The main advantage of GLP-1 is that it has a blood sugar-dependent incretin secretion effect, which avoids the risk of hypoglycemia that often exists in diabetes treatment. In addition to regulating blood sugar, GLP-1 can also prevent the degeneration of pancreatic β cells, stimulate the proliferation and differentiation of β cells, and improve the course of diabetes from the source. In addition, GLP-1 also has the effects of inhibiting gastric acid secretion, delaying gastric emptying, suppressing appetite, etc., and has a partial weight loss effect. Currently, many long-acting GLP-1 drugs have been launched on the market, such as liraglutide, semaglutide and dulaglutide. Although GLP-1 drugs have a safe hypoglycemic effect, if a better weight loss effect is required, it is generally necessary to increase the dosage, and high-dose administration of GLP-1 drugs is prone to gastrointestinal side effects, poor tolerance, and a narrow therapeutic window. Therefore, there is still a need for safer and more tolerable therapeutic agents that can effectively reduce weight and control blood sugar. GCG is a hormone produced in the α cells of the pancreas. It acts on the liver under stress conditions such as cold and hunger, breaking down glycogen in the liver and raising blood sugar. In addition to its blood sugar-raising effect, GCG also has the effects of promoting lipolysis, fat oxidation, and fever in the body (Diabetologia, 2017, 60, 1851–1861). Long-term administration can increase energy metabolism and show weight loss effects, but these beneficial effects of GCG on energy metabolism have not been applied due to its inherent blood sugar-raising effect.

OXM is an endogenous dual agonist of GLP-1 receptor and GCG receptor in the human body. Its agonist activity on GLP-1 receptor and GCG receptor is weaker than the natural ligands of each receptor (natural GLP-1 or GCG). The acute physiological effects of OXM include inhibiting gastric emptying, food intake, gastric and pancreatic exocrine secretion, increasing resting energy expenditure, etc., which can produce weight loss. Experiments have shown that peripheral administration of OXM in animals and humans can reduce body weight and reduce food intake, and can increase metabolic rate and activity-related energy expenditure in obese subjects. In addition, in clinical practice, high-dose administration of OXM is not prone to common gastrointestinal side effects such as nausea and vomiting while reducing weight. The above experiments confirm that therapies based on OXM or GLP-1/GCG receptor dual agonists have potential application value in the treatment of metabolic syndrome.

The currently reported polypeptide GLP-1/GCG receptor dual agonists can be divided into four categories based on sequence structure: GCG, OXM, GLP-1 or exendin-4. The published patent documents are: CN201911103118.6, CN201780013643.1, CN201680021972.6, CN201580030150.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 WO2011/117416, WO 2012/177443, WO 2012/177444, WO 2012/150503, WO2013/004983, WO 2013/092703, WO 2014/041195 and WO 2014/041375, etc.

In addition, some studies have described GLP-1/GCG/GIP receptor triple agonists that can activate not only GLP-1 receptors and GCG receptors, but also glucose-dependent insulinotropic polypeptide (GIP) receptors, as described by Brian Finan et al. (Nat. Med., 2015, 21, 27-36), Victor A. Gault et al. (Biochem. Pharmacol., 2013, 85, 1655-1662; Diabetologia, 2013, 56, 1417-1424) and described in CN104902919B, WO 2012/088116, etc.

The effects of GLP-1 in amphibians are similar to those of human GLP-1, so structural modification of amphibian GLP-1 is expected to discover new GLP-1 drugs with more efficient and long-lasting hypoglycemic effects. XenGLP-1 is a class of animal-derived GLP-1 analogs discovered in African clawed frogs. Compared with natural GLP-1, XenGLP-1 has better hypoglycemic activity and stability. In addition, compared with GLP-1, OXM and GCG, in addition to being more resistant to degradation by dipeptidyl peptidase (DPP-IV), XenGLP-1 also shows much more stability against degradation by neutral endopeptidase (NEP). XenGLP-1 is a highly efficient agonist of the GLP-1 receptor, but it does not activate the GCG receptor. XenGLP-1 has many glucose regulatory effects observed with natural 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, slower gastric emptying, reduced food intake and weight, and promotion of β-cell proliferation and restoration of islet function (Biochem.Pharmacol., 2017, 142, 155–167; FASEB J., 2019, 33, 7113-7125). These effects are not only beneficial for diabetics, but also for patients with obesity. Patients with obesity have a higher risk of developing hypertension, hyperlipidemia, diabetes, NAFLD, NASH, musculoskeletal and cardiovascular diseases.

