Gipr and glp-1r co-agonist polypeptides and uses thereof

Abstract

Provided are GIPR and GLP-1R co-agonist polypeptides, or pharmaceutically acceptable salts, amides or esters thereof, and uses of the above polypeptides, salts, amides or esters, including treating type II diabetes, obesity or overweight, etc.

Description

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202410580912.4, filed May 11, 2024, and Chinese Patent Application No. 202510512619.9, filed April 23, 2025, the entire contents of which are incorporated herein by reference and used for all purposes. Technical Field

[0003] This application relates to the field of biopharmaceuticals, and more specifically, to GIPR and GLP-1R co-stimulatory peptides, or pharmaceutically acceptable salts, amides or esters thereof, and the use of such peptides, salts, amides or esters. Background Technology

[0004] According to the American Diabetes Association's 2018 classification and diagnostic guidelines for diabetes, diabetes is mainly divided into the following types: Type 1 diabetes is caused by the autoimmune system mistakenly attacking and destroying pancreatic beta cells, leading to an absolute lack of insulin; Type 2 diabetes (T2D) occurs against a background of insulin resistance, where beta cells gradually reduce insulin production; Gestational diabetes mellitus (GDM) is diabetes first diagnosed during pregnancy; and there are also diabetes caused by other specific factors, such as monogenic diabetes syndromes, pancreatic exocrine diseases (such as cystic fibrosis and pancreatitis), and drug or chemical-induced diabetes (such as diabetes after steroid treatment for HIV / AIDS or organ transplantation). Among these types, T2D is the most common, formerly known as adult-onset diabetes, accounting for more than 90% of all diabetes cases. Currently, an increasing number of children are being diagnosed with T2D, which may be related to the rising rate of childhood obesity. As the most common metabolic disease, the number of T2D patients has been steadily increasing over the past half-century, showing a trend of spreading from the US and Europe to the Western Pacific region, including Asia and Africa. According to current models, nearly 700 million people worldwide will be affected by this disease by 2045. Unlike type 1 diabetes caused by congenital insulin insufficiency, the occurrence and development of T2D are mainly related to insulin resistance.

[0005] Obesity is a complex disease caused by a combination of factors, and excessive accumulation of body fat has adverse effects on health. Currently, obesity is increasing rapidly, forming a large-scale epidemic that shows no signs of slowing down in the short term. Obesity is a risk factor for non-communicable diseases such as diabetes and heart disease, seriously impacting people's quality of life and life expectancy.

[0006] Obesity is also one of the main causes of type 2 diabetes (T2D). The progression from obesity to diabetes generally follows this pattern: obesity → impaired glucose tolerance → T2D → uncontrollable hyperglycemia → diabetic complications. Therefore, for the treatment of diabetes accompanied by obesity, in addition to lowering blood sugar, weight loss is also a necessary consideration.

[0007] The glucagon-like peptide-1 receptor (GLP-1R) belongs to the G protein-coupled receptor B cluster and is a member of the glucagon receptor subfamily. Its structure is distinctive, including a relatively long extracellular N-terminal region (ECD), approximately 100-150 amino acids long, containing seven transmembrane domains (TMDs), and a relatively short intracellular C-terminal region (ICD). The extracellular region consists of two loop-connected inverted β-sheets, containing six conserved cysteine ​​residues forming three disulfide bonds, and a flexible α-helix.

[0008] GLP-1R is widely distributed in body tissues, including the pancreas, stomach, small intestine, heart, kidneys, lungs, and brain. In pancreatic β-cells, GLP-1R primarily promotes insulin secretion, enhances β-cell regeneration, inhibits apoptosis, and reduces glucagon release. In tissues such as the gastrointestinal tract, GLP-1R slows gastrointestinal motility, inhibits gastric juice secretion, delays gastric emptying, and enhances satiety by binding to its agonists. In the nervous system, GLP-1R agonists can penetrate the brain, protecting nerve cells from apoptosis and enhancing learning and memory abilities by activating GLP-1R. Furthermore, activating GLP-1R can help with weight loss by controlling food intake. In the cardiovascular system, studies knocking out the GLP-1R gene in mice have shown that this leads to a decrease in resting heart rate and an increase in left ventricular diastolic pressure. In addition, activating GLP-1R can reduce oxidative stress and inhibit cardiomyocyte apoptosis.

[0009] The glucose-dependent insulinotropic polypeptide receptor (GIPR) is also a G protein-coupled receptor with seven transmembrane domains. The N-terminus of GIPR contains a glycosylated sequence and a third cytoplasmic loop, while the C-terminus is rich in threonine and serine residues, which are potential phosphorylation sites. GIPR is expressed in various organs and tissues, such as the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, heart, pituitary gland, bone, lung, and spleen. In the pancreatic islets, only α and β cells express GIPR. GIPR increases intracellular cAMP and calcium levels by activating G proteins. 2+GIPR signals regulate the expression of downstream genes through signaling pathways such as PI3K, PAK, and PKB. After food intake, GIPR signaling promotes insulin secretion and lowers blood glucose levels. GIPR also affects the storage and metabolism of adipocytes, as well as glucose synthesis and release in the liver. Studies have shown that GIPR plays an important role in regulating insulin secretion, blood glucose, and lipid metabolism, and is crucial for maintaining normal metabolic status.

[0010] In recent years, the importance of the GIPR-GIP pathway has received increasing attention. The GIPR signaling pathway participates in various physiological processes, including pancreatic islet proliferation, energy metabolism regulation, and intestinal function control, and is closely related to lipid accumulation and insulin resistance in adipose tissue. However, the downstream signaling pathways activated by GIPR-GIP require further investigation. In adipocytes, GIPR-GIP enhances lipoprotein lipase (LPL) activity and promotes fat accumulation by activating CREB, TORC2, or insulin, and promoting PKB phosphorylation. GIP can also promote IL-6 expression in adipose tissue, inducing insulin resistance. GIPR-GIP reduces caspase3 and bax gene activity through the MAPK, Akt, and FoxO1 pathways, exerting pro-proliferative and anti-apoptotic effects. There are also reports indicating that activation of the GIPR-GIP signaling pathway can promote leptin resistance in obese individuals. GIP induces central leptin resistance by activating the Epac / Rapl signaling pathway in the hypothalamus, inhibiting the activation of leptin receptor signaling pathway molecules, promoting the expression of negative regulators of the leptin signaling pathway, and inhibiting leptin-induced activation of opioid-melanocyte-stimulating corticotropin-promoting neurons. Furthermore, GIPR-GIP enhances GIPR expression by upregulating TCF4 through the Akt signaling pathway, thereby improving β-cell proliferation and insulin secretion, and maintaining glycemic homeostasis.

[0011] In 2022, Eli Lilly launched telpokines, the world's first dual GLP-1R / GIPR receptor agonist. Telpokines is a clinically effective peptide that targets both GIPR and GLP-1R for lowering blood sugar and reducing weight. While it increases GIPR activation, it may affect GLP-1R activation.

[0012] Given the promising prospects of GLP-1R / GIPR dual receptor agonists, the development of more drugs of this kind is urgently needed in this field. Invention Overview

[0014] The first aspect of this application relates to a GIPR and GLP-1R co-activating polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, said polypeptide having the general formula sequence shown in SEQ ID No:1:

[0015] R1X1X2GluX4X5X6X7X8X9X 10 X11 X 12 X 13 LeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:1), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 X 13 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 and X 33 Independently, R1 can be any natural amino acid residue, non-natural amino acid residue, or absent; and / or R2 can be any peptide or absent; and / or R2 can be -NH2, any peptide, or absent.

[0016] The second aspect of this application relates to pharmaceutical compositions comprising the GIPR and GLP-1R co-activating peptides as described in the first aspect, or pharmaceutically acceptable salts, amides, or esters thereof, and pharmaceutically acceptable carriers, diluents, and / or excipients.

[0017] The third aspect of this application relates to methods for preventing, treating, and / or alleviating metabolic diseases or conditions, the methods comprising administering to a patient or subject in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect.

[0018] The fourth aspect of this application relates to a method for weight loss, the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect.

[0019] The fifth aspect of this application relates to a method for reducing food intake, the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect.

[0020] The sixth aspect of this application relates to a method for improving blood glucose control, the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect.

[0021] The seventh aspect of this application relates to the use of the polypeptides as described in the first aspect, or pharmaceutically acceptable salts, amides, or esters thereof, or pharmaceutical compositions as described in the second aspect, in the preparation of a medicament for one or more of the following purposes: (1) prevention, treatment, and / or relief of metabolic diseases or conditions; (2) prevention, treatment, and / or relief of diabetes; preferably, said diabetes is type II diabetes; (3) prevention, treatment, and / or relief of obesity or overweight; (4) weight loss; (5) reduction of food intake; and (6) improvement of glycemic control (e.g., improvement of glucose tolerance).

[0022] Brief description of the attached figures

[0023] Figure 1 The chemical structure of an exemplary molecule of this application, DTM0001, is shown.

[0024] Figure 2 The chemical structure of an exemplary molecule of this application, DTM0002, is shown.

[0025] Figure 3 The chemical structure of an exemplary molecule of this application, DTM0003, is shown.

[0026] Figure 4 and Figure 5 The chemical structural formulas of exemplary molecules DTM0026 and DTM0028 of this application are shown.

[0027] Detailed description of the invention

[0028] The embodiments described below are for the purpose of better illustrating the content of this application, but are not limited to the embodiments described herein. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above-described invention are still within the scope of protection of this application.

[0029] Although various embodiments of this application are described in detail below, it should be understood that this application is not limited to the specific methods, schemes, and reagents described herein, as these methods, schemes, and reagents can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application, which is defined solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0030] The range of values ​​listed herein is intended solely as a shorthand for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were listed separately herein. Unless otherwise indicated herein or otherwise clearly contradictory to the context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended solely to better illustrate the application and does not impose any limitation on the scope of the originally claimed application. No language in this specification should be construed as indicating that any unclaimed element is necessary for the practice of this application.

