RU2848325C1 - Compound with dual agonist activity on glp-1 and gip receptors and use thereof - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions refers to biological pharmaceutics. Disclosed are compounds with dual agonist activity on glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors and use thereof. Compounds are effective in reducing blood glucose level and body weight, also have high stability in plasma and have pharmacokinetic characteristics for subcutaneous injection once a week.

EFFECT: compounds can be used for treating metabolic diseases such as obesity, type II diabetes mellitus and non-alcoholic fatty liver disease.

15 cl, 3 dwg, 20 tbl, 11 ex

Description

The present invention claims priority from Chinese Patent Application No. 202010472577.8, filed on May 29, 2020, and Chinese Patent Application No. 202110335100.X, filed on March 29, 2021.

AREA OF TECHNOLOGY

The present invention relates to the field of biological pharmaceuticals, in particular to a compound and a pharmaceutically acceptable salt thereof that have a dual agonistic effect on human glucagon-like peptide-1 (GLP-1) and human glucose-dependent insulinotropic polypeptide (GIP) receptor, which can be used to treat metabolic diseases such as diabetes and/or obesity.

PRIOR ART

Diabetes is a metabolic disease in which glucose, protein, and lipid metabolism is impaired due to insufficient insulin secretion. Diabetes is primarily classified into insulin-dependent diabetes (type 1) and non-insulin-dependent diabetes (type 2), based on differences in their pathological mechanisms. Non-insulin-dependent diabetes accounts for 90-95% of diabetics worldwide. Non-insulin-dependent diabetes is a long-term, chronic metabolic disorder caused by pancreatic β-cell dysfunction and prolonged insulin resistance. It is primarily characterized by deficient insulin levels and high plasma glucose concentrations. Research has shown that non-insulin-dependent diabetes is associated with a high risk of various complications in patients and can often lead to cardiovascular disease, kidney failure, blindness, amputation, and a host of other complications.

One of the main causes of non-insulin-dependent diabetes is obesity. Obesity is defined as excessive or abnormal accumulation of body fat that is detrimental to health. Obesity can also be defined as a person's body mass index (BMI) greater than or equal to 30 kg/ ^m² , based on their BMI. Increasing obesity can significantly increase the risk of cardiovascular disease, diabetes, musculoskeletal disorders, and certain cancers. Furthermore, an increased BMI also increases the risk of certain non-communicable diseases.

Due to the huge number of patients and the significant economic burden caused by diabetes and its complications, the development of safe and effective medications for the treatment of diabetes has always been a focus for many research institutes and pharmaceutical companies. Currently, marketed diabetes medications primarily include chemically synthesized small-molecule oral hypoglycemic agents, such as biguanides, sulfonamides, insulin sensitizers, and α-glucosides, and biologically synthesized injectable hypoglycemic agents, such as recombinant insulin and its derivatives. Although these medications are clinically effective in controlling plasma glucose levels in diabetic patients, their long-term use is often associated with adverse effects, such as weight gain, which in turn leads to an increased risk of potential cardiovascular events and decreased compliance. Considering the potential pathological relationship between diabetes and obesity and the potential risk of complications caused by obesity, the development of a drug that can not only effectively control blood glucose levels but also appropriately reduce the weight of diabetic patients has several implications for the effective treatment of diabetes and the reduction of the potential risk of complications, and therefore is the best direction for clinical development.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal regulatory peptide containing 30 or 31 amino acid residues. GLP-1 secretion is primarily regulated by small intestinal L cells in response to nutrient absorption and fluctuations in blood glucose levels in vivo. After a meal, small intestinal L cells secrete large amounts of GLP-1 to enhance pancreatic endocrine function. GLP-1 polypeptide exerts its physiological functions of blood glucose control and appetite suppression in vivo primarily through the activation of GLP-1 receptors distributed on the cell membrane surface. The mechanism of GLP-1 action for blood glucose control in vivo is primarily through the activation of GLP-1 receptors distributed on pancreatic β cells to promote insulin biosynthesis and secretion. Meanwhile, GLP-1 polypeptide can inhibit glucagon secretion, gastric emptying, and food intake in the presence of high blood glucose levels in the body, and enhance glucose degradation in the body through specific neurological effects. Notably, the physiological function of GLP-1 polypeptide to stimulate insulin secretion is controlled by plasma glucose concentration, so GLP-1 polypeptide does not cause severe and prolonged hypoglycemia compared with other drugs for the treatment of diabetes. In addition, it has been reported in the literature that GLP-1 polypeptide and its analogs have a direct stimulating effect on the growth, differentiation, and proliferation of β-cells in experimental animals, indicating that GLP-1 polypeptide and its analogs can protect pancreatic islets, delay the development of diabetes, and inhibit β-cell apoptosis. GLP-1 polypeptide also has a potential effect on the inhibition of gastrin secretion and food-stimulated gastric acid secretion. These characteristics suggest that GLP-1 polypeptide also has a physiological effect in preventing peptic ulcers. GLP-1 polypeptide can also activate GLP-1 receptors distributed throughout the central nervous system and brain to increase satiety, reduce food intake, and achieve the physiological effect of maintaining or reducing body weight. Thus, the extensive mechanisms of action and physiological functions of GLP-1 polypeptide and its analogs make GLP-1 polypeptide an ideal therapeutic option for the treatment of non-insulin-dependent diabetes and diabetic obesity.

The physiological functions of the GLP-1 polypeptide, such as blood glucose control and weight loss, are promising for the treatment of non-insulin-dependent diabetes/diabetic obesity. Natural GLP-1 has poor druggability and is easily degraded by dipeptidyl peptidase-IV (DPP-IV) in vivo, thus having a half-life of only 1-2 minutes in humans. In the face of this difficulty, the pharmaceutical industry has designed long-acting GLP-1 analogs and their derivatives through site-directed mutagenesis of amino acids at the enzymatic cleavage site, modification of the fatty acids of the polypeptide backbone, and coupling of the GLP-1 polypeptide to various protein/polymeric high-molecular compounds. Long-acting GLP-1 analogs currently on the market and widely used clinically include liraglutide (once-daily subcutaneous injection) and dulaglutide and semaglutide (once-weekly subcutaneous injection).

Clinical side effects of GLP-1 polypeptide and its derivatives primarily include nausea, vomiting, and diarrhea, all of which are gastrointestinal in nature. Furthermore, GLP-1 polypeptide and its derivatives have been found to cause tachycardia in subjects and, in some cases, increase the risk of pancreatitis in patients. Therefore, the dosage of GLP-1 polypeptide and its derivatives is limited by the side effects they cause, so their clinical use cannot ensure complete blood glucose control and weight loss in patients.

Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are both incretins that play a key physiological role in blood glucose metabolism in the body. GIP is primarily composed of 42 amino acid residues in the body and is secreted by K cells in the duodenum and adjacent jejunum in response to plasma glucose levels. GIP polypeptides exert their physiological effects by binding to GIP receptors distributed in pancreatic β-cells, adipose tissue, and the central nervous system. Like GLP-1, GIP can stimulate insulin secretion from pancreatic β-cells, thereby reducing plasma glucose concentrations, and can protect pancreatic β-cells, thereby controlling glucose metabolism in the body. Furthermore, the physiological functions of GIP polypeptide include activation of GIP receptors in adipose tissue, thereby promoting fat metabolism. Intraventricular injection of GIP polypeptide in mice can reduce food intake and body weight, suggesting that GIP polypeptide also has a physiological role in weight loss. Studies have shown that in patients with non-insulin-dependent diabetes, the incretin action of GIP polypeptide is significantly weakened, resulting in the absence or loss of its incretin effect in these patients. Studies have shown that the inhibitory properties of GIP polypeptide produced by these diabetic patients are significantly reduced when blood glucose levels return to normal.

Therefore, there is a clinical need for a method for treating non-insulin-dependent diabetes using GIP polypeptide in combination with a clinically effective hypoglycemic drug to restore tolerance in non-insulin-dependent diabetic patients to GIP polypeptide, and in combination with the incretin effect of GIP polypeptide to obtain a stronger clinical hypoglycemic effect.

The present invention aims to provide a GLP-1 analog derivative with agonist activity at the human GIP receptor, which exerts dual agonist activity on both the human GLP-1 receptor and the human GIP receptor. Furthermore, certain compounds of the present invention exhibit greater efficacy in lowering blood glucose levels and reducing body weight compared to GLP-1 receptor agonists known in the art. Certain compounds of the present invention exhibit exceptionally high plasma stability and exhibit pharmacokinetic properties suitable for once-weekly subcutaneous injection in humans.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a GLP-1 analogue having the general formula (I) or a pharmaceutically acceptable salt thereof:

R ₁ -X ₁ -X ₂ -Glu-Gly-Thr-Phe-Thr-Ser-Asp-X ₁₀ -Ser-X ₁₂ -X ₁₃ -X ₁₄ -X ₁₅ -X ₁₆ -X ₁₇ -X ₁₈ -X ₁₉ -X ₂₀ -Glu-Phe-X ₂₃ -X ₂₄ -Trp-Leu-X ₂₇ -X ₂₈ -X ₂₉ -X ₃₀ -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R ₂

(I) (SEQ ID NO: 19),

Where:

R ₁ is hydrogen (H), alkyl, acetyl, formyl, benzoyl, trifluoroacetyl, pGlu, or absent;

R ₂ is -NH ₂ , -OH, or absent;

X ₁ , X ₂ , X ₁₀ , X ₁₂ , X _{13 , X 14} , X ₁₅ , X ₁₆ , X ₁₇ , X ₁₈ , X ₁₉ , X ₂₀ , X ₂₃ , X ₂₄ , X ₂₇ , X ₂₈ , X ₂₉ and X ₃₀ are independently selected from the group consisting of any natural amino acid residues, any non-natural amino acid residues and peptide fragments consisting of natural amino acid residues and/or _non -natural amino acid residues.