Summary of the invention

The purpose of the present invention is to provide a novel polypeptide compound with dual agonist effects on GLP-1/GCG receptors. The polypeptide is a variant designed based on the XenGLP-1 sequence, which retains the therapeutic effect of XenGLP-1 on diabetes and has the beneficial effects of GCG on lipid metabolism and energy metabolism, thereby producing a synergistic effect on sugar, lipid and energy metabolism. It has greater potential than a single receptor agonist in the preparation of drugs for treating metabolic syndrome, such as obesity, diabetes, NAFLD, NASH and other diseases.

To achieve the above-mentioned purpose, the technical solution of the present invention is as follows:

A class of GLP-1/GCG receptor dual agonist polypeptide compounds, the amino acid sequence general formula of the class of GLP-1 receptor/GCG receptor dual agonist polypeptide compounds is:

His-Xaa ₁ -Xaa ₂ -Gly-Thr-Tyr-Thr-Asn-Asp-Val-Xaa ₃ -Xaa ₄ -Tyr-Xaa ₅ -Xaa ₆ -Xaa ₇ -Xaa ₈ -Xaa ₉ -Ala-Xaa ₁₀ - Xaa ₁₁ -Phe-Ile-Glu-Trp-Leu-Xaa ₁₂ -Xaa ₁₃ -Gly-Xaa ₁₄ -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Xaa ₁₅

in:

Xaa ₁ is derived from Ser, D-Ser or Aib;

Xaa ₂ is derived from Glu or Gln;

Xaa ₃ is taken from Thr or Ser;

Xaa ₄ is derived from Glu, Lys, or Lys with a modified side chain;

Xaa ₅ is derived from Leu, Lys, or Lys with a modified side chain;

Xaa ₆ is derived from Glu or Asp;

Xaa ₇ is derived from Glu or Ser;

Xaa ₈ is derived from Glu or Arg;

Xaa ₉ is derived from Ala or Arg;

Xaa ₁₀ is derived from Lys or Gln;

Xaa ₁₁ is derived from Glu or Asp;

Xaa ₁₂ is derived from Ile or Lys;

Xaa ₁₃ is derived from Lys or Asn;

Xaa ₁₄ is derived from Lys or Gly;

Xaa ₁₅ is derived from -NH ₂ or a Lys with a modified side chain;

wherein the side chain of the modified Lys is selected from Lys(γ-Glu-CO-(CH ₂ ) _n -CH ₃ ) or Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) _n -COOH),

The structural formula of Lys (γ-Glu-CO-(CH ₂ ) _n -CH ₃ ) is shown below:

The structural formula of Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) _n -COOH) is shown below:

Wherein, n is a natural number, and 12≤n≤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-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(2) SEQ ID NO: 2

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(3) SEQ ID NO: 3

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(4) SEQ ID NO: 4

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(5) SEQ ID NO: 5

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(6) SEQ ID NO: 6

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(7) SEQ ID NO:7

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(8) SEQ ID NO: 8

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(9) SEQ ID NO: 9

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(10) SEQ ID NO: 10

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(11) SEQ ID NO: 11

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(12) SEQ ID NO: 12

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(13) SEQ ID NO: 13

His-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

(14) SEQ ID NO: 14

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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

(15) SEQ ID NO: 15

His-Aib-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

(16) SEQ ID NO: 16

His-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂

(17) SEQ ID NO: 17

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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH) -NH ₂

(18) SEQ ID NO: 18

His-Aib-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂

The present invention also provides a class of pharmaceutically acceptable salts of GLP-1/GCG receptor dual agonist polypeptide compounds.

Preferably, the salt is a salt formed by a GLP-1/GCG 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, hexanoic 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, camphor Sulfonic acid, digluconic acid, nicotinic acid, pamoic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxyethanesulfonic acid, aminosulfonic acid, dodecylsulfuric acid, trifluoromethanesulfonic acid, naphthalene disulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, stearic acid, stearic acid, oxalic acid, lactic acid, succinic acid, malonic acid, hemisulfuric acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphoric acid, glucoheptanoic acid, aspartic acid, thiocyanic acid or sulfosalicylic acid.

The present invention also provides a pharmaceutical composition of a GLP-1/GCG receptor dual agonist polypeptide compound, which comprises: any of the above-mentioned GLP-1/GCG receptor dual agonist polypeptide compounds or pharmaceutically acceptable salts thereof as an effective raw material, and a pharmaceutically acceptable carrier or diluent.

The present invention also provides a medicament containing the above-mentioned GLP-1/GCG receptor dual agonist polypeptide compound, wherein the medicament is any capsule, tablet, spray, inhalant, injection, patch, emulsion, film, powder or compound preparation mentioned in pharmacy, and the medicament is composed of the GLP-1/GCG receptor dual agonist polypeptide compound and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents.