[0031] Numerous references are cited throughout this specification. Every reference cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer's instructions, guidance, etc.), is incorporated herein by reference in its entirety.

[0032] the term

[0033] Throughout this specification and claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply inclusion of the stated member, integer, or step, or a group of members, integers, or steps, but not to exclude any other member, integer, or step, or a group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps, or a group of members, integers, or steps may be excluded, i.e., the subject matter is to include the stated member, integer, or step, or a group of members, integers, or steps.

[0034] Unless otherwise indicated herein or clearly contradicted by the context, the terms “a” and “an” as used in the context of describing this application (especially in the context of the claims) and similar references shall be interpreted to cover both the singular and the plural.

[0035] As used herein, the terms “agonize,” “agonism,” and “agonizing” refer to an increase in GLP-1R and / or GIPR signaling. The terms “activation” and “agonism” are used interchangeably.

[0036] In this application, when used in conjunction with amino acids, the term “natural” refers to 20 common amino acids (i.e., alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)) as well as selenocysteine, pyrrolidone (PYL), and pyrrolidone-carboxylysine (PCL).

[0037] In this article, all natural amino acids without an specified optical isomer are understood to refer to the L-isomer, unless otherwise stated.

[0038] As used herein, when used in conjunction with amino acids, the term "non-natural" is intended to refer to an amino acid that is not naturally encoded or found in the genetic code of any organism. It can, for example, be a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azacyclobutanecarboxylic acid, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmodium, 2,2'-diaminopimelic acid, and 2,3-diamino... Propionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, n-valine, n-leucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, piperidinic acid, and thioproline.

[0039] The term "amino acid residue" includes amino acids in which a hydrogen atom has been removed from the amino group and / or a hydroxyl group has been removed from the carboxyl group and / or a hydrogen atom has been removed from the thiol group. In imprecisely, the term "amino acid residue" may sometimes be used interchangeably with "amino acid" in this document. Throughout this document, the abbreviations for each amino acid in the sequence refer to the corresponding amino acid residue.

[0040] In this article, the term "Aib" refers to 2-aminoisobutyric acid, and when it forms a polypeptide, it represents 2-aminoisobutyryl.

[0041] In this article, the term "AEEA" refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid, which, when used as a group in a compound, represents 2-[2-(2-aminoethoxy)ethoxy]acetyl.

[0042] In this article, the term "γ-Glu" refers to γ-glutamyl.

[0043] The term "modification" of a peptide as used in this application includes any alteration made to the peptide, such as changes in peptide length, amino acid sequence, chemical structure, co-translational modifications, or post-translational modifications. In some cases, the peptide of this application comprises one or more modified amino acid residues. The types of modifications are well known in the art. Modifications that can be used in this application include amidation, fatty acid modification, methylation, myristylation, PEGylation, fluorine modification, biotinylation, fluorescent labeling, cyclization, carboxylation, acetylation, phosphorylation, glycosylation, or other known peptide modifications.

[0044] As used herein, any term “treat,” “treating,” or “treatment” relating to a disease, disorder, or condition means to reduce or improve a disease, disorder, or condition (i.e., to slow or stop the development or progression of a disease, disorder, or condition or at least one of its clinical symptoms); or to reduce or improve at least one physical parameter or biomarker associated with a disease, disorder, or condition, including those physical parameters or biomarkers that the patient may not be able to identify.

[0045] As used herein, any term “prevent,” “preventing,” or “prevention” relating to a disease, disorder, or condition means preventive treatment of the disease, disorder, or condition; or delaying the onset or progression of the disease, disorder, or condition.

[0046] As used herein, a subject, patient, or individual is considered "needed" for treatment if the subject would benefit biologically, medically, or in terms of quality of life from the treatment. In this text, the terms "subject," "patient," and "individual" are used interchangeably in certain contexts and have the same meaning.

[0047] The term "effective amount" refers to an amount that will elicit a biological or medical response in a subject. As a non-limiting example, such an effective amount described herein may, for example, activate GLP-1R and / or GIPR activity, improve one or more symptoms, alleviate one or more conditions, slow, reduce or delay the progression of a disease, disorder or condition, or prevent a disease, disorder or condition.

[0048] The metabolic diseases described herein have the same meaning as those described in this application.

[0049] The term "body mass index" or "BMI" for a human patient or subject is defined as weight in kilograms divided by the square of height in meters; therefore, BMI has the unit kg / m². 2 .

[0050] The term "obesity" means excess adipose tissue. When energy intake exceeds energy expenditure, excess calories are stored in adipose tissue, and if this net positive balance continues, obesity results; that is, weight balance has two components, and an abnormality on either side (intake or expenditure) can lead to obesity. In this context, obesity is best considered as any degree of excess adipose tissue that poses health risks. The difference between normal and obese individuals can only be approximated, but the health risks associated with obesity are likely a continuous process with increasing adipose tissue. The term "obesity disorder" refers to a metabolic disease affecting the whole body caused by the excessive accumulation of adipose tissue in the body. In this article, "obesity" and "obesity disorder" may be used interchangeably in some cases.

[0051] The WHO defines "overweight" as an individual having a body weight greater than 25 kg / m². 2 And less than 30kg / m 2 BMI is a symptom of obesity. In some cases, the terms "overweight" and "pre-obesity" have the same meaning.

[0052] The WHO defines "obesity" as an individual having a body mass index (BMI) of 30 kg / m² or higher. 2 Obesity is a symptom of BMI. According to the WHO definition, obesity can be classified as follows: the term "Group I obesity" refers to a BMI equal to or greater than 30 kg / m². 2 But below 35kg / m 2 The symptoms; the term "Class II obesity" refers to a BMI equal to or greater than 35 kg / m². 2 But below 40kg / m2 The symptoms; the term "Class III obesity" refers to a BMI equal to or greater than 40 kg / m². 2 symptoms.

[0053] In this article, in some cases, the term "obesity" refers to a subject who is otherwise healthy and has a body weight of 30 kg / m² or greater. 2 The body mass index (BMI) status, or subjects with at least one comorbidity having a BMI greater than or equal to 27 kg / m². 2 The subject's BMI status. "Obese subjects" are those with a BMI greater than or equal to 30 kg / m². 2 Subjects with a body mass index (BMI) who are otherwise healthy, or who have a body mass index (BMI) greater than or equal to 27 kg / m² 2 Subjects with a BMI of 25 kg / m² and at least one comorbidity. "Overweight," "pre-obese," or "at risk of obesity" is defined as subjects with a BMI of 25 kg / m². 2 to less than 30kg / m 2 Subjects with a BMI who are otherwise healthy, or have a BMI of 25 kg / m² 2 to less than 27kg / m 2 Subjects with a BMI and at least one comorbidity.

[0054] The increased risk of obesity in Asians occurs at lower body mass index (BMI). In Asian countries, "obesity" is defined as a body mass index (BMI) of 25 kg / m² or higher in subjects with at least one obesity-inducing or obesity-related comorbidity. 2 The BMI status, the comorbidity requiring weight loss or improvement through weight loss. In Asian countries, "obese subject" is defined as having a BMI greater than or equal to 25 kg / m². 2 Subjects with a BMI greater than 23 kg / m² and at least one obesity-induced or obesity-related comorbidity requiring or being improved by weight loss. In Asian countries, “overweight,” “pre-obese,” or “subjects at risk of obesity” are defined as those with a BMI greater than 23 kg / m². 2 to less than 25kg / m 2 Subjects with a BMI.

[0055] As used in this article, the term “obesity” is intended to include all of the above definitions of obesity.

[0056] As used herein, the term "overweight" is intended to include all of the above definitions of overweight. Obesity-induced or obesity-related comorbidities include, but are not limited to, type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwick syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver; cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic conditions, osteoarthritis, low back pain, menstrual disorders, and infertility. In particular, comorbidities include: hypertension, hyperlipidemia, dyslipidemia, impaired glucose tolerance, cardiovascular disease, sleep apnea, diabetes, and other obesity-related conditions.

[0057] The term "weight loss" refers to the reduction of body weight in people and / or animals. Weight loss may be in line with concerns about improving health, fitness levels, and / or appearance. The terms "weight loss," "weight reduction," and "weight loss" have the same meaning and are used interchangeably.

[0058] The term "diabetes" as used in this article includes insulin-dependent diabetes mellitus (IDDM, also known as type 1 diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, also known as type 2 diabetes), a metabolic disease. Type 1 diabetes, or insulin-dependent diabetes, is the result of an absolute deficiency of insulin (the hormone that regulates glucose utilization). Type 2 diabetes, or diabetes that does not depend on insulin (i.e., non-insulin-dependent diabetes), often occurs in the presence of normal or even elevated levels of insulin and appears to be the result of tissues being unable to respond properly to insulin.

[0059] The term “improved glycemic control” includes, but is not limited to, improving glucose tolerance in subjects, reducing intraperitoneal plasma glucose, postprandial plasma glucose and / or glycosylated hemoglobin (HbA1c) levels in patients or subjects.

[0060] In this article, the term "HbA1c" or "glycated hemoglobin" refers to the product of the combination of hemoglobin in red blood cells and carbohydrates (mainly glucose) in serum through a non-enzymatic reaction. The non-enzymatic reaction that forms glycated hemoglobin is continuous, slow, and irreversible. Therefore, the level of glycated hemoglobin is determined by past, rather than immediate, blood glucose concentrations, and is unrelated to factors such as fasting, insulin injection, or the use of hypoglycemic drugs before the test. It is generally believed that glycated hemoglobin concentration can effectively reflect the average blood glucose level over the past 8–12 weeks. Glycated hemoglobin is composed of HbA1a, HbA1b, and HbA1c, with HbA1c accounting for approximately 70% and having a relatively stable structure. Clinically, it is commonly used as a monitoring indicator for diabetes control, and its concentration should be expressed as a percentage of adult hemoglobin.

[0061] As used herein, unless otherwise stated, the amino acids in the term "general formula sequence" or other polypeptide sequences are arranged in order from the N-terminus to the C-terminus.