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the group consisting of the amino acid residues Tyr and His; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the group consisting of the amino acid residues Val and Tyr; X ₁₂ is selected from the group consisting of the amino acid residues Ser and Ile; X ₁₃ is selected from the group consisting of the amino acid residues Tyr and Ala; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg, Glu, Gly, Lys and Aib; X ₁₇ is selected from the group consisting of the amino acid residues Glu, Ile and Gln; X ₁₈ is selected from the group consisting of the amino acid residues Ala, Aib and His; X ₁₉ is selected from the group consisting of the amino acid residues Ala, Aib and Gln; X ₂₀ is selected from the group consisting of the amino acid residues Gln, Glu and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Ala, Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Val and Leu; X ₂₈ is selected from the group consisting of the amino acid residues Arg and Ala; X ₂₉ is selected from the group consisting of the amino acid residues Gly and Gln; and X ₃₀ is selected from the group consisting of the amino acid residues Gly and Lys.

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the group consisting of the amino acid residues Tyr and His; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the group consisting of the amino acid residues Val and Tyr and Y1; X ₁₂ is selected from the group consisting of the amino acid residues Ser and Ile and Y1; X ₁₃ is selected from the group consisting of the amino acid residues Tyr and Ala and Y1; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle and Y1; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg, Glu, Gly, Lys and Aib and Y1; X ₁₇ is selected from the group consisting of the amino acid residues Glu, Ile and Gln and Y1; X ₁₈ is selected from the group consisting of the amino acid residues Ala, Aib and His; X ₁₉ is selected from the group consisting of the amino acid residues Ala, Aib and Gln; X ₂₀ is selected from the group consisting of the amino acid residues Gln, Glu and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Ala, Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Val and Leu; X ₂₈ is selected from the group consisting of the amino acid residues Arg and Ala; X ₂₉ is selected from the group consisting of the amino acid residues Gly and Gln; and X ₃₀ is selected from the group consisting of the amino acid residues Gly and Lys; Y1 is a substituted Lys, Orn, Dap, Dab or Cys residue, in particular with a modified group on the side chain of the Lys, Orn, Dap, Dab or Cys residue. In some embodiments, Y1 is a Lys, Orn, Dap, Dab, or Cys residue with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-( _CH2 ) _c -COOH; wherein a is an integer from 1 to 3 (reference may be made to 1, 2, or 3); b is 1 or 2; c is an integer from 10 to 30 (reference may be made to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the amino acid residue Aib; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the amino acid residue Gln; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the amino acid residue Asn; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a side chain connected to a substituent of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the amino acid residue Aib; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the amino acid residue Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the amino acid residue Gln; X ₂₃ is selected from the amino acid residue Val; X ₂₄ is selected from the amino acid residue Asn; X ₂₇ is selected from the amino acid residue Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from Y1; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from Y1; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from Y1; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the amino acid residue Aib; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the amino acid residue Gln; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the amino acid residue Asn; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from Y1; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the group consisting of the amino acid residues Aib and D-Ala; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from the group consisting of the amino acid residues Leu and Nle; X ₁₅ is selected from the amino acid residue Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from Y1; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the group consisting of the amino acid residues Gln and Lys; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the group consisting of the amino acid residues Asn and Gln; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is a Lys, Orn, Dap, Dab or Cys residue with a substituent on the side chain, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a -(y-Glu) _b -CO-(CH ₂ ) _c -COOH; where a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein each of X ₁₀ , X ₁₂ , X ₁₃ , X ₁₄ , X ₁₆ and X ₁₇ is independently selected from Y1; wherein Y1 is a Lys, Orn, Dap, Dab or Cys residue with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO- (CH ₂ ) _c -COOH; a is an integer from 1 to 3; b is 1 or 2; c is an integer from 10 to 30.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein a is 2, b is 1 or 2, and c is an integer from 16 to 20 (reference may be made to 16, 17, 18, 19, or 20).

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein a is 2, b is 1 or 2, and c is 16, 18, or 20.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₀ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₂ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₃ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₄ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₆ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein X ₁₇ is Y1; Y1 is Lys with a side chain substituent, wherein the substituent has a structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} _a- (y-Glu) _b -CO-(CH ₂ ) _c -COOH; a is 2; b is 1 or 2; c is 16 or 18.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein the side chain amino group of the Lys residue in Y1 is covalently linked to a substituent to form an amide bond.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein Y1 is K(-OEG-OEG-yGlu-C18-OH) or K(-OEG-OEG-yGlu-C20-OH), wherein K(-OEG-OEG-yGlu-C18-OH) has the structure shown below:

; and K(-OEG-OEG-yGlu-C20-OH) has the structure shown below:

.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein Y1 is K(-OEG-OEG-yGlu-C18-OH) or K(-OEG-OEG-yGlu-C20-OH), where:

K(-OEG-OEG-yGlu-C18-OH) has the structure shown below:

;

K(-OEG-OEG-yGlu-C20-OH) has the structure shown below:

In some embodiments of the present invention, there is also provided a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein in Y1 the ε-amine of the Lys residue is covalently linked to the substituent by an amide bond, and the α-amine of the Lys residue is linked to the peptide chain.

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the amino acid residue Aib; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the amino acid residue Gln; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the amino acid residue Asn; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is K(-OEG-OEG-yGlu-C18-OH) or K(-OEG-OEG-yGlu-C20-OH), where K(-OEG-OEG-yGlu-C18-OH) has the structure shown below:

; and K(-OEG-OEG-yGlu-C20-OH) has the structure shown below:

.

In some embodiments, the present invention provides a GLP-1 analog having the general formula (I) or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from the amino acid residue Tyr; X ₂ is selected from the amino acid residue Aib; X ₁₀ is selected from the amino acid residue Tyr; X ₁₂ is selected from the amino acid residue Ile; X ₁₃ is selected from the amino acid residue Tyr; X ₁₄ is selected from Y1; X ₁₅ is selected from the group consisting of the amino acid residues Asp and Glu; X ₁₆ is selected from the group consisting of the amino acid residues Arg and Lys; X ₁₇ is selected from the amino acid residue Ile; X ₁₈ is selected from the amino acid residue Ala; X ₁₉ is selected from the amino acid residue Ala; X ₂₀ is selected from the amino acid residue Gln; X ₂₃ is selected from the group consisting of the amino acid residues Ile and Val; X ₂₄ is selected from the amino acid residue Asn; X ₂₇ is selected from the group consisting of the amino acid residues Ile and Leu; X ₂₈ is selected from the amino acid residue Ala; X ₂₉ is selected from the amino acid residue Gly; X ₃₀ is selected from the amino acid residue Gly; Y1 is K(-OEG-OEG-yGlu-C18-OH) or K(-OEG-OEG-yGlu-C20-OH), where K(-OEG-OEG-yGlu-C18-OH) has the structure shown below:

;

K(-OEG-OEG-yGlu-C20-OH) has the structure shown below:

.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analog is shown in the general formula (II) (SEQ ID NO: 20):

H-YAibEGTFTSDYSIYX ₁₄ X ₁₅ X ₁₆ IAAQEFX ₂₃ NWLX ₂₇ AGGPSSGAPPPS-NH ₂ (II), where X ₁₄ is K or L, X ₁₅ is D or E, X ₁₆ is K or R, X ₂₃ is V or I, and X ₂₇ is I or L.

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analog is selected from the group consisting of compounds designated by numbers 1-18:

SEQ ID NO Subsequence 1 H-YAibEGTFTSDYSIYKDKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 2 H-YAibEGTFTSDYSIYKDRIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 3 H-YAibEGTFTSDYSIYKDKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 4 H-YAibEGTFTSDYSIYKDRIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 5 H-YAibEGTFTSDYSIYKDKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 6 H-YAibEGTFTSDYSIYKDRIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 7 H-YAibEGTFTSDYSIYKDKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 8 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 9 H-YAibEGTFTSDYSIYLEKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 10 H-YAibEGTFTSDYSIYLEKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 11 H-YAibEGTFTSDYSIYLEKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 12 H-YAibEGTFTSDYSIYKEKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 13 H-YAibEGTFTSDYSIYKERIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 14 H-YAibEGTFTSDYSIYKEKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 15 H-YAibEGTFTSDYSIYKERIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 16 H-YAibEGTFTSDYSIYKEKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 17 H-YAibEGTFTSDYSIYKERIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 18 H-YAibEGTFTSDYSIYKEKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ .

In some embodiments, the present invention also provides a GLP-1 analog having the general formula (I), or a pharmaceutically acceptable salt thereof, wherein the GLP-1 analog is selected from the group consisting of compounds designated by numbers 1#-18# below:

No. Subsequence 1# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 2# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 3# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 4# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 5# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 6# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 7# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 8# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 9# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 10# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLIAGGPSSGAPPPS-NH ₂ 11# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLLAGGPSSGAPPPS-NH ₂ 12# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 13# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 14# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 15# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 16# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 17# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 18# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ .

In some embodiments, the GLP-1 analog of the present invention is selected from the group consisting of compounds designated as 7#, 12#, 13#, 14#, 15#, 16#, 17#, and 18# in Fig. 3.

In some embodiments of the present invention, there is also provided a pharmaceutical composition comprising:

1) a GLP-1 analogue having the general formula (I), or a pharmaceutically acceptable salt thereof, and

2) a pharmaceutically acceptable excipient or pharmaceutical carrier.

In some specific embodiments, the pharmaceutical composition may comprise from 0.01 to 99% by weight of a GLP-1 analog in a unit dose, or the pharmaceutical composition may comprise 0.1-2000 mg, and in some specific embodiments, 1-1000 mg of a GLP-1 analog in a unit dose.

In some embodiments of the present invention, there is also provided the use of a GLP-1 analogue having the general formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing them, in the preparation of a medicament for the treatment of non-insulin-dependent diabetes/type II diabetes, insulin-dependent diabetes, obesity, non-alcoholic fatty liver disease, liver steatosis, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes.

In some embodiments of the present invention, the use of a GLP-1 analogue having the general formula (I) or a pharmaceutically acceptable salt thereof is proposed as a medicinal product.

In some embodiments of the present invention, there is provided the use of a GLP-1 analogue having the general formula (I) or a pharmaceutically acceptable salt thereof as a medicament for the treatment of non-insulin-dependent diabetes/type II diabetes, insulin-dependent diabetes, obesity, non-alcoholic fatty liver disease, liver steatosis, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes.

In some embodiments of the present invention, there is provided a method for treating non-insulin-dependent diabetes/type II diabetes, insulin-dependent diabetes, obesity, non-alcoholic fatty liver disease, liver steatosis, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes, which comprises administering to a subject in need thereof a GLP-1 analogue having the general formula (I) and a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same.

The present invention relates to a compound capable of simultaneously activating the GLP-1 receptor and the GIP receptor, and in some embodiments, the GLP-1 analog has greater agonist activity with respect to the GLP-1R than with respect to the GIP receptor.

In some specific embodiments, a GLP-1 analog of the present invention has a ratio of GLP-1R to GIP receptor agonist activity of (1-10):1, (1.1-10):1, (1.1-9.5):1, (1.1-9):1, (1.1-8.5):1, (1.1-8):1, (1.1-7.5):1, (1.1-7):1, (1.1-6.5):1, (1.1-6):1, (1.2-10):1, (1.2-9.5):1, (1.2-9):1, (1.2-8.5):1, (1.2-8):1, (1.2-7.5):1, (1.2-7):1, (1.2-6.5):1, (1.2-6):1, (1.3-10):1, (1.3-9.5):1, (1.3-9):1, (1.3-8.5):1, (1.3-8):1, (1.3-7.5):1, (1.3-7):1, (1.3-6.5):1, (1.3-6):1, (1.4-10):1, (1.4-9.5):1, (1.4-9):1, (1.4-8.5):1, (1.4-8):1, (1.4-7.5):1, (1.4-7):1, (1.4-6.5):1, (1.4-6):1, (1.5-10):1, (1.5-9.5):1, (1.5-9):1, (1.5-8.5):1, (1.5-8):1, (1.5-7.5):1, (1.5-7):1, (1.5-6.5):1, (1.5-6):1, (2-10):1, (2-9.5):1, (2-9):1, (2-8.5):1, (2-8):1, (2-7.5):1, (2-7):1, (2-6.5):1, (2-6):1, (2.5-10):1, (2.5-9.5):1, (2.5-9):1, (2.5-8.5):1, (2.5-8):1, (2.5-7.5):1, (2.5-7):1, (2.5-6.5):1, (2.5-6):1, (3-10):1, (3-9.5):1, (3-9):1, (3-8.5):1, (3-8):1, (3-7.5):1, (3-7):1, (3-6.5):1, (3-6):1, (3.5-10):1, (3.5-9.5):1, (3.5-9):1, (3.5-8.5):1, (3.5-8):1, (3.5-7.5):1, (3.5-7):1, (3.5-6.5):1, (3.5-6):1, (4-10):1, (4-9.5):1, (4-9):1, (4-8.5):1, (4-8):1, (4-7.5):1, (4-7):1, (4-6.5):1, (4-6):1, (4.5-10):1, (4.5-9.5):1, (4.5-9):1, (4.5-8.5):1, (4.5-8):1, (4.5-7.5):1, (4.5-7):1, (4.5-6.5):1, (4.5-6):1, (5-10):1, (5-9.5):1, (5-9):1, (5-8.5):1, (5-8):1, (5-7.5):1, (5-7):1, (5-6.5):1, (5-6):1, (5-5.5):1, (5.1-5.5):1, (5.2-5.4):1, (5.2-5.3):1, or any range or exact value therebetween, such as about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about 10:1. The above ratio is the normalized ratio of the in vitro assay data of the corresponding agonist activity. For example, the corresponding agonist activity can be determined using the cAMP-Gs kinetic kit. In this context, the expression (1-10):1 and the expression from 1:1 to 10:1 have the same meaning.

In another embodiment, the present invention relates to the aforementioned GLP-1 analog and a pharmaceutically acceptable salt thereof. The GLP-1 analog described herein is an amphoteric compound that can exhibit both acidic and basic properties. The GLP-1 analog described herein can be reacted with acidic or basic compounds to form salts by those skilled in the art using well-known methods.

A pharmaceutical composition containing a GLP-1 analogue according to the present invention can be used to treat patients in need of such treatment via parenteral administration. Subcutaneous, intramuscular, or intravenous injection may be selected for parenteral administration. The polypeptide compound with dual agonist activity according to the present invention can also be administered transdermally, optionally via an iontophoretic patch, or transmucosally.

The GLP-1 analog provided in this specification was synthesized by a solid-phase synthesis method. As an example, the synthetic vector is Rink amide resin MBHA (Xi'an sunresin Tech Ltd.). During the synthesis, the α-amino group of the amino acid derivative used is protected with Fmoc (fluorenylmethoxycarbonyl) group. As an example, the following protecting groups are selected for the amino acid side chain according to the difference in functional groups: the mercapto group of the cysteine side chain, the amino groups of asparagine and glutamine side chains and the imidazolyl group of the histidine side chain are protected with Trt (trityl); the guanidyl group of the arginine side chain is protected with Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl); the indolyl group of the side chain of tryptophan and the amino group of the side chain of lysine are protected with Boc (tert-butyloxycarbonyl); and the carboxyl group of the side chains of aspartic acid and glutamic acid, the hydroxyl group of the side chain of threonine, the phenolic group of the side chain of tyrosine and the hydroxyl group of the side chain of serine are protected with t-Bu (tert-butyl). As an example, in the synthetic process, the carboxyl group of the C-terminal amino acid residue of the polypeptide is first condensed with the insoluble polymeric amide resin of Rink and MBHA to form an amide bond; then, the Fmoc protecting group on the α-amino group is removed using an N,N-dimethylformamide (DMF) solution containing 20% 4-methylpiperidine; and then the solid phase support is condensed in excess with the next amino acid derivative in the polypeptide sequence to form an amide bond to extend the peptide chain. The "condensation → wash → deprotection → wash → next amino acid condensation" procedures are repeated to enable the synthesis of the desired length of the polypeptide chain; finally, a mixed solution of trifluoroacetic acid:water:triisopropylsilane (e.g., 90:5:5, v:v:v) is reacted with the resin to cleave the polypeptide from the solid phase support, and the mixture is precipitated using 5 times the volume of frozen methyl tert-butyl ether to obtain a solid crude GLP-1 analog product. The crude solid polypeptide product is dissolved in a mixed solution of acetonitrile/water containing 0.1% trifluoroacetic acid and purified and separated using a reversed-phase preparative C-18 chromatography column to obtain a pure GLP-1 analog product.

According to some embodiments, the present invention also relates to a kit of components that comprises:

- a GLP-1 analogue or a pharmaceutically acceptable salt thereof according to the present invention; and

- an additional therapeutic agent selected from any one of, or a combination of: an antiobesity agent, an antidiabetic agent, an antihypertensive agent, and a lipid-lowering agent; wherein each of the GLP-1 analogue or a pharmaceutically acceptable salt thereof and the additional therapeutic agent is contained in a separate container. In some embodiments of the invention, the GLP-1 analogue or a pharmaceutically acceptable salt thereof and the additional therapeutic agent are administered to the subject separately or in combination (e.g., simultaneously or sequentially).