The present invention also provides the use of the GLP-1/GCG receptor dual agonist polypeptide compound, its pharmaceutically acceptable salt, its pharmaceutical composition or its medicament of the present invention in the preparation of a medicament for treating a metabolic disease or condition. In a specific aspect, the metabolic disease or condition is diabetes, NAFLD, NASH, hyperlipidemia or obesity. In a specific aspect, diabetes is type 1 diabetes, T2DM or gestational diabetes. In a specific aspect, the medicament is used to treat more than one metabolic disease or condition, for example, diabetes 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 present invention has the following beneficial effects:

Compared with the existing GLP-1 receptor agonists, the GLP-1/GCG receptor dual agonist polypeptide compound of the present invention has the effects of promoting weight loss and preventing weight gain while more effectively lowering blood sugar, reversing insulin resistance, and regulating lipid metabolism, and has unexpected beneficial effects compared with existing drugs. The polypeptide compound of the present invention has higher agonist activity on GLP-1 receptors and GCG receptors than the natural ligands of each receptor, and has lower agonist activity on GIP receptors. The polypeptide compound provided by the present invention has stable chemical properties, is not easily degraded by DPP-IV and NEP in the body, is not easily filtered by glomeruli, and has significantly improved stability of the compound, and has pharmacokinetic characteristics that support once-daily or once-weekly administration. The polypeptide compound provided by the present invention has improved biophysical properties, and its solubility at neutral pH and pH 4.5 is higher than that of natural GLP-1 and GCG, and has properties that are beneficial to preparations. The polypeptide compound provided by the present invention has low immunogenicity characteristics, and its therapeutic effect on metabolic diseases such as T2DM, obesity, NAFLD, NASH and hyperlipidemia is better than existing marketed drugs. Therefore, the polypeptide compound provided by the present invention is suitable as an active ingredient of drugs for treating metabolic diseases, such as diabetes, obesity, hyperlipidemia, NAFLD, NASH, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the long-term hypoglycemic effect of each test substance after a single administration in db/db mice in a non-fasting state;

FIG2 shows the hypoglycemic effect of each test substance in an oral glucose tolerance test in DIO mice after long-term administration for 21 days;

FIG3 shows the in vitro immunogenicity of each test substance.

DETAILED DESCRIPTION

The following abbreviations are used throughout this specification:

English abbreviation Chinese

Glycine

Ser Serine

Ala Alanine

Thr Threonine

Val

Ile Isoleucine

Leu Leucine

Tyr Tyrosine

Phe Phenylalanine

His

Proline

Asp Aspartic acid

Met Methionine

Glu Glutamate

Trp Tryptophan

Lys

Arg Arginine

Asn Asparagine

Gln Glutamine

Cys Cysteine

Aib α-aminoisobutyric acid

AEEA 8-Amino-3,6-dioxanoic acid

DCM Dichloromethane

DMF Dimethylformamide

Fmoc 9-fluorenylmethoxycarbonyl

Boc tert-Butyloxycarbonyl

DMSO Dimethyl sulfoxide

DIC N,N’-Diisopropylcarbodiimide

HOBT 1-Hydroxy-benzotriazole

Alloc Allyloxycarbonyl

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

Mtt 4-Methyltrityl

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

TFA Trifluoroacetic acid

EDT ethylene dimercaptan

HPLC High Performance Liquid Chromatography

LC-MS Liquid chromatography-mass spectrometry

DMEM Dulbecco'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 SEQ ID NO:1 polypeptide compound

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

(1) Swelling of resin

Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 mmol/g and put it into a 25 mL reactor. Wash the resin once with 7 mL of DCM and methanol alternately, wash the resin twice with 7 mL of DCM, then swell the resin with 7 mL of DCM for 1 h, and finally wash the resin three times with 7 mL of DMF.

(2) Removal of Fmoc protecting group from resin

The swollen resin was transferred to a PSI200 peptide synthesizer, and 7 mL of 20% piperidine/DMF (v/v) was added to react at room temperature for 5 min. The deprotection solution was filtered off, and the resin was washed once with 7 mL of DMF. Then, 7 mL of 20% piperidine/DMF (v/v) deprotection solvent was added to react with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF, each time for 1.5 min, to obtain a Rink resin with the Fmoc protecting group removed.

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

Fmoc-Ser(Boc)-OH (0.4 mmol) was weighed and dissolved in 3 mL 10% DMF/DMSO (v/v). 2 mL DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added. After pre-activation for 30 min, the activated amino acid was added to the reactor and reacted at room temperature for 2 h. After filtering the reaction solution, the resin was washed 4 times with 7 mL DMF. Kaiser reagent was used to detect whether the reaction coupling was complete. If not, coupling was repeated twice.