[0062] In general, this application provides various uses of GIPR and GLP-1R co-stimulatory peptides or pharmaceutically acceptable salts, amides or esters thereof, combinations or compositions comprising said GIPR and GLP-1R co-stimulatory peptides or pharmaceutically acceptable salts, amides or esters thereof, and combinations or compositions comprising said peptides or pharmaceutically acceptable salts, amides or esters thereof, including but not limited to the prevention, treatment and / or relief of type II diabetes, obesity and overweight.

[0063] The following contains a description of certain elements of this application, which can be listed in specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The differentiated descriptions of embodiments and exemplary embodiments should not be construed as limiting this application to only the explicitly described embodiments. This specification should be understood to support and cover embodiments combining the explicitly described embodiments with any number of disclosed and / or exemplary elements. Furthermore, unless the context otherwise indicates, any permutation and combination of the elements described in this application should be considered as disclosed in this specification.

[0064] Specifically, the first aspect of this application relates to a GIPR and GLP-1R co-activating polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, said polypeptide having the general formula sequence shown in SEQ ID No:1:

[0065] R1X1X2GluX4X5X6X7X8X9X 10 X 11 X 12 X 13 LeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:1), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X11 X 12 X 13 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 and X 33 Independently, R1 can be any natural amino acid residue, non-natural amino acid residue, or absent; and / or R2 can be any peptide or absent; and / or R2 can be -NH2, any peptide, or absent.

[0066] In some embodiments, the polypeptide has a general formula sequence shown in any one of SEQ ID No:2-21. In the general formula sequence shown in any one of SEQ ID No:2-21, variables common to the general formula sequence shown in SEQ ID No:1 (e.g., R1, R2, X) are also included. n (etc.) In the absence of a specific reference, the definition of the variable can be found in the general formula sequence shown in SEQ ID No:1.

[0067] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:2:

[0068] X1X2GluX4X5X6X7X8X9X 10 X 11 X 12 X 13 LeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSer (SEQ ID No:2), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12X 13 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 and X 33 Independently, it can be any natural amino acid residue, non-natural amino acid residue, or none at all.

[0069] In some embodiments, X1 is His, Phe, or Tyr; and / or X2 is Aib or Ala; and / or X4 is Gly or Ala; and / or X5 is Thr, Asn, or Ser; and / or X6 is Phe or His; and / or X7 is Thr or Val; and / or X8 is Thr or Ser; and / or X9 is Asp or Glu; and / or X... 10 For Tyr or Phe; and / or the X mentioned above. 11 For Thr or Ser; and / or the X 12 For Ile or Asn; and / or the X 13 For Tyr, Gln, Ala, or Gly; and / or the X mentioned above. 15 For Asp, Glu, or Gln; and / or the X 16 For Glu or Lys; and / or the X 17 For Gln, Leu, or Ile; and / or the X mentioned above. 18 For Ala, His, or Gly; and / or the aforementioned X 19 Gln or Asn; and / or the X 20 For Arg, Gln, or Lys; and / or the X 21 For Asp, Glu, or Ala; and / or the X 22 For Phe or Tyr; and / or the X 23 For Ile or Val; and / or the X 24 Gln, Lys, or Asn; and / or the X 25 For Trp or Tyr; and / or the X 26 For Leu or Ala; and / or the X mentioned above. 27 Val or Ile; and / or the X 33 For Ser or Lys.

[0070] In some implementations, X1 is His, Phe, or Tyr. X1 being His, Phe, or Tyr can activate GLP-1R and GIPR. In some implementations, X1 is Tyr.

[0071] In some embodiments, X2 is Aib or Ala. This site on both GLP-1 and GIP is a cleavage site for dipeptidyl peptidase-4 (DPP-4). In some embodiments, X2 is Aib, and substitution of X2 with Aib completely prevents the degradation of DPP-4, while Aib substitution has almost no effect on its activation of GLP-1R and GIPR activities.

[0072] In some embodiments, X4 is Gly or Ala. In some embodiments, X4 is Gly, and the mutation of X4 from Gly to Ala slightly reduces its activity in activating GLP-1R and GIPR.

[0073] In some embodiments, X5 is Thr, Asn, or Ser. In some embodiments, X5 is Thr, and replacing Asn with Thr in X5 significantly enhances its activity in activating GLP-1R and GIPR.

[0074] In some embodiments, X6 is Phe or His. In some embodiments, X6 is Phe, and replacing Phe with His slightly reduces its activity in activating GLP-1R and GIPR.

[0075] In some embodiments, X7 is Thr or Val. In some embodiments, X7 is not Ile, as introducing Ile into this site results in a decrease in its GLP-1R activation activity. In some embodiments, X7 is Thr.

[0076] In some embodiments, X8 is Thr or Ser. In some embodiments, X8 is Ser.

[0077] In some embodiments, X9 is Asp or Glu. In some embodiments, X9 is Asp, and replacing Asp with Glu slightly reduces its activity in activating GLP-1R and GIPR.

[0078] In some implementations, the X 10 For Tyr or Phe. In some implementations, the X 10 For Tyr.

[0079] In some implementations, the X 11 For Thr or Ser. In some implementations, the X 11 For Ser.

[0080] In some implementations, the X 12 For Ile or Asn. In some embodiments, the X 12 For Ile, X 12 The Ile mutation to Ala can significantly reduce its insulin-promoting effect. Compared with Ala, the use of Ile at this position does not weaken its activation of GLP-1R activity, but rather enhances its activation of GIPR activity.

[0081] In some implementations, the X 13 It can be Tyr, Gln, Ala, or Gly. In some implementations, X... 13 For Tyr, X 13 Replacing Tyr with Gln slightly reduces its activity in activating GLP-1R and GIPR.

[0082] In some implementations, the X 15 It is Asp, Glu, or Gln. In some embodiments, X... 15 For Glu, X 15 Mutations of Glu to Ala or Gly can negatively impact the activity of its activating receptor.

[0083] In some implementations, the X 16 For Glu or Lys. In some embodiments, the X 16 For Glu, X 16 Replacing Glu with Glu increases its affinity for GLP-1R and GIPR to some extent. In some embodiments, the X 16 For Lys, X 16 Replacing GLP-1 with Lys can increase affinity and GLP-1 activity to some extent.

[0084] In some implementations, the X 17 It is Gln, Leu, or Ile. In some embodiments, X 17 For Ile, X 17 The Ile is important for activating GIPR activity. In some embodiments, the X 17 Gln is used, and Gln is important for activating GLP-1R activity. In some embodiments, X... 17 For Ile.

[0085] In some implementations, the X 18 It can be Ala, His, or Gly. In some implementations, the X... 18 For Ala, X 18 This enhances Ala's ability to activate both GLP-1R and GIPR.

[0086] In some implementations, the X 19 It is Gln or Asn. In some implementations, the... 19 For Gln. In some implementations, X 19 For Asn, X 19 Replacing Gln with Asn slightly reduces its activity in activating GLP-1R and GIPR.

[0087] In some implementations, the X 20 It is Arg, Gln, or Lys. In some embodiments, the X 20 For Arg, X 20 Arg enhanced its activity in activating GLP-1R.

[0088] In some implementations, the X 21 For Asp, Glu, or Ala. In some implementations, the X... 21 For Glu. In some implementations, the X 21 Asp can form hydrogen bonds with Arg on GIPR, which plays an important role in maintaining GIP activity.

[0089] In some implementations, the X 22 For Phe or Tyr. In some implementations, the X 22 For Phe, X 22 Replacing Phe with Tyr significantly reduces its activity in activating GIPR targets.

[0090] In some implementations, the X 23 For Ile or Val. In some implementations, the X 23 For Ile.

[0091] In some implementations, the X 24 It is Gln, Lys, or Asn. In some embodiments, X... 24 For Gln, X 24 Gln can enhance its activity in activating GLP-1R.

[0092] In some implementations, the X 25 For Trp or Tyr. In some implementations, the X 25 For Trp.

[0093] In some implementations, the X 26 For Leu or Ala. In some implementations, the X 26 For Leu. In some implementations, the X 26 Not for His, X26 Replacing Leu with His significantly reduces its activity in activating GLP-1R and GIPR.

[0094] In some implementations, the X 27 It is Val or Ile. In some embodiments, the X 27 It is Val.

[0095] In some implementations, the X 33 For Ser or Lys. In some implementations, X 33 For Ser.

[0096] In some implementations, X5 is Thr; and / or X 10 For Tyr; and / or the X 22 For Phe; and / or the X mentioned above 26 For Leu.

[0097] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:3:

[0098] R1X1X2GluX4ThrX6X7X8X9TyrX 11 X 12 X 13 LeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 PheX 23 X 24 X 25 LeuX 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:3), wherein X1, X2, X4, X6, X7, X8, X9, X 11 X 12 X 13 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 23 X 24 X 25 X 27 and X 33Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that do not exist or have the aforementioned definitions; R1 and R2 have the aforementioned definitions.

[0099] In some implementations, X4 is Gly; and / or X6 is Phe; and / or X9 is Asp; and / or X... 13 For Tyr.

[0100] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:4:

[0101] R1X1X2GluGlyX5PheX7X8AspX 10 X 11 X 12 TyrLeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:4), wherein X1, X2, X5, X7, X8, X 10 X 11 X 12 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that do not exist or have the aforementioned definitions; R1 and R2 have the aforementioned definitions.

[0102] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:5:

[0103] R1X1X2GluGlyThrPheX7X8AspTyrX 11 X 12 TyrLeuX 15 X 16 X 17 X 18 X 19 X 20 X 21 PheX 23 X 24 X 25 LeuX27AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:5), wherein X1, X2, X7, X8, X 11 X 12 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 23 X 24 X 25 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that do not exist or have the aforementioned definitions; R1 and R2 have the aforementioned definitions.

[0104] In some implementations, the X 12 For Ile; and / or the X 15 For Glu; and / or the X 16 For Glu; and / or the X 18 For Ala; and / or the X mentioned above 20 For Arg; and / or the X 24 It is Gln.