In some embodiments, the pharmaceutical composition of the present invention and an administration device (e.g., a syringe, pen, or auto-injector) are provided in combination. As an example, the pharmaceutical composition of the present invention is pre-filled into an administration device for self-administration of the composition by a subject at home. As another example, the pharmaceutical composition of the present invention and the administration device are provided separately.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the effect of the compound of the present invention on the rate of body weight change in diet-induced obese mice.

Fig. 2 shows the effect of the compound of the present invention on the daily food intake of diet-induced obese mice.

Fig. 3 shows the structures of typical compounds according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains.

The amino acid sequences of the present invention contain standard single-letter or three-letter codes for twenty amino acids, and all amino acid residues of the present invention are preferably in the L-configuration unless otherwise specifically stated. Furthermore, Aib refers to α-aminoisobutyric acid, D-Ala refers to D-alanine, Orn refers to ornithine, Dap refers to 2,3-diaminopropionic acid, and Dab refers to 2,4-diaminobutyric acid.

The term "agonist" is defined as a substance that has an activating effect on the GLP-1 receptor or the GIP receptor.

The term "dual GLP-1/GIP agonist" as used in the context of the present invention refers to a substance or ligand that can activate the GLP-1 receptor and the GIP receptor.

As used herein, the term "treat, cure, or treat" includes inhibiting, alleviating, stopping, or reversing the progression or severity of an existing symptom or condition.

The term "natural amino acids" refers to the 20 common amino acids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

The term "unnatural amino acids" refers to amino acids that are not encoded in nature or are not found in the genetic code of any organism. For example, unnatural amino acids may be entirely synthetic compounds. Examples of unnatural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-serine phosphate, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine (Orn), D-ornithine, D-arginine, para-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline. In addition, the term also includes derivatives obtained by chemical modification of the C-terminal carboxyl group (or the N-terminal amino group and/or the side chain functional group) of a natural amino acid (or unnatural amino acid).

The term "alkyl" refers to a saturated aliphatic hydrocarbon group that is a linear or branched group containing from 1 to 20 carbon atoms, for example, an alkyl group containing from 1 to 8 carbon atoms, for example, an alkyl group containing from 1 to 6 carbon atoms, for example, an alkyl group containing from 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and various branched isomers thereof, etc. Alkyl may be, for example, a lower alkyl containing from 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Alkyl may be substituted or unsubstituted, and when substituted, substitution with a substituent may be made at any available site on the compound, wherein the substituent may be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, and carboxylate group. Substituted alkyl according to the present invention may be methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl, or hydroxy-substituted alkyl.

The expressions "X is selected from the group consisting of A, B, or C," "X is selected from the group consisting of A, B, and C," "X is A, B, or C," "X is A, B, and C," and the like all carry the same meaning, i.e., X may be any one or more of A, B, and C.

"Modification" of an amino acid, as disclosed herein, refers to the substitution, addition, or deletion of an amino acid, including the substitution or addition of any one or more of the 20 naturally occurring amino acids.

The term "natural GLP-1" refers to a naturally occurring molecule of the glucagon or exendin peptide families, wherein: the glucagon peptide family is encoded by the preproglucagon gene and includes three small peptides with high homology, i.e., glucagon (1-29), GLP-1 (1-37), and GLP-2 (1-33); and exendins are peptides expressed in lizards and, like GLP-1, are insulinotropic. In some embodiments, the term "natural GLP-1" also refers to human GLP-1 (7-37) and human GLP-1 (7-36).

The term "GLP-1 analogue" refers to a substance having up to 25, up to 24, up to 23, up to 22, up to 21, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or 1 amino acid modification or chemical modification compared to natural GLP-1 (in particular, human GLP-1(7-37) and human GLP-1(7-36)), wherein the amino acid modification may be an amino acid substitution, addition and/or deletion; and the chemical modification may be a chemical modification with a group selected from amide, carbohydrate, alkyl, acyl, ester, polyethyleneglycol (PEG), sialylating group, glycosylating group and the like.

The term "substitution" of an amino acid, as described herein, refers to the replacement of one amino acid residue with another amino acid residue.

The term "polyethylene glycol" or "PEG" refers to a mixture of polycondensates _of ethylene oxide and water, which is present in linear or branched form and is represented by the general formula H( _OCH2CH2 ) _nOH , where n is at least 9. Unless otherwise specified, this term includes polymers of polyethylene glycol having an average total molecular weight selected from the group consisting of 5,000-40,000 daltons.

The term "fatty acid" refers to a carboxylic acid with a long aliphatic tail (chain), which may be saturated or unsaturated. Fatty acids, as used herein, are carboxylic acids with a linear or branched aliphatic group of C4-C30.

The term "peptide" as used herein encompasses a category of peptides having modified amino and carboxyl termini. For example, an amino acid chain containing a terminal carboxylic acid substituted with an amide group is also included in the amino acid sequence designated as a natural amino acid.

All hydrogen atoms described in the present invention may be replaced by their isotopes (protium, deuterium and tritium), and any hydrogen atom in the compound of the present invention to which the present invention pertains may also be replaced by an isotopic atom.

The term "optional" or "optionally" means that the event or circumstance described subsequently may, but need not, occur, and that such description includes instances in which the event or circumstance does or does not occur. For example, "a heterocyclyl group optionally substituted with alkyl" means that alkyl may be present, but need not be, and that the description includes instances in which the heterocyclyl group is or is not substituted with alkyl.

The term "substituted" means that one or more, preferably up to 5 and more preferably 1-3, hydrogen atoms in the group are independently replaced by a substituent. The substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) whether a substitution is possible or impossible without much effort. For example, it may be unstable when an amino or hydroxy group, which has a free hydrogen, is bonded to a carbon atom containing an unsaturated (e.g., olefinic) bond.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds disclosed herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, with other components including, for example, physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration, facilitating absorption of the active ingredient, thereby achieving biological activity.

The term "agonist activity" refers to the ability of a compound of the present invention to activate the human GIP receptor and the human GLP-1 receptor. In some instances, "agonist activity" is embodied in a relatively active form and, in particular, refers to the ratio of the activating ability of a compound of the present invention for GLP-1R to the activating ability for the GIP receptor.

The term "pharmaceutically acceptable salt" refers to salts of a compound of the present invention that are safe and effective for administration to a mammal and possess the desired biological activity.

Semaglutide is a once-weekly polypeptide drug with GLP-1 agonist activity developed by Novo Nordisk in Denmark, which is currently approved and marketed in the United States, Japan, and the European Union.

LY3298176 is a once-weekly polypeptide drug with dual GIP receptor/GLP-1 receptor agonist activity developed by Eli Lilly, which is currently in Phase III clinical trials in several countries. The structure is as follows:

YAibEGTFTSDYSIAibLDKIAQKAFVQWLIAGGPSSGAPPPS-NH ₂ , where fatty acid modified to K at position 20.

Examples

The following specific embodiments are presented here only to illustrate the present invention in more detail and are not intended to limit the present invention. Experimental procedures without specific conditions indicated in the examples of the present invention were generally carried out according to conventional conditions or according to conditions recommended by the manufacturer of the starting materials or commercial products. Reagents not specifically indicated by a source are commercially available conventional reagents.

1. Experimental reagent

Table 1. Reagents and sources No. Reagent Source 1 Rink's MBHA amide resin Xi'an sunresin Tech Ltd. 2 HCTU (O-(6-chloro-1-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) Highfine Tech Ltd., Sunzhou 3 Fmoc-Aib-OH GL Biochem 4 Fmoc-L-Lys(Mtt)-OH GL Biochem 5 N,N-dimethylformamide SinoPharm 6 Dichloromethane SinoPharm 7 Trifluoroacetic acid SinoPharm 8 Triisopropylsilane Sigma-Aldrich 9 Hexafluoroisopropanol Sigma-Aldrich 10 Acetonitrile Merck-Millipore 11 Diisopropylethylamine Sigma-Aldrich 12 4-methylpiperidine TCI Chemicals 13 Methyl tert-butyl ether TCI Chemicals 14 Boc-L-Tyr(tBu)-OH GL Biochem 15 Fmoc-NH-PEG ₂ -COOH GL Biochem 16 Fmoc-L-Glu-OtBu GL Biochem 17 HOOC-(CH ₂ ) ₁₈ -COOtBu ChinaPeptides Co., Ltd, Suzhou 18 4-methylmorpholine TCI Chemicals

2. Experimental devices

Table 2. Instruments and sources No. device Source 1 Analytical ultra-performance liquid chromatograph H-CLASS WATERS 2 Agilent 1290-6530 Ultra Performance Liquid Chromatograph/Mass Spectrometer Agilent 3 Labconco Multifunctional Lyophilizer Thermo Fisher Scientific 4 Preparative high-performance liquid chromatograph Prep150 WATERS 5 Prelude-X Automatic Polypeptide Synthesizer Protein Technology Inc 6 Multichannel high-speed centrifuge Sigma