(4) Peptide chain extension

According to the sequence of the peptide chain, the above deprotection and coupling steps are repeated to connect the corresponding amino acids in sequence until the peptide chain is synthesized. Among them, the 12th Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH. In this example, the Fmoc-Lys(Dde)-OH protection strategy is adopted, and the N-terminal His uses Boc-His(Boc)-OH.

(5) Modification of Lys side chain

After the peptide chain was synthesized, 7 mL of 2% hydrazine hydrate/DMF (v/v) was added to selectively remove the Dde protecting group of Lys at position 12. After the Dde protecting group was removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt were added and shaken for 2 h. Then, the Fmoc protecting group was removed using the same method as above, and 0.4 mmol of palmitic acid, 0.4 mmol of DIC and 0.44 mmol of HOBt were added for condensation reaction for 2 h. After the reaction was complete, the resin was washed 4 times with 7 mL of DMF.

(6) Peptide cleavage

The obtained resin with polypeptide was transferred to a round-bottom bottle, and the resin was cut with 5 mL of cutting agent Reagent R (TFA/thioanisole/phenol/EDT, 90:5:3:2, V/V). The reaction was kept at 30°C in an oil bath for 2 h, and the cutting solution was poured into 40 mL of ice ether. After refrigerated centrifugation, the crude product was washed three times with 15 mL of ice ether and finally dried with nitrogen to obtain the crude peptide.

(7) Peptide purification

The crude target polypeptide was dissolved in water, filtered with a 0.25 μm microporous filter membrane, and then purified by Shimadzu preparative reverse phase HPLC system. The chromatographic conditions were C18 reverse phase preparative column (250 mm × 20 mm, 12 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8 mL/min; detection wavelength was 214 nm. A linear gradient (20% B ~ 70% B/30 min) was used for elution, and the target peak was collected. After removing methanol, lyophilization was performed to obtain 0.14 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.

Example 2

Synthesis of SEQ ID NO:2 polypeptide compound

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.16 g of pure product.

Example 3

Synthesis of SEQ ID NO:3 polypeptide compound

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.13 g of pure product.

Example 4

Synthesis of SEQ ID NO:4 polypeptide compound

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

(1) Swelling of resin

Weigh 0.262 g (0.1 mmol equivalent) of Rink Amide MBHA resin with a loading of 0.382 mmol/g and put it into a 25 mL reactor. Wash the resin once with 7 mL of DCM and methanol alternately, wash the resin twice with 7 mL of DCM, then swell the resin with 7 mL of DCM for 1 h, and finally wash the resin three times with 7 mL of DMF.

(2) Removal of Fmoc protecting group from resin

The swollen resin was transferred to a PSI200 peptide synthesizer, and 7 mL of 20% piperidine/DMF (v/v) was added to react at room temperature for 5 min. The deprotection solution was filtered off, and the resin was washed once with 7 mL of DMF. Then, 7 mL of 20% piperidine/DMF (v/v) deprotection solvent was added to react with the resin for 15 min. Finally, the resin was washed 4 times with 7 mL of DMF, each time for 1.5 min, to obtain a Rink resin with the Fmoc protecting group removed.

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

Fmoc-Ser(Boc)-OH (0.4 mmol) was weighed and dissolved in 3 mL 10% DMF/DMSO (v/v). 2 ml DIC/HOBt (0.4 mmol/0.44 mmol) condensing agent was added. After pre-activation for 30 min, the activated amino acid was added to the reactor and reacted at room temperature for 2 h. After filtering the reaction solution, the resin was washed 4 times with 7 mL DMF. Kaiser reagent was used to detect whether the reaction coupling was complete. If not, coupling was repeated twice.

(4) Peptide chain extension

According to the sequence of the peptide chain, the above deprotection and coupling steps are repeated to connect the corresponding amino acids in sequence until the peptide chain is synthesized. Among them, the 12th Lys can be Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Mtt)-OH or Fmoc-Lys(ivDde)-OH. In this example, the Fmoc-Lys(Dde)-OH protection strategy is adopted, and the N-terminal His uses Boc-His(Boc)-OH.