[0105] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:6:

[0106] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX 25 X26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:6), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 13 X 17 X 19 X 21 X 22 X 23 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0107] In some implementations, the X 12 For Ile; and / or the X 15 For Glu; and / or the X 16 For Lys; and / or the X 18 For Ala; and / or the X mentioned above 20 For Arg; and / or the X 24 It is Gln.

[0108] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:7:

[0109] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluLysX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:7), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 13 X 17 X 19 X 21X 22 X 23 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0110] In some implementations, the X 12 For Ile; and / or the X 15 For Glu; and / or the X 16 For Glu; and / or the X 18 For Ala; and / or the X mentioned above 20 For Lys; and / or the X 24 It is Gln.

[0111] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:8:

[0112] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluGluX 17 AlaX 19 LysX 21 X 22 X 23 GlnX 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:8), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 13 X 17 X 19 X 21 X 22 X 23 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0113] In some implementations, the X 12 For Ile; and / or the X15 For Glu; and / or the X 16 For Glu; and / or the X 18 For Ala; and / or the X mentioned above 20 For Arg; and / or the X 24 For Lys.

[0114] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:9:

[0115] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 LysX 25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 9), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 13 X 17 X 19 X 21 X 22 X 23 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0116] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:10:

[0117] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX 25X 26 X 27 AlaGlyGlyProSerLysGlyAlaProProProSerR2 (SEQ ID No:10), wherein X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 13 X 17 X 19 X 21 X 22 X 23 X 25 X 26 X 27 And independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or either absent or having the foregoing definition; R1 and R2 have the foregoing definition.

[0118] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:11:

[0119] R1X1X2GluX4ThrX6X7X8X9TyrX 11 IleX 13 LeuGluGluX 17 AlaX 19 ArgX 21 PheX 23 GlnX 25 LeuX 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 11), wherein X1, X2, X4, X6, X7, X8, X9, X 11 X 13 X 17 X 19 X 21 X 23 X 25 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0120] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:12:

[0121] R1X1X2GluGlyX5PheX7X8AspX10 X 11 IleTyrLeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX 25 X 26 X27AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 12), wherein X1, X2, X5, X7, X8, X 10 X 11 X 17 X 19 X 21 X 22 X 23 X 25 X 26 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0122] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:13:

[0123] R1X1X2GluGlyThrPheX7X8AspTyrX 11 IleTyrLeuGluGluX 17 AlaX 19 ArgX 21 PheX 23 GlnX 25 LeuX 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 13), wherein X1, X2, X7, X8, X 11 X 17 X 19 X 21 X 23 X 25 X 27 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0124] In some embodiments, X1 is Tyr; and / or X2 is Aib; and / or X7 is Thr; and / or X8 is Ser; and / or X... 11 For Ser; and / or the X 17 Gln; and / or the X 19 For Gln and / or the X 21 For Glu; and / or the X 23 For Ile; and / or the X 25 For Trp; and / or the X 27 It is Val.

[0125] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:14:

[0126] R1TyrAibGluX4X5X6ThrSerX9X 10 SerX 12 X 13 LeuX 15 X 16 GlnX 18 GlnX 20 GluX 22 IleX 24 TrpX 26 ValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:14), wherein X4, X5, X6, X9, X 10 X 12 X 13 X 15 X 16 X 18 X 20 X 22 X 24 X 26 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0127] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:15:

[0128] R1TyrAibGluX4ThrX6ThrSerX9TyrSerX 12 X 13 LeuX 15 X 16GlnX 18 GlnX 20 GluPheIleX 24 TrpLeuValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:15), wherein X4, X6, X9, X 12 X 13 X 15 X 16 X 18 X 20 X 24 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0129] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:16:

[0130] R1TyrAibGluGlyX5PheThrSerAspX 10 SerX 12 TyrLeuX 15 X 16 GlnX 18 GlnX 20 GluX 22 IleX 24 TrpX 26 ValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:16), wherein X5, X 10 X 12 X 15 X 16 X 18 X 20 X 22 X 24 X 26 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0131] In some embodiments, the GIPR and GLP-1R co-activating polypeptide has the general formula sequence shown in SEQ ID No:17:

[0132] R1TyrAibGluX4X5X6ThrSerX9X10 SerIleX 13 LeuGluGluGlnAlaGlnArgGluX 22 IleGlnTrpX 26 ValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:17), wherein X4, X5, X6, X9, X 10 X 13 X 22 X 26 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0133] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:18:

[0134] R1TyrAibGluGlyThrPheThrSerAspTyrSerX 12 TyrLeuX 15 X 16 GlnX 18 GlnX 20 GluPheIleX 24 TrpLeuValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:18), wherein the X 12 X 15 X 16 X 18 X 20 X 24 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0135] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:19:

[0136] R1TyrAibGluX4ThrX6ThrSerX9TyrSerIleX 13 LeuGluGluGlnAlaGlnArgGluPheIleGlnTrpLeuValAlaGlyGlyProSerX 33GlyAlaProProProSerR2 (SEQ ID No: 19), wherein X4, X6, X9, X 13 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0137] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide has the general formula sequence shown in SEQ ID No:20:

[0138] R1TyrAibGluGlyX5PheThrSerAspX 10 SerIleTyrLeuGluGluGlnAlaGlnArgGluX 22 IleGlnTrpX 26 ValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:20), wherein X5, X 10 X 22 X 26 and X 33 Independently, R1 and R2 are any natural amino acid residues, non-natural amino acid residues, or residues that are absent or have the aforementioned definitions.

[0139] In some embodiments, the GIPR and GLP-1R co-excitation peptide has the general formula sequence shown in SEQ ID No:21:

[0140] R1TyrAibGluGlyThrPheThrSerAspTyrSerIleTyrLeuGluGluGlnAlaGlnArgGluPheIleGlnTrpLeuValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No:21), wherein the X 33 Independently, it can be any natural amino acid residue, non-natural amino acid residue, or either absent or having the foregoing definition; R1 and R2 have the foregoing definition. In a further embodiment, X 33 For Lys.

[0141] In some embodiments of the general formula sequence SEQ ID No:1-21 above, the X 16 X 20 X 24 and / or X 33The Lys is a fatty acid modified. In a further embodiment, the Lys further comprises fatty acid modification.

[0142] In some embodiments, the N-terminus, C-terminus, and / or amino acid residues within the polypeptide sequence further include chemical modifications. In some embodiments, the chemical modifications include one or more of the following: amidation, fatty acid modification, methylation, myristylation, PEGylation, fluorine modification, biotinylation, fluorescent labeling, cyclization, carboxylation, acetylation, phosphorylation, and glycosylation. In some embodiments, the chemical modifications include amidation and / or fatty acid modification. In some embodiments, the chemical modifications are both amidation and fatty acid modification.

[0143] In some embodiments, the chemical modification occurs at the C-terminus. In some embodiments, the C-terminal chemical modification includes or is an amidation modification. In some embodiments, the amidation modification is a primary amide modification.

[0144] In some embodiments, the chemical modification occurs at amino acid residues within the polypeptide sequence. In some embodiments, the chemical modification includes or is a fatty acid modification. In some embodiments, one or more Lys residues in the polypeptide contain a fatty acid modification.

[0145] In some embodiments, the chemical modification includes or is an amidation modification at the C-terminus (e.g., a primary amide modification) and a fatty acid modification occurring within the polypeptide sequence (e.g., one or more Lys residues contain a fatty acid modification).

[0146] In some embodiments, the fatty acid modification occurs for X 16 X 20 X 24 or X 33 One or more amino acid residues in X. In some embodiments, the fatty acid modification occurs at X. 16 X 20 X 24 or X 33 One of them. In some embodiments, the fatty acid modification occurs for X. 16 X 20 X 24 or X 33 On the lysine residue.

[0147] In some embodiments, the fatty acid modification has -(AEEA) a -(γ-Glu) b -CO-(CH2) nThe structure is -COOH, where a, b, and n are natural numbers. In some implementations, 12 ≤ n ≤ 20. In some implementations, a is 1, 2, or 3. In some implementations, b is 1 or 2.

[0148] In some embodiments, the fatty acid modification has the following characteristics: -AEEA-AEEA-γ-Glu-CO-(CH2). n The structure is -COOH, where n is a natural number and 12 ≤ n ≤ 20. In some embodiments, the fatty acid modification has the structure -AEEA-AEEA-γ-Glu-CO-(CH2). n The structure is -COOH, where n is a natural number, and n is 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the fatty acid modification has -AEEA-AEEA-γ-Glu-CO-(CH2). 16 The structure is -COOH. In some embodiments, the fatty acid modification has the structure -AEEA-AEEA-γ-Glu-CO-(CH2). 18 The structure of -COOH.

[0149] In some embodiments, the fatty acid modification is linked to the ε-amino group of the Lys residue. In some embodiments, R1 and / or R2 are absent in the general formula sequence. In some embodiments, R1 is absent. In some embodiments, R2 is absent. In some embodiments, both R1 and R2 are absent.

[0150] In some embodiments, the GIPR and GLP-1R co-excitation peptides involved in this application have the sequences shown in any one of SEQ ID No. 22-41, 43-73, 76, 78-80, 82-86, 88-98, 101-131, or the sequence of the peptide is composed of the sequences shown in any one of SEQ ID No. 22-41, 43-73, 76, 78-80, 82-86, 88-98, 101-131.

[0151] In some embodiments, the GIPR and GLP-1R co-excitation polypeptides involved in this application comprise sequences selected from Table 1A or consist of sequences selected from Table 1A.

[0152] Table 1A

[0153]

[0154] In some embodiments, the GIPR and GLP-1R co-excitation peptides involved in this application contain or have sequences identical to those shown in Table 1, except that the A (Ala) at the second position is replaced by Aib. In some other embodiments, the sequences containing the A (Ala) at the second position replaced by Aib are shown in Table 1B.