Example 1. Chemical synthesis of Compound 18#

1. Synthesis of the polypeptide skeleton

Rink amide resin MBHA (degree of substitution: 0.48 mmol/g, 0.1 mmol) was taken and placed in a polypropylene reaction tube for solid-phase polypeptide synthesis; N,N-dimethylformamide (DMF, 10 mL) was added to swell the resin for 10 min under nitrogen purge; DMF was removed in vacuo, and fresh DMF (10 mL) was added to wash the resin; After repeated washing of the resin, solid-phase polypeptide synthesis was performed twice on a Prelude-X automated polypeptide synthesizer using the Fmoc/tBu strategy, in which 10 equivalents of amino acid residues activated by HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and 4-methylmorpholine (the molar ratio of HCTU to 4-methylmorpholine amino acid residues was 1:2:1) were reacted with DMF at room temperature for 25 min for coupling. Removal of the N-terminal Fmoc-protecting group was performed in 2 reactions (10 min each) at room temperature using a DMF solution containing 20% 4-methylpiperidine. During the synthesis of the polypeptide backbone, the N-terminal amino acid residue was constructed using Boc-L-Tyr(tBu)-OH and subjected to secondary condensation, which was necessary to improve the quality of the crude peptide.

2. Selective removal of the Mtt resin-peptide protecting group and side chain modification with fatty acids

After the polypeptide backbone (or called resin-peptide) was extended, a mixed solution (10 ml) of dichloromethane containing 30% hexafluoroisopropanol was added, and the mixture was shaken at room temperature for 45 min, and then the mixed solution was removed; a mixed solution (10 ml) of methylene chloride containing 30% hexafluoroisopropanol was added, and the mixture was shaken at room temperature for 45 min, and then the mixed solution was removed. After completion of the reaction, the resin was washed 6 times with DMF. The lysine side chain at position 14 was extended using a Prelude-X automated polypeptide synthesizer with an additional coupling/deprotection cycle involving the amino acid components Fmoc-NH-PEG ₂ -COOH and Fmoc-L-Glu-OtBu. All couplings were carried out in DMF at room temperature for 25 min using 10 equivalents of amino acid residues activated with HCTU and 4-methylmorpholine (the molar ratio of HCTU to 4-methylmorpholine to amino acid residues was 1:2:1). The removal of the N-terminal Fmoc-protecting group was carried out by 2 reactions (10 min each) at room temperature using a DMF solution containing 20% 4-methylpiperidine. After the finally obtained resin was washed three times with DCM and DMF separately, a mixed solution (8 mL) of DMF containing 10 equivalents of HOOC-( _CH2 ) _18- COOtBu, 10 equivalents of HCTU, and 20 equivalents of diisopropylethylamine (DIEA) was added, and the mixture was reacted at room temperature for 4 hours to complete the side chain modification with fatty acids.

3. Splitting of the product

The resin-peptide obtained in the previous step was washed sequentially three times with DMF and DCM and dried under vacuum, followed by the addition of freshly prepared digestion buffer (trifluoroacetic acid:triisopropylsilane:water = 90:5:5, v:v:v), and the mixture was shaken at room temperature for 3-4 h. After completion of the reaction, the mixture was filtered and the resin was washed twice with trifluoroacetic acid. The filtrates were combined before the addition of a large amount of frozen methyl tert-butyl ether to precipitate the solid. The mixture was centrifuged and the supernatant was discarded, yielding the crude polypeptide compound 18#.

4. Purification by reversed-phase liquid chromatography

The crude polypeptide of compound 18# was dissolved in a mixed solvent containing 0.1% trifluoroacetic acid, 20% acetonitrile and 20% acetic acid/water, and the solution was filtered through a 0.22 μm membrane; the filtrate was separated using a WATERS Prep150 LC reversed-phase high performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid, 10% acetonitrile and water) and B (0.1% trifluoroacetic acid, 90% acetonitrile and water). The chromatography column was an X-SELECT OBD C-18 reversed-phase chromatography column, and during the purification process, the detection wavelength of the chromatograph was set to 220 nm, and the flow rate was 15 mL/min. The related product fractions were collected and lyophilized to obtain the pure polypeptide product of compound 1# in 18% yield. The purity of the pure polypeptide product was determined by a combination of analytical high-performance liquid chromatography and ultra-performance liquid chromatography/mass spectrometry with a purity of 92.81%. The molecular structure of compound 18# was H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ , with the structural formula shown as structure 18# in Fig. 3.

Example 2. Chemical synthesis of other compounds

The compounds in Table 3 were synthesized using the experimental protocol of Example 1.

Table 3. Compounds of the present invention Connection number Molecular structure 1# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 2# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 3# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 4# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 5# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 6# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DRIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 7# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 8# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 9# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 10# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLIAGGPSSGAPPPS-NH ₂ 11# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLLAGGPSSGAPPPS-NH ₂ 12# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 13# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 14# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 15# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 16# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 17# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLLAGGPSSGAPPPS-NH ₂

The purity of the compounds was determined using a combination of analytical high performance liquid chromatography and ultra performance liquid chromatography/mass spectrometry with the purity of some compounds shown in Table 4 below.

Table 4. Combination of analytical high-performance liquid chromatography and liquid chromatography/mass spectrometry for the determination of purity and molecular weight of compounds 8#-11# Connection number Purity 8# 96.30% 9# 93.28% 10# 94.56% 11# 92.18%

Biological assessment

The present invention is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

1. Experimental reagent

Table 5. Reagents used in this experiment and sources No. Reagent Source 1 DMEM/F12 Gibco 11330032 2 Casein Sigma C3400-500G 3 3-isobutyl-1-methylxanthine Sigma I7018-250MG 4 cAMP - Gs Dynamic kit - 20,000 tests Cisbio 62AM4PEC 5 Corning® 384-well low volume plate Sigma CLS4514-50EA 6 96-well V-plate (PS) Oxygen WIPP02280 7 Countess® Cell Counting Chambers Invitrogen C10228 8 puromycin ThermoFisher A1113803 9 Hygromycin B Sigma A1720 10 PBS Gibco 10010023 11 0.25% trypsin-EDTA (1×), phenol red ThermoFisher 25200-114 12 Gibco™ Fetal Bovine Serum, Australian Origin, Qualified ThermoFisher 10099-141 13 Glucose Sigma G8270-100G

2. Experimental devices

Table 6. The apparatus used in this experiment and sources No. device Source 1 _CO2 incubator Thermo 311 2 Biological safety cabinet BOXUN BSC-1300IIA2 3 Refrigeration centrifuge Eppendorf 5702R 4 Haier Double Door Refrigerator HaierBCD-268TN 5 Cell counter Life Technologies Countess II 6 Medicine storage box Haier hyc-940 7 Refrigerator at -20°C Haier DW-25L262 8 Refrigeration centrifuge 5810R Eppendorf 5810R 9 Automatic dispenser (Multidrop) Thermo 5840300 10 Microplate reader BioTek H ₁ MFD 11 Bacterial _CO2 incubator BOXUN BC-J80S 12 Active glucometer. Roche

Example 3. Evaluation of the agonist activity of the compounds of the present invention in relation to the glucagon-like peptide-1 receptor (GLP-1R)

1. Purpose of the experiment

This test example is intended to determine the agonist activity of the compounds of the present invention against the glucagon-like peptide-1 receptor (GLP-1R).

2. Experimental procedures

Cryopreserved stable CHO-K1/GLP-1R/CRE-luc cell strains (which can be obtained by conventional methods in the art) were removed from the liquid nitrogen reservoir, quickly thawed in a 37°C water bath, resuspended in DMEM/F12 medium (Gibco cat. No. 11330032) and centrifuged, and the cells were washed once, resuspended in assay buffer, i.e., DMEM/F12 medium containing 0.1% casein (Sigma cat. No. C3400), adjusted for cell density with assay buffer, and seeded in a 384-well plate (Sigma cat. No. CLS4514) at a density of 2500 cells/5 µl/well. Then, 2.5 μl of IBMX working solution (Sigma cat. No. I7018) prepared in buffer (final concentration of IBMX was 0.5 mM) and 2.5 μl of gradient-diluted polypeptide samples were added to each well, and the plate was centrifuged at 1000 rpm for 1 min, shaken for 30 s to mix well, and left to incubate at room temperature for 30 min. Detection was performed using the Cisbio cAMP-Gs Dynamic kit (Cisbio cat. No. 62AM4PEC), and cAMP-d2 and anti-cAMP-Eu3 ⁺ -cryptate were separately diluted in a 20-fold gradient and mixed well with cAMP lysis and detection buffer. 5 μl of diluted cAMP-d2 solution was added to each well, then 5 μl of diluted anti-cAMP-Eu ³⁺ -cryptate solution was added, and the mixture was shaken for 30 seconds to mix well and incubated at room temperature for 1 h in the dark.