(5) Modification of Lys side chain

After the peptide chain is synthesized, 7 mL of 2% hydrazine hydrate/DMF (v/v) is added to selectively remove the Dde protecting group of Lys at position 12. After the Dde protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt are added, and the reaction is shaken for 2 hours. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt are added again, and the reaction is shaken for 2 hours. After the Fmoc protecting group is removed, 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt are added, and the reaction is shaken for 2 hours. After removing the Fmoc protecting group, 0.4 mmol of mono-tert-butyl octadecane dioate, 0.4 mmol of DIC and 0.44 mmol of HOBt were added for condensation reaction for 2 h. After the reaction was complete, the resin was washed 4 times with 7 mL of DMF.

(6) Peptide cleavage

The resin with the polypeptide obtained above was transferred to a round-bottom bottle, and the resin was cut with 5 mL of cutting agent Reagent R (TFA/thioanisole/phenol/EDT, 90:5:3:2, V/V). The reaction was kept at 30°C in an oil bath for 2 h, and the cutting solution was poured into 40 mL of ice ether. After refrigerated centrifugation, the crude product was washed three times with 15 mL of ice ether and finally dried with nitrogen to obtain the crude peptide.

(7) Peptide purification

The crude target polypeptide was dissolved in water, filtered with a 0.25 μm microporous filter membrane, and then purified by Shimadzu preparative reverse phase HPLC system. The chromatographic conditions were C18 reverse phase preparative column (250 mm × 20 mm, 12 μm); mobile phase A: 0.1% TFA/water (V/V), mobile phase B: methanol (V/V); flow rate was 8 mL/min; detection wavelength was 214 nm. A linear gradient (20% B ~ 80% B/30 min) was used for elution, and the target peak was collected. After removing methanol, lyophilization was performed to obtain 0.18 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS.

Example 5

Synthesis of SEQ ID NO:5 polypeptide compound

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.17 g of pure product.

Example 6

Synthesis of SEQ ID NO:6 polypeptide compound

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.15 g of pure product.

Example 7

Synthesis of SEQ ID NO:7 polypeptide compound

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.15 g of pure product.

Example 8

Synthesis of SEQ ID NO:8 polypeptide compound

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.16 g of pure product.

Example 9

Synthesis of SEQ ID NO:9 polypeptide compound

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-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-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.14 g of pure product.

Example 10

Synthesis of SEQ ID NO:10 polypeptide compound

His-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.17 g of pure product.

Embodiment 11

Synthesis of SEQ ID NO:11 polypeptide compound

His-D-Ser-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.16 g of pure product.

Example 12

Synthesis of SEQ ID NO:12 polypeptide compound

His-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Lys-Tyr-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) 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-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.14 g of pure product.

Example 13

Synthesis of SEQ ID NO:13 polypeptide compound

His-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.13 g of pure product.

Embodiment 14

Synthesis of SEQ ID NO:14 polypeptide compound

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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.15 g of pure product.

Embodiment 15

Synthesis of SEQ ID NO:15 polypeptide compound

His-Aib-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂

The synthesis method was the same as in Example 1, and the target peak was collected and freeze-dried to obtain 0.16 g of pure product.

Example 16

Synthesis of SEQ ID NO:16 polypeptide compound

His-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.15 g of pure product.

Embodiment 17

Synthesis of SEQ ID NO:17 polypeptide compound

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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH) -NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.16 g of pure product.

Embodiment 18

Synthesis of SEQ ID NO:18 polypeptide compound

His-Aib-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂

The synthesis method was the same as that in Example 4, and the target peak was collected and freeze-dried to obtain 0.15 g of pure product.

Embodiment 19

Determination of the agonist activity of peptide compounds on GLP-1 receptor, GCG receptor and GIP receptor

The agonistic effect of the polypeptide compound on the receptor was determined by a functional assay that measures the cAMP response of HEK-293 cell lines that stably express the human GLP-1 receptor, GCG receptor, or GIP receptor. Cells stably expressing the three receptors were divided into T175 culture flasks and grown overnight in culture medium (DMEM/10% FBS) to near confluence, then the culture medium was removed and the cells were washed with PBS without calcium and magnesium, and then treated with Accutase enzyme for protease. The detached cells were washed and resuspended in assay buffer (20mM HEPES, 0.1% BSA, 2mM IBMX, 1× HBSS), and the cell density was determined, and 25 μL aliquots were dispensed into the wells of a 96-well plate. For measurement, 25 μL of a solution of the test polypeptide compound in assay buffer was added to the wells and then incubated at room temperature for 30 minutes. The cAMP content of the cells was determined based on homogeneous time-resolved fluorescence (HTRF) using Cisbio's kit. After addition of HTRF reagent diluted in lysis buffer (kit component), the plates were incubated for 1 hour and the fluorescence ratio at 665/620 nm was measured. The in vitro potency of agonists was quantified by measuring the concentration that causes 50% activation of the maximal response ( _EC50 ).