[0155] Table 1B

[0156]

[0157] In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application is formed by C-terminal amidation of the GIPR and GLP-1R co-activating peptide of this application, for example, the sequence shown in any one of SEQ ID No. 76, 78-80, 82-86, and 88-98 in Table 3 below. In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application is formed by amidation of the GIPR and GLP-1R co-activating peptide sequence selected from sequences identical to those in Table 1A or sequences identical to those shown in Table 1A except that A (Ala) at position 2 is replaced by Aib.

[0158] In some embodiments, the GIPR and GLP-1R co-excitation peptides involved in this application comprise modified sequences selected from or composed of modified sequences from Table 2.

[0159] Table 2

[0160]

[0161]

[0162] In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application is formed by the C-terminal amidation of the GIPR and GLP-1R co-activating peptide of this application, for example, the sequence shown in any one of SEQ ID Nos. 101-121 and 128-130. In some embodiments, the amidation modification is a primary amide modification.

[0163] The aforementioned {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} indicates that the lysine residue at this site is represented by -AEEA-AEEA-γ-Glu-CO-(CH2). 16-COOH is chemically modified by conjugation with the ε-amino group of the lysine side chain. The aforementioned {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} indicates that the lysine residue at this site is modified by conjugation with -AEEA-AEEA-γ-Glu-CO-(CH2). 18 -COOH is chemically modified by conjugating with the ε-amino group of the lysine side chain.

[0164] Figures 1 to 5 The chemical structures of five exemplary molecules of this application are shown.

[0165] In some embodiments, the GIPR and GLP-1R co-excitatory polypeptides involved in this application, or pharmaceutically acceptable salts, amides, or esters thereof, contain or have sequences that are variants of sequences selected from Table 2, wherein, relative to sequences selected from Table 2, {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} or {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} is replaced by amino acid residues modified with other fatty acids, wherein "other fatty acids" refers to long-chain fatty acids other than -AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl and -AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl. In some embodiments, the GIPR and GLP-1R co-excitation polypeptide comprises or has sequences selected from the sequences in Table 2, wherein, relative to the sequences selected from Table 2, {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} or {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} is replaced by fatty acid-modified arginine residues. In some embodiments, the GIPR and GLP-1R co-excitation polypeptide comprises or has sequences selected from the sequences in Table 2 wherein {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} or {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} is replaced by {R(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} or {R(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)}. In some embodiments, the GIPR and GLP-1R co-excitation peptide contains or has sequences that are variants of sequences selected from Table 2, wherein {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} is replaced by {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} relative to sequences selected from Table 2.In some embodiments, the GIPR and GLP-1R co-excitation peptide contains or has sequences that are variants of sequences selected from Table 2, wherein {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} is replaced by {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} relative to sequences selected from Table 2.

[0166] In some embodiments, the GIPR and GLP-1R co-activating polypeptides involved in this application, or their pharmaceutically acceptable salts, amides, or esters, contain or have sequences that are variants selected from the sequences in Table 2, wherein the fatty acid modifications used by the variants relative to the sequences selected from Table 2 are replaced by other fatty acid modifications. In some embodiments, the other fatty acid modification is HOOC(CH2). n CO-, where n is an integer selected from 10-24, more preferably an integer selected from 16-20. The fatty acid modification is selected from HOOC(CH2). 14 CO-, HOOC(CH2) 15 CO-, HOOC(CH2) 16 CO-, HOOC(CH2) 17 CO-, HOOC(CH2) 18 CO-, HOOC(CH2) 19 CO-, HOOC(CH2) 20 CO-, HOOC(CH2) 21 CO- or HOOC(CH2) 22 CO-. In some embodiments, the other fatty acid modifications may be COOH(CH2). 14～20 CO-γ-Glu-AEEA-AEEA-.

[0167] In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application is formed by C-terminal amidation of the GIPR and GLP-1R co-activating peptide of this application. In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application comprises or consists of sequences selected from Table 2 after C-terminal amidation. In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-activating peptide of this application comprises or consists of sequences from Table 2 after C-terminal amidation to form a C-terminal primary amide.

[0168] In some embodiments, the GIPR and GLP-1R co-agonistic peptides involved in this application, or pharmaceutically acceptable salts, amides, or esters thereof, contain or have sequences that are variants selected from the sequences in Table 2, wherein the variants are C-terminally linked to a modification fragment relative to the sequences selected from Table 2, the modification fragment being selected from {NH2}amidation, {-CHO}peptide aldehyde, {-ol} alcohol peptide, {CMK} chloromethyl ketone, {FMK} fluoromethyl ketone, {Cya} mercaptoethylamine, {pNA} p-nitroaniline, {-ONP} p-nitrophenol, {AMC} 7-amino-4-methylcoumarin, {AFC}, -OMe (C-terminus), -OE t (C-terminal), -OBzl (C-terminal), -OtBu (C-terminal), {-OSu} hydroxysuccinimide ester, -NHMe (C-terminal), -NHEt (C-terminal), -NH isopentylamino (C-terminal), NH(C2H)6 (C-terminal), -NHPh (C-terminal), {NHEt(O)EtNH-Fmoc}2,2'-oxodiethylamine-Fmoc, {NHEt(EtNH-Myr)2}, -NH(OMe)Me (C-terminal), -TBzl (C-terminal), -NHNH2 (C-terminal), -ED (C-terminal)-NH-CH2CH2-NH2 or -BD (C-terminal)-NH-CH2CH2CH2CH2-NH2NH2 groups.

[0169] In some embodiments, the GIPR and GLP-1R co-activating peptides involved in this application, or pharmaceutically acceptable salts, amides, or esters thereof, contain or have sequences selected from the sequences in Table 2, wherein the variants are further modified relative to the sequences selected from Table 2 at the N-terminal first and / or second amino acid to reduce sensitivity to DPP-4 cleavage. More specifically, in some embodiments, the N-terminal first and / or second amino acid is replaced with a DPP-4 resistant amino acid. In some embodiments, the N-terminal first and / or second amino acid of the sequences in Table 2 is replaced with an amino acid selected from D-serine, D-alanine, glycine, N-methylserine, and ε-aminobutyric acid.

[0170] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide involved in this application has the general formula sequence shown in SEQ ID No:132:

[0171] X1AlaGluGlyThrPheThrSerAspTyrSerIleX 13 LeuX 15 X 16 X 17 AlaGlnX 20 X21 PheX 23 X 24 TrpLeuX 27 AlaGlyGlyProSerSerGlyAlaProProProSerR2 (SEQ ID No:132), wherein X1 is selected from Tyr, Phe, or His; X 13 Selected from Ala or Tyr; X 15 Selected from Asp or Glu;X 16 Selected from Lys or Glu;X 17 Selected from Ile or Gln; X 20 Selected from Lys or Arg; X 21 Selected from Ala, Glu, or Asp; X 23 Selected from Val or Ile; X 24 Selected from Gln or Asn; X 27 Selected from Val or Ile.

[0172] In some embodiments, the polypeptide has a sequence shown in any one of SEQ ID No. 22, 24, 38-41, 76, 79, 95-98, or the sequence of the polypeptide consists of a sequence shown in any one of SEQ ID No. 22, 24, 38-41, 76, 79, 95-98.

[0173] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide involved in this application has the general formula sequence shown in SEQ ID No:133:

[0174] X1X2GluGlyThrPheThrSerAspTyrSerIleTyrLeuX 15 X 16 GlnAlaGlnX 20 X 21 PheX 23 X 24 TrpLeuX 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 133), where X1 is Tyr, Phe, or His; X2 is Ala or Aib; X 15 For Glu or Asp; X 16 For Glu or Lys; X 20 For Arg or Lys; X 21 For Glu, Ala, or Asp;X 23 For Ile or Val; X 24 For Gln, Lys, or Asn; X27 Val or Ile; X 33 For Ser or Lys.

[0175] In some embodiments of the general formula sequence shown in SEQ ID No:133, X 16 X 20 X 24 and / or X 33 For Lys.

[0176] In some embodiments of the general formula sequence shown in SEQ ID No:133, the Lys comprises a fatty acid modification -(AEEA). a -(γ-Glu) b -CO-(CH2) n -COOH, where a, b, and n are natural numbers, and 12 ≤ n ≤ 20. In some embodiments, n is 16 or 18, a is 2, and b is 1.

[0177] In some embodiments of the general formula sequence shown in SEQ ID No: 133, the polypeptide has any one of the sequences shown in SEQ ID No. 47-51, 61, 62, 65, 66, 71, 73, 131, 101-105, 116, 117, 120, 121, 128, 129, 130, 122-127, or the sequence of the polypeptide consists of any one of the sequences shown in SEQ ID No. 47-51, 61, 62, 65, 66, 71, 73, 131, 101-105, 116, 117, 120, 121, 128, 129, 130, 122-127.

[0178] In some embodiments, the GIPR and GLP-1R co-excitation polypeptide involved in this application has the general formula sequence shown in SEQ ID No: 134:

[0179] TyrX2GluGlyThrPheThrSerAspTyrSerIleTyrLeuGluX 16 GlnAlaGlnArgX 21 PheIleX24TrpLeuValAlaGlyGlyProSerX 33 GlyAlaProProProSerR2 (SEQ ID No: 134), where X2 is Ala or Aib; X 16 For Glu or Lys; X 21 For Glu or Asp; X 24 For Gln, Lys, or Asn; X 33 For Ser or Lys.

[0180] In some embodiments of the general formula sequence shown in SEQ ID No:134, X 16 X 24 and / or X 33 For Lys.

[0181] In some embodiments of the general formula sequence shown in SEQ ID No:133, the Lys comprises a fatty acid side chain modification -(AEEA). a -(γ-Glu) b -CO-(CH2) n -COOH, where a, b, and n are natural numbers, and 12 ≤ n ≤ 20. In some embodiments, n is 16 or 18, a is 2, and b is 1.

[0182] In some embodiments of the general formula sequence shown in SEQ ID No: 133, the polypeptide has the sequence shown in any one of SEQ ID No. 38, 95, 47-49, 71, 73, 131, 101, 102, 103, 128, 129, 130, 122-127, or the sequence of the polypeptide consists of the sequence shown in any one of SEQ ID No. 38, 95, 47-49, 71, 73, 131, 101, 102, 103, 128, 129, 130, 122-127.