3. Data processing

HTRF signal readings were performed using a Biotek Synergy H1 microplate reader at an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm/620 nm × 10,000) was calculated and fitted nonlinearly to sample concentrations in GraphPad Prism 6 using a four-parameter equation to obtain _EC50 values with specific data shown in Table 7 below.

Example 4. Evaluation of the agonist activity of the compounds of the present invention in relation to the glucose-dependent insulinotropic polypeptide receptor (GIP receptor)

1. Purpose of the experiment

This example is intended to determine the agonist activity of the compounds of the present invention at the glucose-dependent insulinotropic polypeptide receptor (GIP receptor).

2. Experimental procedures

Wild-type CHO-K1 cells were harvested, and the cell suspension was adjusted to the appropriate density, seeded in a 6-well plate at 2 ml/well, and placed in an incubator at 37°C with 5% _CO2 for overnight culture adhesion. The transfection mixture (hGIP receptor plasmid, Fugene HD (Promega cat. No. E2311) and OptiMEM (Gibco cat. No. 31985070)) was mixed well and left to stand at room temperature for 15 min, added to the respective cell wells in a volume of 100 μl, and transfected for 24 h to ensure overexpression of the hGIP receptor on the surface of CHO-K1 cells. After completion of transient transfection, cells in the 6-well plate were collected, washed once with assay buffer, i.e., DMEM/F12 medium (Gibco cat.# 11330032) containing 0.1% casein (Sigma cat.# C3400), adjusted for cell density using assay buffer, and seeded in a 384-well plate (Sigma cat.# CLS4514) at a density of 5000 cells/5 µl/well. Then, 2.5 μl of IBMX working solution (Sigma cat. No. I7018) prepared in buffer (final concentration of IBMX was 0.5 mM) and 2.5 μl of gradient-diluted polypeptide samples were added to each well, and the plate was centrifuged at 1000 rpm for 1 min, shaken for 30 s to mix well, and left to incubate at room temperature for 30 min. Detection was performed using the Cisbio cAMP-Gs Dynamic kit (Cisbio cat. No. 62AM4PEC), and cAMP-d2 and anti-cAMP-Eu3 ⁺ -cryptate were separately diluted in a 20-fold gradient and mixed well with cAMP lysis and detection buffer. 5 μl of diluted cAMP-d2 solution was added to each well, then 5 μl of diluted anti-cAMP-Eu ³⁺ -cryptate solution was added, and the mixture was shaken for 30 seconds to mix well and incubated at room temperature for 1 h in the dark.

3. Data processing

The HTRF signal was read using a Biotek Synergy H1 microplate reader at an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm/620 nm × 10,000) was calculated and fitted nonlinearly to sample concentrations in GraphPad Prism 6 using a four-parameter equation to obtain _EC50 values with specific values shown in Tables 7 and 8 below.

Table 7. Results of determination of agonistic activity against human GLP-1R and human GIP receptor Compound Human GLP-1R activity
(EC ₅₀ nM) Human GIP receptor activity
(EC ₅₀ nM) Natural GLP-1 0.010 ND Natural GIP ND 0.011 Semaglutide 0.024 >10 LY3298176 0.13 0.056 7# 0.021 0.11

Table 8. Results of determination of agonistic activity against human GLP-1R and human GIP receptor Compound Human GLP-1R activity
(EC ₅₀ nM) Human GIP receptor activity
(EC ₅₀ nM) Natural GLP-1 0.006 ND Natural GIP ND 0.006 Semaglutide 0.014 >10.0 LY3298176 0.078 0.031 9# 0.049 0.040 10# 0.065 0.056 12# 0.030 0.170 13# 0.017 0.130 14# 0.013 0.130 15# 0.015 0.230 16# 0.029 0.095 17# 0.022 0.110 18# 0.013 0.060

4. Conclusion on the results of the experiment

Due to the design of the polypeptide backbone and subsequent site-specific modification of the fatty acids, the compounds of the present invention exhibit more potent agonist activity at the GLP-1/GIP receptor than many polypeptides with dual agonist activity at the GLP-1/GIP receptor in the art and, thus, have better potential for the treatment of metabolic diseases. Furthermore, LY3298176 exhibits preferential activity at the GIP receptor, in contrast to compounds 12#-18# of the present invention, which exhibit preferential activity at the GLP-1R.

Example 5. Stability testing of some compounds of the present invention

Plasma stability is important for therapeutic polypeptide drugs, as they are likely to be sensitive to polypeptide hydrolases and protein hydrolases in plasma. The half-life and efficacy of polypeptides that are unstable in plasma will be affected.

1. Purpose of the experiment

This experiment is aimed at testing the stability of some compounds of the present invention in human blood plasma.

2. Experimental procedures

5 μl of each sample at concentrations of 20 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 500 ng/ml, 1000 ng/ml, 2000 ng/ml, 5000 ng/ml, and 10000 ng/ml were added to 45 μl of human plasma. The content of compounds in the samples was determined by LC-MS and a standard curve was generated. 5 μl of the polypeptide solution at a concentration of 1 mg/ml were added to 45 μl of human plasma. Five samples were prepared for each test compound, and samples were collected at 0 min, 30 min, 60 min, 120 min, and 240 min, respectively, and the content of the retained compound was determined by LC-MS. At the 0 min content as a standard (100%), the relative content of the retained compounds in the samples at other time points was calculated. The LC-MS method for detecting the compounds was as follows: 5% acetonitrile solution was prepared as solution A, 95% acetonitrile solution was prepared as solution B, the solution gradient was generated at a flow rate of 0.6 mL/min according to the time points and solution proportions shown in Table 9, and 15 μL of the sample was injected and the compound content was determined using a Raptor Biphenyl 2.7 μm detection column, see Table 9.

Table 9. Test time points and solution proportions Time (min) A (%) B (%) 0.20 95.0 5.00 1.70 5.00 95.0 2.00 5.00 95.0 2.01 95.0 5.00 2.50 95.0 5.00

3. Experimental results

Plasma stability data for some compounds of the present invention are provided in Table 10 below.

Table 10. Results of plasma stability tests of compounds Compound Relative content of compounds retained in plasma (%) 0 min 30 min 60 min 120 min 240 min LY3298176 100.00 102.89 114.76 117.59 113.35 7# 100.00 101.45 101.66 103.28 102.15

Conclusion

In the study, it was found that compound 7# of the present invention has similar stability (relative content greater than 90%) in human blood plasma compared to compound LY3298176 at the 4-hour time point.

Example 6. Pharmacokinetic properties of some compounds of the present invention in mice

Plasma stability is one of the factors influencing the pharmacokinetics of polypeptide drugs. The pharmacokinetics of polypeptide drugs in vivo are also influenced by factors such as absorption and clearance of polypeptide drugs in vivo.

1. Purpose of the experiment

This experiment aims to evaluate the pharmacokinetic behavior of the compounds of the present invention in Balb/c mice (plasma) after a single intravenous injection by using mice as experimental animals.

2. Experimental procedures

Seven- to nine-week-old male Balb/c mice weighing 18-30 g were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. After compound 7# was prepared in a buffer containing 20 mM citric acid (pH 7.0), compound 7# was intravenously administered to mice at a dose of 30 nmol/kg body weight through the tail vein, and 0.2 ml of blood was separately collected at time points of 0 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 32 h. The collected blood of mice was centrifuged at 6000 rpm for 6 min at 4°C to separate plasma. The content of compound 7# in the plasma of mice was analyzed using the experimental procedures of Example 3.3.

3. Experimental results

Using the above experimental techniques, the specific data are shown in Table 11 below.

Table 11. Pharmacokinetics of a single intravenous injection in mice (plasma) Pharmacokinetic parameters Units of measurement Connection 7# T _1/2 h 13.0 AUC _Inf h•ng/ml 16133

4. Conclusion on the results of the experiment

It was found that compound 7# of the present invention has good pharmacokinetic properties after intravenous injection to mice, indicating that this compound is useful in the treatment of diseases, for example, it can support subcutaneous injection once a week in humans.

Example 7. Pharmacokinetic properties of some compounds of the present invention in mice

1. Purpose of the experiment

This experiment aims to evaluate the pharmacokinetic behavior of the compounds of the present invention in Balb/c mice (plasma) after a single subcutaneous injection by using mice as experimental animals.

2. Experimental procedures

Seven- to nine-week-old male Balb/c mice weighing 18-30 g were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. After compound 7# was prepared in a buffer containing 20 mM citric acid (pH 7.0), compound 7# was subcutaneously administered to mice at a dose of 30 nmol/kg body weight through the left side of the abdomen, and 0.2 ml of blood was separately collected at time points of 0 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 32 h. The collected blood of mice was centrifuged at 6000 rpm for 6 min at 4°C to separate plasma. The content of compound 7# in the plasma of mice was analyzed according to the experimental procedures of Example 5.2.

3. Experimental results

Using the above experimental techniques, the specific data are shown in Table 12 below.