The test data (nM) in the examples of the present patent application are shown in the following Table 1. Although the test data are stated with a certain number of significant figures, it should not be considered that the data have been determined to be accurate to the number of significant figures.

Table 1: EC ₅₀ values of peptide compounds for human GLP-1 receptor, GCG receptor and GIP receptor (expressed in nM)

As shown in Table 1, all peptide compounds have higher agonist activity on GLP-1 receptor than natural GLP-1, and most of them also have higher agonist activity on GCG receptor than natural GCG. Meanwhile, all peptide compounds show weaker agonist activity on GIP receptor.

Embodiment 20

Solubility and stability testing of peptide compounds

Before testing the solubility and stability of the polypeptide compound, its purity was first determined using HPLC. Then, based on the determined % purity, 10 mg of the polypeptide compound was dissolved in 1 mL of solution in different buffer systems and gently stirred for 2 hours. After centrifugation at 4500 rpm for 20 minutes, the supernatant was taken for HPLC analysis to determine the peak area. Then, the relative concentration of the test sample solution was calculated by comparing with the corresponding sample standard solution. For the stability test, an aliquot of the supernatant obtained by solubility was stored at 40°C for 7 days, and then the sample was centrifuged at 4500 rpm for 20 minutes, and the supernatant was analyzed by HPLC to determine the peak area. By comparing the peak area before the start of the stability experiment (t ₀ ) and the peak area after storage for 7 days (t ₇ ), the "% remaining peptide" was obtained. Calculated according to the following formula: % remaining peptide = [(peak area t ₇ ) × 100]/peak area t ₀ , stability is expressed as "% remaining peptide", and the calculation results are shown in Table 2 below.

Table 2: Solubility and stability of peptide compounds

As shown in Table 2, the polypeptide compound of the present invention has greatly improved solubility under the injection pH conditions acceptable to the body compared with natural GLP-1 and GCG, and has characteristics that are beneficial to the preparation. The polypeptide compound of the present invention also has high solubility at pH 4.5, which may allow for co-formulation in combination therapy with insulin or insulin derivatives. In addition, the polypeptide compound of the present invention also has high stability under pH 4.5 and neutral pH conditions.

Embodiment 21

Stability of peptide compounds to DPP-IV and NEP enzymes

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

Table 3: Half-life of polypeptide compounds in DPP-IV enzyme or NEP enzyme system (expressed in h)

As shown in the results of Table 3, the half-life of the polypeptide compound of the present invention in the DPP-IV enzyme solution and the NEP enzyme solution system is more than 8 hours, indicating that it can effectively withstand the degradation of DPP-IV and NEP enzymes.

Embodiment 22

Pharmacokinetic properties of peptide compounds in rats

Rats were given 50 nmol/kg of subcutaneous (s.c.) injections, and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 36 and 48 hours after administration. Plasma samples were analyzed by LC-MS after protein precipitation with acetonitrile. Pharmacokinetic parameters and half-life were calculated using WinonLin5.2.1 (non-compartmental model) (Table 4).

Table 4: Pharmacokinetic profile of peptide compounds in rats

sample T _1/2 (h) C _max (ng/mL) Liraglutide 2.3 489 Semaglutide 9.2 519 SEQ ID NO:8 3.9 539 SEQ ID NO:9 4.6 551 SEQ ID NO:11 11.5 541 SEQ ID NO:12 12.6 561

As shown in the results of Table 4, the in vivo half-life of the polypeptide compound of the present invention is significantly prolonged, and it has pharmacokinetic characteristics that support once-daily or once-weekly administration.

Embodiment 23

Effects of peptide compounds on blood glucose in diabetic mice (db/db mice)

Male db/db mice were randomly divided into groups, with 6 mice in each group. The blank group was subcutaneously injected with normal saline (10 mg/kg), and the drug-treated group was divided into 6 groups. The mice were free to eat and drink during the experiment. The mice were subcutaneously injected with 25nmol/kg of liraglutide, semaglutide, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 12 in a non-fasting state. The blood glucose level of each group of mice was measured with a blood glucose meter at 0h before administration, and 4, 6, 24 and 48h after administration.

As shown in the results of FIG. 1 , the results of the hypoglycemic experiment in db/db mice showed that the polypeptide compound of the present invention exhibited a long-acting hypoglycemic activity superior to the positive control drugs liraglutide and semaglutide.