[0183] This application also relates to polypeptide compositions comprising GIPR and GLP-1R co-activating peptides as described in the first aspect, or pharmaceutically acceptable salts, amides, or esters thereof. In some embodiments, the polypeptide composition comprises two or more of the GIPR and GLP-1R co-activating peptides, or pharmaceutically acceptable salts, amides, or esters thereof.

[0184] A second aspect of this application relates to pharmaceutical compositions comprising the GIPR and GLP-1R co-activating peptides as described in the first aspect, or pharmaceutically acceptable salts, amides, or esters thereof, and pharmaceutically acceptable carriers, diluents, and / or excipients. In some embodiments, the pharmaceutical composition comprises the aforementioned peptide composition.

[0185] In some embodiments, the pharmaceutical composition is used for one or more of the following purposes: (1) prevention, treatment, and / or relief of metabolic diseases or conditions; (2) prevention, treatment, and / or relief of diabetes; preferably, the diabetes is type 2 diabetes; (3) prevention, treatment, and / or relief of obesity or overweight; (4) weight loss; (5) reduction of food intake; (6) improvement of glycemic control (e.g., improvement of glucose tolerance). In some embodiments, the metabolic disease or condition is diabetes. In some embodiments, the diabetes is type 2 diabetes. In some embodiments, the metabolic disease or condition is type 2 diabetes.

[0186] The third aspect of this application relates to methods for preventing, treating, and / or alleviating metabolic diseases or conditions, the methods comprising administering to a patient or subject in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect.

[0187] In some embodiments, the metabolic disease or condition is diabetes. In some embodiments, the diabetes is type 2 diabetes.

[0188] In some implementations, the metabolic disease or condition is obesity or overweight.

[0189] The fourth aspect of this application relates to a method for weight loss, the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect. In some embodiments, the individual is obese or overweight. In some embodiments, the individual is diabetic, such as a type 2 diabetic. In some embodiments, the individual is obese or overweight and has diabetes, such as type 2 diabetes.

[0190] The fifth aspect of this application relates to a method for reducing food intake, the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect. In some embodiments, the individual is obese or overweight. In some embodiments, the individual is diabetic, such as a type 2 diabetic. In some embodiments, the individual is obese or overweight and has diabetes, such as type 2 diabetes.

[0191] The sixth aspect of this application relates to a method for improving glycemic control (e.g., improving glucose tolerance), the method comprising administering to an individual in need an effective amount of a polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition as described in the second aspect. In some embodiments, the individual is obese or overweight. In some embodiments, the individual has diabetes, such as type 2 diabetes. In some embodiments, the individual is obese or overweight and has diabetes, such as type 2 diabetes.

[0192] The seventh aspect of this application relates to the use of the polypeptides as described in the first aspect, or pharmaceutically acceptable salts, amides, or esters thereof, or pharmaceutical compositions as described in the second aspect, in the preparation of a medicament for one or more of the following purposes: (1) prevention, treatment, and / or relief of metabolic diseases or conditions; (2) prevention, treatment, and / or relief of diabetes; preferably, said diabetes is type II diabetes; (3) prevention, treatment, and / or relief of obesity or overweight; (4) weight loss; (5) reduction of food intake; and (6) improvement of glycemic control (e.g., improvement of glucose tolerance).

[0193] The following supplementary descriptions supplement the inventions described in aspects one through seven above. Where there is no conflict, the supplementary descriptions below apply to the various technical solutions described in aspects one through seven.

[0194] GIPR and GLP-1R co-stimulatory peptides, or their pharmaceutically acceptable salts, amides, or esters.

[0195] This application provides general formulas and examples of various GIPR and GLP-1R co-activating peptides, or pharmaceutically acceptable salts, amides, or esters thereof. One of the revealed or anticipated beneficial effects may be enhanced activation of GLP-1R while maintaining GIPR activation activity, which is beneficial for enhancing the effect in lowering blood glucose levels and further enhancing the effect in weight loss.

[0196] The GIPR and GLP-1R co-excitation peptides provided in this application can be synthesized using methods known in the art.

[0197] The GIPR and GLP-1R co-stimulatory peptides of this application may be in the form of pharmaceutically acceptable salts, amides, or esters.

[0198] The salt can be a basic salt, an acidic salt, or a neutral salt. In water, basic salts produce hydroxide ions, and acidic salts produce hydrated hydrogen ions. The synthesis of co-excited polypeptide salts in this application can be achieved by introducing the corresponding counterion into the polypeptide solution. For example, if the polypeptide molecule contains protonable amino acid residues, forming negatively charged anionic groups, this can be achieved by adding cations (such as the metal cation Na). + K + or proton H+ This reacts with the amino acid to form a polypeptide cationic salt. Conversely, if the polypeptide molecule contains deprotonable amino acid residues, forming a positively charged cationic group, it can be deprotonated by adding anion (such as Cl-). - SO4 2- (etc.) react with it to form a polypeptide anionic salt. These counterionic groups may be located within the peptide moiety and / or within the side chain of the co-excited polypeptide in this application.

[0199] Non-limiting examples of anionic groups in the co-excited peptides of this application include side chains (if any) and free carboxyl groups in the peptide moiety. The peptide moiety typically includes a C-terminal free carboxylic acid, and may also include free carboxyl groups on internal acidic amino acid residues, such as Asp and Glu. Non-limiting examples of cationic groups in the peptide moiety include an N-terminal free amino group (if any) and any free amino group on internal basic amino acid residues, such as His, Arg, and Lys.

[0200] The esters of the co-excited polypeptides of this application can be formed, for example, by reacting a free carboxylic acid group with an alcohol or phenol, resulting in the substitution of at least one hydroxyl group with an alkoxy or aryloxy group. The formation of the ester may involve a free carboxyl group at the C-terminus of the peptide, and / or any free carboxyl group in the side chain.

[0201] The amide of the co-excited peptide of this application can be generated, for example, by reacting a free carboxylic acid group with an amine or a substituted amine, or by reacting a free or substituted amino group with a carboxylic acid. The formation of the amide may involve a free carboxyl group at the C-terminus of the peptide, any free carboxyl group in the side chain, a free amino group at the N-terminus of the peptide, and / or any free or substituted peptide amino group in the peptide and / or side chain.

[0202] In some embodiments, the amino acid residues in the GIPR and GLP-1R co-excitatory peptide, or its pharmaceutically acceptable salt, amide, or ester, include chemical modifications. In some embodiments, the chemical modifications include amidation, fatty acid modification, methylation, myristylation, PEGylation, fluorine modification, biotinylation, fluorescent labeling, cyclization, carboxylation, acetylation, phosphorylation, glycosylation, and other modifications known in the art (see, for example, US5856298; US2003-0120045, US2004-0063917, US2005-0220800, US2005-0107591, US2006-0035322, and US2006-0073563; and WO200181405) or combinations thereof. All of the aforementioned chemical modification methods can be implemented using conventional methods in the art.

[0203] In some embodiments, the amino acid residues in the GIPR and GLP-1R co-excitation peptides, or their pharmaceutically acceptable salts, amides, or esters, described in this application include amidation modifications.

[0204] In some embodiments, the amino acid residues in the GIPR and GLP-1R co-excitation peptides described in this application, or their pharmaceutically acceptable salts, amides, or esters, include fatty acid modifications.

[0205] In some embodiments, the chemical modification includes or is a fatty acid modification generated within the polypeptide sequence. In some embodiments, the fatty acid modification is linked to the ε-amino group of a Lys residue. In some embodiments, the fatty acid modification has -(AEEA). a -(γ-Glu) b -CO-(CH2) n The structure of -COOH is given, where a, b, and n are natural numbers, 12 ≤ n ≤ 20; a is 1, 2, or 3; b is 1 or 2. In this paper, "-AEEA-" refers to (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl); "-(AEEA)2" refers to (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2; and "-(AEEA)3-" refers to (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)3. In this paper, "(γ-Glu)2" refers to -γ-Glu-γ-Glu.

[0206] In some embodiments, the fatty acid modification has the following characteristics: -AEEA-AEEA-γ-Glu-CO-(CH2). 16 The structure is -COOH (i.e., -AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl). In some embodiments, the lysine residue is represented by -AEEA-AEEA-γ-Glu-CO-(CH2). 16 -COOH is chemically modified by conjugating with the ε-amino group of the lysine side chain.

[0207] In some embodiments, the fatty acid modification has the following characteristics: -AEEA-AEEA-γ-Glu-CO-(CH2). 18 The structure is -COOH (i.e., -AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl). In some embodiments, the lysine residue is represented by -AEEA-AEEA-γ-Glu-CO-(CH2). 18 -COOH is chemically modified by conjugating with the ε-amino group of the lysine side chain.

[0208] Pharmaceutical Composition

[0209] This application provides a pharmaceutical composition comprising the aforementioned GIPR and GLP-1R co-stimulatory peptides, or pharmaceutically acceptable salts, amides, or esters thereof, and pharmaceutically acceptable carriers, diluents, and / or excipients.

[0210] The pharmaceutical compositions of this application comprise one or more carriers, diluents, and / or excipients, all of which are pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means that the material is non-toxic and, in some exemplary embodiments, does not interact with the active agent of the pharmaceutical composition.

[0211] As used herein, the term "carrier" refers to an organic or inorganic component of natural or synthetic nature, wherein an active component is incorporated to facilitate, enhance, or achieve application. According to this application, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject.

[0212] As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and are not the active ingredient.

[0213] In some embodiments, the composition may be formulated into a unit dosage form suitable for administration to a patient, specifically into a form for administration of a polypeptide drug, and may be administered via oral or parenteral routes such as intradermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, intrapulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, local, sublingual, vaginal, or rectal routes, but is not limited thereto.