Table 12. Pharmacokinetic results for compound 7# in mice Pharmacokinetic parameters Units of measurement Connection 7# T _1/2 h 10.1 AUC _Inf h•ng/ml 14488

4. Conclusion on the results of the experiment

It was found that the compound of the present invention has good pharmacokinetic properties after subcutaneous injection to mice, indicating that this compound is useful in the treatment of diseases, for example, it can support subcutaneous injection once a week in humans.

Example 8. In vivo efficacy of certain compounds of the present invention

1. Purpose of the experiment

This experiment was designed to test the regulatory effect of some compounds of the present invention and compound LY3298176 on blood glucose levels in normal mice after a single subcutaneous injection.

2. Experimental procedures

Male C57BL/6 mice aged 10 to 12 weeks were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. C57BL/6 mice were subcutaneously injected with compound 7# or compound LY3298176 (dose: 10 nmol/kg body weight) and control buffer and then subjected to fasting without water deprivation. After 18 h, a glucose solution at a concentration of 0.2 g/mL was intraperitoneally injected. Blood glucose values were measured by collecting blood from the tail of mice at time points of 0 min, 15 min, 30 min, 60 min, and 120 min according to the experimental design. The specific procedures were as follows: the mouse was physically immobilized with the tail exposed, and the tail was slightly incised, then the tail was squeezed until bleeding, and the blood glucose was measured using a Roche active glucometer after discarding the 1st drop of blood. The area under the blood glucose curve (AUC) was calculated from the results of all points.

3. Experimental results

Using the above experimental techniques, the specific data are shown in Table 13 below.

Table 13. Changes in blood glucose levels in mice after a single subcutaneous injection

Tested compounds Dose Blood glucose (mmol/L, mean ± SD) AUC
(mmol/L⋅h) 0 min 15 min 30 min 60 min 120 min Placebo - 5.3 ± 0.6 20.5±2.0 24.0±1.4 19±1.3 10.9±1.2 34.5±2.4 7# 10 nmol/kg 4.4±0.8 6.7±0.8 6.2±1.3 5.7±1.2 3.8±1.1 10.7±1.8 LY3298176 10 nmol/kg 3.2±0.2 9.1±1.3 8-1.4. 6.4±1.0 4.5±0.7 12.7±1.6

SD - standard deviation

4. Conclusion on the results of the experiment

In this experiment, compound 7# of the present invention exhibits a significant blood glucose lowering effect in normal mice at a dose of 10 nmol/kg body weight, with the area under the blood glucose curve of the compound 7# group decreasing by more than 60% compared with that of the placebo (i.e., the blank vehicle).

Example 9. Efficacy of some compounds of the present invention in reducing body weight

1. Purpose of the experiment

This experiment was designed to test the regulatory effect of numbered compounds on body weight of diet-induced obese mice after subcutaneous administration.

2. Experimental procedures

Male high-fat diet-induced obese C57BL/6 mice (weight 35-55 g, aged 10-12 weeks, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.) were tested. Compound LY3298176 (10 nmol/kg body weight), compound 7# (10 nmol/kg body weight), and compound 18# (three doses of 3 nmol/kg, 10 nmol/kg, and 100 nmol/kg body weight, administered once every 3 days) were separately subcutaneously administered to the diet-induced obese C57BL/6 mice. According to the experimental design, the body weight of each mouse was measured and recorded on day 0, day 3, day 6, and so on until day 27, the average body weight of each group of mice was calculated, and body weight change graphs were plotted using the body weight on day 1 as the standard. Finally, the fat and other internal organs from each part of the mice were removed and weighed, and the viscera/brain to fat ratio was calculated for each part of each mouse. The effect of the drug on fat was determined by comparing the change in the organ/brain to fat ratio between different parts of each group of mice.

3. Experimental results

Based on the above experimental procedures, the specific data are shown in Tables 14-16 below and Fig. 1.

Table 14. Weight-reducing effects of compounds on mice with induced obesity Tested compounds Dose Change in body weight (%, mean ± standard deviation) day 1 day 4 day 7 day 10 day 13 day 16 day 19 day 22 day 25 day 28 Placebo - 0 -0.9±1.6 -2.6±1.5 -3.0±2.4 -3.5±3.7 -2.7±4.8 -2.8±6.7 -2.5±8.4 -1.4±8.9 -2.1±9.6 7# 10 nmol/
kg 0 -11.0±1.0 -17.6±2.6 -22.6±5.9 -22.3±6.3 -22.7±6.9 -25.9±6.2 -23.9±5.6 -23.9±5.6 -25.5±5.3

4. Conclusion on the results of the experiment

In this experiment, at doses of 3 nmol/kg, 10 nmol/kg and 100 nmol/kg, compounds 7# and 18# of the present invention showed significant reduction in body weight in high-fat diet-induced obese mice and showed significant dose-dependence. The body weight of the mice in the test group receiving a dose of 10 nmol/kg of compound 18# was reduced by more than 20.0% on day 27, in contrast, the body weight of mice in the same dose test group of the control compound LY3298176 was reduced by about 13.4%. In addition, the fat content in each part (except for the scapular fat) of the mice in all dose test groups of compound 18# was significantly reduced compared with that in the placebo group (i.e., the blank vehicle).

Example 10. Effect of the compounds of the present invention on the food intake of mice

The food intake of mice in each group was measured daily during the test. The results are shown in Table 17 and Fig. 2.

The average daily food intake of DIO (diet-induced obesity) mice in the control group was 2.5 g throughout the experiment. After subcutaneous injection of compound 18# or LY3298176 at various doses, food intake in all groups was reduced to varying degrees.

On the first day after administration, the food intake of mice in each administration group was significantly reduced, and the food intake of mice in the dose groups of 3 nmol/kg, 10 nmol/kg and 100 nmol/kg of compound 18# was 0.6 g, 0.3 g and 0.2 g, respectively, which was significantly different from that in the model control group (2.5 g) and showed better dose dependence.

The total food intake of mice in the model control group within 5 days after administration was 12.8 g, while the total food intake of mice in the 3 nmol/kg, 10 nmol/kg and 100 nmol/kg dose groups of compound 18# within 5 days after administration was 7.2 g, 3.9 g and 1.8 g, respectively, which was significantly lower than that of the model control group and showed better dose dependence.

The daily food intake of mice in each treatment group began to decrease on the first day and resumed on the second and third days after each treatment. Daily food intake showed a general tendency to recover during treatment. After 28 days of treatment, the total food intake of the three dose groups of compound 18# was 58.2 g, 46.8 g, and 36.7 g, respectively, which was significantly lower than that of the model control group (70.8 g) and demonstrated a better dose-response relationship. Thus, compound 18# can significantly reduce food intake in DIO mice.

Example 11. Beneficial effect of certain compounds of the present invention on glucose metabolism in db/db mice

1. Purpose of the experiment

This experiment was designed to test the effect of numbered compound enhancement on glucose metabolism level in db/db mice after subcutaneous administration.

2. Experimental procedures

C57BL/KsJ-db/db mice were individually subcutaneously injected with vehicle blank (20 mM sodium citrate + 0.05% Tween 80, pH 7.5), LY3298176 (100 nmol/kg body weight), and 18# (three doses of 10 nmol/kg body weight, 30 nmol/kg body weight, and 100 nmol/kg body weight) on days 0, 3, 7, 10, 14, 17, 21, 24, and 27. Each administration group included 10 db/db mice. According to the experimental design, tail vein blood was collected by needle prick on days 0, 7, 14, 21, and 28, and fasting blood glucose levels were determined using a glucometer and glucose test strips. Mice were fasted for 6 hours before blood collection at each time point. Tail vein blood was collected by needle prick on days 3, 10, 17, 24, and 27, and blood glucose levels were randomly determined using a glucometer. Finally, at the end of the experiment on day 28, all animals in the treatment groups were anesthetized with 2-5% isoflurane inhalation, and 100 μL of EDTA-K2 anticoagulated whole blood was collected through the orbit of each mouse and used to determine glycated hemoglobin.

3. Experimental results

Based on the above experimental procedures, the specific data are shown in Tables 18-20 below.

Table 18. Effect of long-term administration of compound 18# on fasting blood glucose levels in db/db mice Introduction group Fasting blood glucose concentration (mmol/L, mean ± SD) day 0 day 7 day 14 day 21 day 28 Blank control 14.25±1.27 17.92±1.33 22.89±1.88 24.95±1.52 25.94±1.32 LY3298176 (100 nmol/kg) 14.35±1.41 7.44±0.76 7.56±0.88 9.42±1.67 9.89±1.28*** #18 (10 nmol/kg) 14.77±1.30 6.05±0.42 6.30±0.46 7.89±0.81 9.41±0.97*** #18 (30 nmol/kg) 14.13±1.32 6.21±0.26 6.40±0.57 7.03±0.52 9.68±1.03*** #18 (100 nmol/kg) 14.67±1.46 5.85±0.33 6.25±0.32 6.13±0.19 7.89±0.41*** ***: p < 0.001 compared to the control group receiving a blank control.