Embodiment 24

Effects of peptide compounds on blood glucose and body weight in diet-induced obese (DIO) mice

Male C57BL/6J mice, weighing about 22g, a total of 42 mice in the model group, were fed with D12492 high-fat diet from Research Diets for 18 weeks to create a DIO mouse model. Six mice in the blank control group were fed with standard mouse feed (control standard diet group). Before the start of administration, each group of DIO mice was randomly divided into 7 groups according to body weight, each with 6 mice, namely, a saline group (control high-fat diet group), a positive control group (liraglutide and semaglutide) and a test sample group (SEQID NO: 8, 9, 11, 12). The control standard diet group and the control high-fat diet group were subcutaneously injected with saline (10 mg/kg) twice a day, liraglutide, SEQ ID NO: 8, SEQ ID NO: 9 groups were subcutaneously injected twice a day (25 nmol/kg), and semaglutide, SEQ ID NO: 11, SEQ ID NO: 12 groups were subcutaneously injected once a day (25 nmol/kg), and the administration cycle was 21 days. The weight changes of mice were recorded every day, and nuclear magnetic resonance (NMR) was used to measure body fat before and at the end of the experiment. After the experiment, each group of mice fasted for 12 hours and then orally administered glucose (1.5 g/kg). A blood glucose meter was used to measure the blood glucose levels of each group of mice at 15, 30, 60 and 120 min after administration of glucose (Table 5).

Table 5: Changes in body weight and body fat of DIO mice during the 3-week dosing period

^*** : P<0.001 compared with the control high-fat diet group; ^### : P<0.001 compared with the liraglutide and semaglutide groups

As shown in the results of Table 5, continuous administration of the polypeptide compound of the present invention to DIO mice for 3 weeks can significantly reduce the body weight and body fat content of the mice, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs liraglutide and semaglutide.

As shown in the results of FIG2 , the results of the hypoglycemic experiment indicate that the polypeptide compound of the present invention exhibits hypoglycemic activity comparable to that of the positive control drugs liraglutide and semaglutide.

Embodiment 25

Effects of polypeptide compounds on glycosylated hemoglobin (HbA1c) and fasting blood glucose in db/db mice

Male db/db mice were randomly divided into groups, with 6 mice in each group. After one week of adaptive feeding, blood was collected from the tail to measure the initial HbA1c value and fasting blood glucose value before the start of treatment. The blank group was given normal saline (10 mg/kg) subcutaneously twice a day, and the drug-treated group was divided into 6 groups, which were subcutaneously injected with 25nmol/kg of liraglutide (twice a day), semaglutide (once a day), SEQ ID NO: 8 (twice a day), SEQ ID NO: 9 (twice a day), SEQ ID NO: 11 (once a day), SEQ ID NO: 12 (once a day). The treatment cycle was 5 weeks. After the treatment, the mice were fasted overnight to measure the fasting blood glucose value, and blood was taken to measure the HbA1c (%) value (Tables 6 and 7).

Table 6: Changes in HbA1c (%) in db/db mice during a 5-week dosing period

Sample (dose) HbA1c%(before treatment) HbA1c% (after treatment) Normal saline 5.3±0.4 6.9±0.7 Liraglutide (25 nmol/kg twice daily) 5.2±0.3 5.3±0.2 SEQ ID NO:8 (25 nmol/kg twice a day) 5.4±0.5 5.2±0.4 SEQ ID NO:9 (25 nmol/kg twice a day) 5.1±0.2 5.1±0.3 Semaglutide (25 nmol/kg once daily) 5.6±0.5 5.8±0.3 SEQ ID NO: 11 (25 nmol/kg once a day) 5.4±0.6 5.3±0.4 SEQ ID NO: 12 (25 nmol/kg once a day) 5.7±0.4 5.6±0.3%

As shown in the results of Table 6, the polypeptide compound of the present invention can inhibit the increase of HbA1c value when continuously administered to db/db mice for 5 weeks, indicating that it has a good blood sugar control effect.

Table 7: Changes in fasting blood glucose in db/db mice during the 5-week dosing period

^*** : P<0.001 compared with the saline group; ^## : P<0.01 compared with the liraglutide and semaglutide groups

As shown in the results of Table 7, continuous administration of the polypeptide compound of the present invention to db/db mice for 5 weeks can significantly reduce the fasting blood glucose value of db/db mice, indicating that it has a high blood glucose control effect, and the effect of the polypeptide compound of the present invention is significantly stronger than that of the positive control drugs liraglutide and semaglutide.