[0214] The dosage and frequency of administration of the pharmaceutical composition of this application are determined based on the type of drug as the active ingredient and various relevant factors, such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

[0215] Metabolic diseases or conditions

[0216] This application provides methods for preventing, treating, and / or alleviating metabolic diseases or conditions, the methods comprising administering to a patient or subject in need an effective amount of the aforementioned GIPR and GLP-1R co-agonist polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or polypeptide composition comprising the thereof. The GIPR and GLP-1R co-agonist polypeptide of this application activates the two incretin receptors GIPR and GLP-1R involved in glycemic control through a dual mechanism of action, achieving the effects of lowering blood sugar and reducing weight.

[0217] In some embodiments, the metabolic disease or condition includes, but is not limited to, diabetes. In some embodiments, the diabetes is type 2 diabetes.

[0218] In some implementations, the metabolic disease or condition includes, but is not limited to, obesity or overweight.

[0219] Type 2 Diabetes Treatment / Prevention and Glycemic Control

[0220] This application provides a method for improving glycemic control, the method comprising administering to an individual in need an effective amount of the aforementioned GIPR and GLP-1R co-activating peptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or peptide composition comprising the thereof.

[0221] In some implementations, the term "glycemic control" refers to maintaining or reducing an individual's HbA1c level. "Hemoglobin A1c" or "HbA1c" refers to glycated hemoglobin and its level, which is formed when hemoglobin binds to glucose in the blood. HbA1c level is a commonly used indicator of glycemic control in individuals with diabetes; a lower HbA1c level generally indicates improved glycemic control.

[0222] In some implementations, improving glycemic control includes reducing the patient's or subject's Hb1Ac level to below 7%, below 6.5%, or below 6.0%.

[0223] In some implementation schemes, improving glycemic control refers to improving an individual's glucose tolerance.

[0224] This application provides methods for the prevention, treatment, and / or relief of type 2 diabetes, the methods comprising administering to a patient or subject in need an effective amount of the aforementioned GIPR and GLP-1R co-activating peptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or peptide composition comprising thereof, which is particularly effective for the treatment of type 2 diabetes.

[0225] In some implementations, the methods for preventing, treating, and / or alleviating type 2 diabetes further include administering one or more additional medications for treating type 2 diabetes.

[0226] In some implementations, the subjects diagnosed with type 2 diabetes in this application are patients with type 2 diabetes diagnosed by the usual clinical diagnostic criteria.

[0227] In some implementations, the fasting blood glucose level of subjects diagnosed with type 2 diabetes in this application may be higher than 125 mg / dL.

[0228] In some implementations, subjects diagnosed with type 2 diabetes in this application may have blood glucose levels higher than 199 mg / dL after a glucose tolerance test.

[0229] In some implementations, the HbA1C level of subjects diagnosed with type 2 diabetes in this application may be greater than 6.4%.

[0230] In some embodiments, the diabetes is obese diabetes. Obese diabetes refers to diabetes with obese symptoms that cause diabetes (especially type 2 diabetes) or obese symptoms in patients with type 2 diabetes. Approximately 80% to 90% of patients with type 2 diabetes have obese symptoms, and these patients are characterized by insulin resistance. Appropriate exercise, diet, and drug therapy can prevent and reduce obese diabetes. In this application, in some cases, obese diabetes is caused by obesity.

[0231] Treatment / prevention of overweight and obesity and weight loss

[0232] This application provides methods for preventing, treating, and alleviating overweight, the methods comprising administering to an individual in need an effective amount of the aforementioned GIPR and GLP-1R co-activating peptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or peptide composition containing the thereof.

[0233] In some implementations, the overweight patient or subject has a weight greater than 25 kg / m². 2 And less than 30kg / m 2 BMI.

[0234] In some implementations, the overweight patient or subject has a weight greater than 25 kg / m². 2 to less than 27kg / m 2 The BMI is high, and the individual has at least one comorbidity.

[0235] In some implementations, the overweight patient or subject is of Asian descent and has a weight greater than 23 kg / m². 2 to less than 25kg / m 2 BMI.

[0236] This application provides methods for preventing, treating, and alleviating obesity, the methods comprising administering to an individual in need an effective amount of the aforementioned GIPR and GLP-1R co-activating polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or polypeptide composition containing the thereof.

[0237] In some embodiments, the obese patient or subject has a weight of 30 kg / m² or greater. 2 BMI.

[0238] In some embodiments, the obese patient or subject has a weight of 27 kg / m² or higher. 2BMI and at least one comorbidity.

[0239] In some implementations, the obese patient or subject is of Asian descent and has a weight of 25 kg / m² or higher. 2 The patient's BMI status and has at least one comorbidity.

[0240] This application provides a method for weight loss, the method comprising administering to an individual in need an effective amount of the aforementioned GIPR and GLP-1R co-activating peptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or peptide composition comprising the thereof.

[0241] This application provides a method for reducing food intake, the method comprising administering to an individual in need an effective amount of the aforementioned GIPR and GLP-1R co-activating peptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition or peptide composition containing the thereof. Prevention, treatment, and relief of obesity, overweight, or weight loss can be achieved by reducing food intake.

[0242] In this document, the term "reduced food intake" refers to the spontaneous reduction in food intake under conditions of sufficient food availability. The GIPR and GLP-1R co-activating peptide of this application, upon binding to GLP-1R, can slow gastrointestinal motility, inhibit gastric juice secretion, delay gastric emptying, and enhance satiety, and can help with weight loss by reducing food intake.

[0243] In one embodiment, the GIPR and GLP-1R co-stimulatory peptides of this application, or pharmaceutically acceptable salts, amides, or esters thereof, or pharmaceutical compositions comprising them, or peptide compositions, are used to treat overweight, obesity, and / or eating disorders with one or more of the following clinical outcomes as treatment goals: reduced food intake, increased energy expenditure, weight loss, appetite suppression, or induction of satiety.

[0244] In one embodiment, the GIPR and GLP-1R co-stimulatory peptides of this application, or their pharmaceutically acceptable salts, amides or esters, or pharmaceutical compositions containing them, may also be combined with one or more other pharmacologically active substances. Example

[0245] Example 1: Universal synthetic route for GIPR and GLP-1R co-excitation peptides

[0246] In the S890 peptide synthesizer, the Fmoc / t-Bu strategy was used to synthesize GIPR and GLP-1R co-excitation peptides.

[0247] The lys residues containing fatty acid side chains and their side chains were analyzed using Fmoc-Lys(AEEA). a-(γ-Glu) b -CO-(CH2) n -COOH (where a, b, and n depend on the required side chain) (purchased from Chengdu Pukang Biotechnology Co., Ltd.);

[0248] Lys without fatty acid side chains was produced using Fmoc-Lys(Boc)-OH as a raw material (purchased from Chengdu Kelong Chemical Co., Ltd.);

[0249] Other amino acid residues were sourced from standard side-chain protected amino acid residue raw materials (purchased from Chengdu Kelong Chemical Co., Ltd.);

[0250] Resins with amides at the C-terminus: Rink Amide MBHA Resin, 1% DVB (divinylbenzene), 100-200 mesh, degree of substitution 0.4-0.5 mmol / g (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.).

[0251] Resins with carboxylic acid at the C-terminus: 2-Chlorotrityl Chloride Resin (dichlororesin), 1% DVB, 100-200 mesh, degree of substitution 0.4-0.5 mmol / g (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.).

[0252] The general preparation process is as follows:

[0253] (1) Ligation of the C-terminal amide resin to the first C-terminal amino acid: 20% piperidine / DMF was added to a reactor containing Rink Amide MBHA Resin, and the reaction was carried out for 20 minutes. The reaction solution was then filtered off, and the resin was washed 6 times with DMF. A mixture of the first C-terminal amino acid was added to the deprotected resin, and the condensation reaction was carried out for 3 hours. The molar ratio of the mixture of the first C-terminal amino acid to the resin was: resin: amino acid: diisopropylcarbodiimide: ethyl 2-oxime cyanoacetate = 1:3:3:3. After the reaction was completed, the resin was filtered, and the resin was washed 6 times with DMF.

[0254] (2) Connection of the C-terminal carboxylic acid resin to the first C-terminal amino acid: A mixture of the first C-terminal amino acid was added to a reactor containing 2-ChlorotritylChloride Resin and reacted for 2 h. The molar ratio of the first C-terminal amino acid mixture to the resin was resin: amino acid: diisopropylethylamine = 1:1:6. After the reaction, the mixture was filtered, and the resin was washed twice with DMF. Then, 10% methanol / DMF was added, and the reaction was carried out for 0.5 h. After the reaction, the mixture was filtered, and the resin was washed six times with DMF.

[0255] (3) Deprotection: Add 20% piperidine / DMF to the reactor containing the resin and react for 20 minutes. Then filter off the reaction solution and wash the resin 6 times with DMF.

[0256] (4) Condensation: An amino acid mixture was added to the deprotected resin, and the condensation reaction was carried out for 3 hours. The molar ratio of the amino acid mixture to the resin was resin: amino acid: diisopropylcarbodiimide: ethyl 2-oxime cyanoacetate = 1:3:3:3. After the reaction was completed, the mixture was filtered, and the resin was washed 6 times with DMF.

[0257] Following the order of the polypeptide sequence from C-terminus to N-terminus, steps (3) and (4) were repeated sequentially to link each amino acid or polypeptide fragment, completing the synthesis of the entire polypeptide. The resin was then washed three times with methanol and dried under vacuum to obtain a polypeptide resin with an amide or carboxylic acid at the C-terminus.

[0258] (5) Cutting: Add a cutting solution (trifluoroacetic acid:water:triisopropylsilane, 95:2.5:2.5 v / v) at a mass-to-volume ratio of 10 to the obtained polypeptide resin, react for 3 h, filter, add 10 times the volume of methyl ether to the filtrate to precipitate the polypeptide, filter to obtain the polypeptide precipitate. After vacuum drying, obtain the crude polypeptide product.

[0259] (6) Purification: The crude component was dissolved in a 10% acetonitrile aqueous solution, and the pH was adjusted to 8-9 with ammonia. The mixture was then separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The RP-HPLC column used was 21 x 250 mm, Kromasil C8, 10 μm; the mobile phase was 100% acetonitrile and a 0.1% TFA / water buffer system; the mobile phase was 0–60 min, with a gradient of 20–50% acetonitrile. Components with a purity >95% were collected and freeze-dried to obtain a lyophilized powder.