Table 19. Effect of long-term administration of compound 18# on fasting blood glucose levels in db/db mice Introduction group Fasting blood glucose concentration
(mmol/L, mean ± SD) day 0 day 10 day 17 24 hours Blank control 23.06±0.97 26.40±0.90 27.64±1.15 30.22±0.74 LY3298176 (100 nmol/kg) 18.60±1.52 17.10±1.96 17.98±1.37 20.70±1.27*** #18 (10 nmol/kg) 20.42±1.56 20.66±1.48 18.91±1.33 21.17±2.07** #18 (30 nmol/kg) 16.73±1.59 15.88±1.86 17.30±1.17 17.43±1.92*** #18 (100 nmol/kg) 9.11±1.25 12.34±1.12 11.89±1.15 11.51±0.95*** **: p < 0.01 compared with the blank solution control group; ***: p < 0.001 compared with the blank solution control group.

Table 20. Effect of long-term administration of compound 18# on glycated hemoglobin levels in db/db mice Introduction group Glycated hemoglobin (%, mean ± SD) Blank control 6.54±0.17 LY3298176 (100 nmol/kg) 4.58±0.23** #18 (10 nmol/kg) 4.71±0.23*** #18 (30 nmol/kg) 4.53±0.17*** #18 (100 nmol/kg) 3.78±0.13*** **: p < 0.01 compared with the blank solution control group; ***: p < 0.001 compared with the blank solution control group.

4. Conclusion on the results of the experiment

In this experiment, at doses of 10 nmol/kg, 30 nmol/kg and 100 nmol/kg, compound 18# of the present invention exhibits an excellent effect in improving the glucose metabolism level in db/db mice and shows a significant dose dependence. The glycated hemoglobin level in the 100 nmol/kg dose group of compound 18# is 3.78% at the end of the experiment, in contrast, the glycated hemoglobin level in the same dose group of the control compound LY3298176 is 4.58%. Thus, the efficacy of compound 18# in improving the glucose metabolism level in db/db mice is significantly better than that of the control compound LY3298176 at the same dose.

--->

SEQUENCE LIST

<110> BEIJING TO JIE BIOPHARMACEUTICAL CO. LTD.

<120> A COMPOUND WITH DUAL RECEPTOR AGONIST ACTIVITY

GLP-1 and GIP and its use

<130> 721055CPCT

<140> PCT/CN2021/096568

<141> 2021-05-28

<150> 202010472577.8

<151> 2020-05-29

<150> 202110335100.X

<151> 2021-03-29

<160> 20

<170> Patent-In 3.5

<210> 1

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 1

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 2

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 2

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 3

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 3

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 4

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 4

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 5

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 5

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 6

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 6

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 7

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 7

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Asp Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 8

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 8

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 9

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 9

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 10

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 10

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 11

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 11

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 12

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 12

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 13

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 13

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 14

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 14

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 15

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 15

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Ile Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 16

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 16

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 17

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 17

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Arg

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 18

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<400> 18

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Lys Glu Lys

1 5 10 15

Ile Ala Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 19

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (1)..(1)

<223> Xaa is selected from the group consisting of Tyr and His

<220>

<221> Option

<222> (2)..(2)

<223> Xaa is selected from the group consisting of Aib and D-Ala

<220>

<221> Option

<222> (10)..(10)

<223> Xaa is selected from the group consisting of Val and Tyr

<220>

<221> Option

<222> (12)..(12)

<223> Xaa is selected from the group consisting of Ser and Ile

<220>

<221> Option

<222> (13)..(13)

<223> Xaa is selected from the group consisting of Tyr and Ala

<220>

<221> Option

<222> (14)..(14)

<223> Xaa is selected from the group consisting of Leu and Nle

<220>

<221> Option

<222> (15)..(15)

<223> Xaa is selected from the group consisting of Asp and Glu

<220>

<221> Option

<222> (16)..(16)

<223> Xaa is selected from the group consisting of Arg, Glu, Gly, Lys and Aib

<220>

<221> Option

<222> (17)..(17)

<223> Xaa is selected from the group consisting of Glu, Ile and Gln

<220>

<221> Option

<222> (18)..(18)

<223> Xaa is selected from the group consisting of Ala, Aib and His

<220>

<221> Option

<222> (19)..(19)

<223> Xaa is selected from the group consisting of Ala, Aib and Gln

<220>

<221> Option

<222> (20)..(20)

<223> Xaa is selected from the group consisting of Gln, Glu and Lys

<220>

<221> Option

<222> (23)..(23)

<223> Xaa is selected from the group consisting of Ile and Val

<220>

<221> Option

<222> (24)..(24)

<223> Xaa is selected from the group consisting of Ala, Asn and Gln

<220>

<221> Option

<222> (27)..(27)

<223> Xaa is selected from the group consisting of Val and Leu

<220>

<221> Option

<222> (28)..(28)

<223> Xaa is selected from the group consisting of Arg and Ala

<220>

<221> Option

<222> (29)..(29)

<223> Xaa is selected from the group consisting of Gly and Gln

<220>

<221> Option

<222> (30)..(30)

<223> Xaa is selected from the group consisting of Gly and Lys

<400> 19

Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Glu Phe Xaa Xaa Trp Leu Xaa Xaa Xaa Xaa Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 20

<211> 39

<212> Protein

<213> Artificial sequence

<220>

<221> Option

<222> (2)..(2)

<223> Xaa represents Aib

<220>

<221> Option

<222> (14)..(14)

<223> Xaa represents Lys or Leu

<220>

<221> Option

<222> (15)..(15)

<223> Xaa is Asp or Glu

<220>

<221> Option

<222> (16)..(16)

<223> Xaa is Lys or Arg

<220>

<221> Option

<222> (23)..(23)

<223> Xaa represents Val or Ile

<220>

<221> Option

<222> (27)..(27)

<223> Xaa represents Ile or Leu

<400> 20

Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Xaa Xaa Xaa

1 5 10 15

Ile Ala Ala Gln Glu Phe Xaa Asn Trp Leu Xaa Ala Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

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Claims (41)

1. A GLP-1 analogue or a pharmaceutically acceptable salt thereof that has dual agonist activity at the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic polypeptide) receptor, wherein the GLP-1 analogue is one of the following: 7# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)DKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 9# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 10# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLIAGGPSSGAPPPS-NH ₂ 11# H-YAibEGTFTSDYSIYLEK(OEG-OEG-yGlu-C20-OH)IAAQEFINWLLAGGPSSGAPPPS-NH ₂ 12# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 13# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLIAGGPSSGAPPPS-NH ₂ 14# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 15# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFINWLIAGGPSSGAPPPS-NH ₂ 16# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFINWLLAGGPSSGAPPPS-NH ₂ 17# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)ERIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ 18# H-YAibEGTFTSDYSIYK(OEG-OEG-yGlu-C20-OH)EKIAAQEFVNWLLAGGPSSGAPPPS-NH ₂ , where the substituent on lysine OEG-OEG-yGlu-C20-OH has the structure of the formula {[2-(2-amino-ethoxy)-ethoxy]-acetyl} ₂ -(y-Glu) ₁ -CO-(CH ₂ ) ₁₈ -COOH. 2. A GLP-1 analogue or a pharmaceutically acceptable salt thereof that has a dual agonistic effect on the GLP-1 receptor and the GIP receptor, wherein the GLP-1 analogue is selected from the group consisting of compounds 7#, 12#, 13#, 14#, 15#, 16#, 17# and 18#, where The 7# connection has a structure as shown below: , The 12# connection has a structure as shown below: , The 13# connection has a structure as shown below: , The 14# connection has a structure as shown below: , The 15# connection has a structure as shown below: , The 16# connection has a structure as shown below: , The 17# connection has a structure as shown below: , And The 18# connection has a structure as shown below: . 3. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 7#, having a structure as shown below: . 4. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 12#, having a structure as shown below: . 5. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 13#, having a structure as shown below: . 6. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 14#, having a structure as shown below: . 7. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 15#, having a structure as shown below: . 8. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 16#, having a structure as shown below: . 9. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 17#, having a structure as shown below: . 10. The GLP-1 analogue or pharmaceutically acceptable salt thereof according to claim 2, wherein the GLP-1 analogue is compound 18#, having a structure as shown below: . 11. A pharmaceutical composition that has a dual agonistic effect on the GLP-1 receptor and the GIP receptor, comprising: 1) a GLP-1 analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1-10, and 2) a pharmaceutically acceptable excipient or pharmaceutical carrier. 12. The use of a GLP-1 analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1-10 or a composition according to claim 11 for the treatment of non-insulin-dependent diabetes, insulin-dependent diabetes, obesity, non-alcoholic fatty liver disease, liver steatosis, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, dyslipidemia associated with insulin resistance, and/or dyslipidemia associated with diabetes. 13. The use according to paragraph 12, intended for the treatment of insulin resistance. 14. The use according to claim 12, intended for the treatment of dyslipidemia associated with diabetes. 15. The use according to claim 12, intended for the treatment of dyslipidemia associated with insulin resistance.