Embodiment 26

Immunogenicity of peptide compounds

Immunogenicity experiments for inducing 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 a final concentration of ~3×10 ⁶ cells/mL, and then stimulated by adding liraglutide, semaglutide, SEQ ID NO:8, SEQID NO:9 to AIMV medium. The 24-well plates were cultured in a carbon dioxide incubator (5%) at 37°C 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. The cultures were treated with [3H]-thymidine, cultured for another 18 hours, and the counts per minute (cpm) of each well were measured. The stimulation index (SI) was calculated by dividing the proliferation response (cpm) of the test well of each donor by the proliferation response of the medium treatment (cpm), and an SI greater than 2.0 was considered positive. The percentage of donor responses was calculated by dividing the number of donors with a positive response over the entire time course (5-8 days) by the total number of donors tested.

As shown in the results of FIG3 , the donor reaction ratio of the polypeptide compound of the present invention is lower than that of liraglutide and semaglutide, indicating that the polypeptide compound of the present invention has low immunogenicity.

Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Sequence Listing

<110> Jiangsu Normal University

<120> GLP-1/glucagon receptor dual agonists and their applications

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 1

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 2

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 2

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 3

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 3

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 4

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 4

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 5

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 5

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 6

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 6

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 7

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 7

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 8

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 8

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 9

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 9

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 10

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 10

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 11

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 11

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 12

<211> 39

<212> PRT

<213> Artificial Sequence

<400> 12

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Lys Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 13

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 13

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210> 14

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 14

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210> 15

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 15

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210> 16

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 16

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210> 17

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 17

His Ser Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210> 18

<211> 40

<212> PRT

<213> Artificial Sequence

<400> 18

His Xaa Gln Gly Thr Tyr Thr Asn Asp Val Ser Lys Tyr Leu Asp Ser

1 5 10 15

Arg Arg Ala Gln Asp Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

Claims (7)

1. GLP-1/GCG receptor dual agonist polypeptide compound, characterized in that the amino acid sequence of the GLP-1/GCG receptor dual agonist polypeptide compound is one of the following formulas: (7) His-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂ (8) 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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂ (9) His-Aib-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-Lys(γ-Glu-CO-(CH ₂ ) ₁₄ -CH ₃ )-NH ₂ (10) His-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂ (11) 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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH) -NH ₂ (12) His-Aib-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-Lys(AEEA-AEEA-γ-Glu-CO-(CH ₂ ) ₁₆ -COOH)-NH ₂ 2. A pharmaceutically acceptable salt of the GLP-1/GCG receptor dual agonist polypeptide compound according to claim 1. 3. The pharmaceutically acceptable salt of the GLP-1/GCG receptor dual agonist polypeptide compound according to claim 2, wherein the salt is a GLP-1/GCG receptor dual agonist polypeptide compound and the following A salt formed from 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, pectic acid, butyric acid, hexanoic acid 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, parapeptic acid, propionic acid, persulfuric acid, picric acid, 3-phenylpropionic acid, pivalic acid, itaconic acid, 2-hydroxy Ethanesulfonic acid, sulfamic acid, dodecyl sulfate, trifluoromethanesulfonic acid, naphthalenedisulfonic acid, 2-naphthalenesulfonic acid, citric acid, mandelic acid, ascorbic acid, stearic acid, stearic acid, oxalic acid, lactic acid , succinic acid, malonic acid, hemisulfonic acid, malic acid, maleic acid, alginic acid, fumaric acid, D-gluconic acid, glycerophosphate, glucoheptanoic acid, aspartic acid, thiocyanic acid, sulfohydrate Cylic acid. 4. A pharmaceutical composition containing a GLP-1/GCG receptor dual agonist polypeptide compound, which is characterized in that it includes: a GLP-1/GCG receptor dual agonist polypeptide compound as claimed in claim 1 or a pharmaceutical composition as claimed in claim 2. an acceptable salt, and a pharmaceutically acceptable carrier or diluent. 5. A medicament containing a GLP-1/GCG receptor dual agonist polypeptide compound according to claim 1, which is characterized in that it includes: a GLP-1/GCG receptor dual agonist polypeptide compound and pharmaceutically acceptable pharmaceutical excipients, carrier or diluent. 6. The pharmaceutical preparation according to claim 5, characterized in that the pharmaceutical preparation is a pharmaceutical capsule, tablet, spray, inhalant, injection, patch, emulsion, film, powder or compound preparation, The pharmaceutical preparation consists of a GLP-1/GCG receptor dual agonist polypeptide compound and pharmaceutically acceptable pharmaceutical excipients, carriers or diluents. 7. The GLP-1/GCG receptor dual agonist polypeptide compound, its pharmaceutically acceptable salt, and its pharmaceutical composition or its medicament in the preparation of drugs for the treatment of metabolic diseases or disorders described in claim 1 application; The metabolic disease or condition is diabetes, NAFLD, NASH, hyperlipidemia or obesity.