[0260] (7) Analysis and detection: The purity of the polypeptide lyophilized powder was determined to be 98.5% by analytical HPLC (Agilent, 1260 Infinity II) and the molecular weight was determined by LC-MS (Thermo Fisher Scientific, OE240), which was consistent with the theoretical calculation value.

[0261] Example 2: Detection of GLP-1R / GIPR agonist activity of test molecules

[0262] Functional activities were determined in HEK-293 clonal cell lines expressing GIPR and luciferase or GLP-1R and luciferase, respectively. Cell lines expressing each receptor were treated with different concentrations of each peptide working solution in 100 μl of DMEM medium (Gibco Cat#12800) supplemented with 1% fetal bovine serum (FBS) (Gibco Cat#10099141C). After incubation at 37°C for 4 h, cells were lysed using Glo lysis buffer (Promega, Cat#E2661), followed by the addition of an equal volume of Stead-Glo Lucifferase buffer substrate from a luciferase reporter gene assay system (Promega, Cat#E2520). Luciferase catalyzes substrate oxidation in the presence of ATP, and this oxidation process emits biofluorescence, which was read using a microplate reader.

[0263] Fluorescence values ​​were fitted using GraphPad Prism 8 software (Ver 8.3.0), with the X-axis representing the concentration of the test molecule (nM) and the Y-axis representing the fluorescence value. The fitting formula was based on the Hill equation: Y = Bottom + (X^Hill slope) * (Top - Bottom) / (X^HillSlope + EC50^HillSlope). (Where Top is the level of Y at the maximum concentration; Bottom is the level of Y at a concentration of 0. The Hill slope is the Hill coefficient, describing the shape of the curve, i.e., the sensitivity of Y to changes in X). EC50 values ​​obtained from luciferase reporter cell lines treated with different test molecules were calculated. 50 The values ​​are shown in Tables 3 and 4.

[0264] Candidate test molecules with superior agonistic activity at the GLP-1R end and comparable or superior agonistic activity at the GIPR end, compared to the corresponding telpopeptide control group (main chain peptide or modified peptide), are considered to have good prospects.

[0265] Table 3. GIPR / GLP-1R agonistic activity of unmodified peptides (EC) 50

[0266]

[0267] Note: EC 50 Lower values ​​indicate higher activity; "*" represents the control group; "-NH2" indicates C-terminal amidation of the peptide; "-" in the results indicates no activity; Tir-0* and Tir-Aib*

[0268] Indicates a telpoeptide backbone derivative

[0269] Table 4. GIPR / GLP-1R agonist activity of modified peptides (EC) 50

[0270]

[0271]

[0272] Note: EC 50 The lower the value, the higher the activity; "*" indicates the control group; "-NH2" indicates C-terminal amidation of the peptide;

[0273] Example 3: Effect of GIPR and GLP-1R co-excitation peptides on weight loss in diet-induced obese (DIO) mice

[0274] Based on the GIPR / GLP-1R agonist activity test results of Example 2, DTM0001 to DTM0003 were selected for the next in vivo experimental test, and telpolide (SEQ ID No: 100) was used as a control molecule.

[0275] The in vivo weight-loss efficacy of DTM0001 to DTM0003 was evaluated in diet-induced obesity (DIO) mice (C57 / B16 mice purchased from Vital River were induced into DIO mice by a high-fat diet) mice (60% of the calories came from fat).

[0276] In this study, 24-week-old male diet-induced obese (DIO) C57 / B16 mice were used. Mice were housed in a facility with a temperature controlled at 20–24°C and a 12-hour light / dark cycle, with free access to food and water. After 3 weeks of acclimatization, mice were randomly assigned to experimental groups based on their body weight (n = 9 / group, 3 mice per cage), thus ensuring similar starting average body weight for each group. The average body weight of mice in each group on the day of grouping was 46 g.

[0277] Excipient controls (PBS, 100 mM phosphate-buffered saline, pH 7.2–7.4), DTM0001 to DTM0003 (30 nmol / kg) or telpolide (30 nmol / kg) were dissolved in the excipients and administered to free-feeding DIO mice via subcutaneous injection (SC) every three days for 15 days. SC injections were performed on days 1, 4, 7, 10, and 13. Mouse body weight was measured daily throughout the study. The absolute rate of change in mouse body weight was calculated using the following formula:

[0278]

[0279] One-way ANOVA and subsequent Dunnett's multiple comparison test were used for statistical comparisons between groups. The results showed that DTM0001 to DTM0003 all had significant weight-loss effects, with DTM0003 showing a better rate of weight change on day 7 than telpotetide (p<0.05). The weight loss rates of DTM0003, DTM0001, DTM0002, and telpotetide on day 15 in DIO mice were -25.76±6.18, -23.73±2.09, -23.60±3.08, and -24.94±4.12, respectively.

[0280] Table 5. Percentage of body weight change in DIO mice (change in body weight from initial weight %)

[0281]

[0282] Note: ***p<0.001 vs PBS # p<0.05 vs. telpolide. Results are expressed as mean ± standard deviation of the rate of change in body weight of 9 mice in each group.

[0283] Example 4: In vitro agonist activity assay of hGLP-1R and hGIPR

[0284] Based on the test results of Examples 2 and 3, the superior properties of DTM0001 to DTM0003 were confirmed. Combining the structural commonality analysis of DTM0001 to DTM0003, SYDT0020 (SEQ ID No: 95) was selected as a candidate parent chain for further modification, resulting in DTM0026, DTM0028, and DTM0030 (as shown in Table 6 below). The six test molecules in Table 6 were subjected to in vitro hGLP-1R and hGIPR agonist activity tests, with telpolide (SEQ ID No: 100) used as a control molecule.

[0285] Table 6

[0286]

[0287] The in vitro agonistic activity of the above six test molecules on the human GLP-1 receptor (hGLP-1R) was evaluated in HEK293T cells expressing hGLP-1R using cAMP assay; the in vitro agonistic activity of the above six test molecules on the human GIP receptor was evaluated in HEK293 cells expressing hGIPR receptor.

[0288] Test method:

[0289] 1. Cell resuscitation and preparation:

[0290] After resuscitating stable GLP-1R or GIPR cell lines, add Hank's balanced salt solution, centrifuge, discard the supernatant, and resuspend the cells in experimental buffer (detection buffer 1 (5mM hydroxyethylpiperazine ethanesulfonic acid, 0.5mM 3-isobutyl-1-methylxanthine, 1*Hank's balanced salt solution, 0.1% casein) or detection buffer 2 (5mM hydroxyethylpiperazine ethanesulfonic acid, 0.5mM 3-isobutyl-1-methylxanthine, 1*Hank's balanced salt solution, 0.1% casein, 1% human serum albumin)). Adjust the viable cell density to 1×10⁻⁶ cells / year. 5 / mL.

[0291] 2. Compound dilution and addition:

[0292] Using detection buffer 1 (5 mM hydroxyethylpiperazine ethanesulfonic acid, 0.5 mM 3-isobutyl-1-methylxanthine, 1*Hank's balanced salt solution, 0.1% casein) and detection buffer 2 (5 mM hydroxyethylpiperazine ethanesulfonic acid, 0.5 mM 3-isobutyl-1-methylxanthine, 1*Hank's balanced salt solution, 0.1% casein, 1% human serum albumin), telpoeptide and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, and DTM0030 were diluted to working concentrations (2× starting concentration). 40 μL of the diluted compound was pipetted into an Echo Qualified 384-well plate, and 5 μL of the serially diluted 2× test molecule and telpoeptide was added to the reaction plate (Optiplate-384) using a Bravo V11 automated liquid handling platform. In the GLP-1R cAMP assay, the initial concentrations of telpolide and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, and DTM0030 in the reaction system of detection buffer 1 were 100, 1000, 20, 20, 1000, 20, and 20 nM, respectively; and the initial concentration of all of them in the reaction system of detection buffer 2 was 10000 nM. In the GIPR cAMP assay, the initial concentrations of telpolide and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, and DTM0030 in the reaction system of detection buffer 1 were 200, 10000, 500, 500, 10000, 200, and 200 nM, respectively; and the initial concentrations in the reaction system of detection buffer 2 were 10000, 30000, 10000, 10000, 30000, 10000, and 10000 nM, respectively. All were serially diluted 4-fold, for a total of 10 sites, in duplicate wells. The high control group consisted of 20 nM GLP-1(7-37) (human glucagon-like peptide-1(7-37) MCE, HY-P0055A) and 200 nM GIP (human glucose-dependent insulinotropic peptide, MCE, HY-P0276A), while the low control group consisted of the detection buffer. cAMP standards (Revvity, 62AM4PEJ) were prepared at an initial concentration of 800 nM and serially diluted 4-fold for 10 spots, and added to the reaction plate (Optiplate-384) at a rate of 10 μL per well.

[0293] 3. cAMP detection:

[0294] Add 10 μL of either Detection Buffer 1 or Detection Buffer 2 containing cells to the reaction plate and incubate at room temperature for 30 min. Add 10 μL of cAMP detection solution (Revvity, 62AM4PEJ) to the corresponding well of the reaction plate and incubate at room temperature in the dark for 1 hour. Read the ratio of emitted light at 665 nm to emitted light at 615 nm using an EnVision microplate reader.

[0295] 4. Data Analysis:

[0296] a) Calculate the actual cAMP level (nM) for each sample well using the cAMP standard curve.

[0297] b) Formula for calculating the activity percentage of sample wells:

[0298] Activity % = (Average cAMP level in sample wells - Average cAMP level in low control group) / (Average cAMP level in high control group - Average cAMP level in sample wells) * 100%

[0299] c) Fit the model using the "log(agonist) vs. response -- Variable slope" model in GraphPad Prism 5.0 to calculate EC. 50 .

[0300] EC2 assays were performed in assay buffer 1 and assay buffer 2 to determine the EC2 activity of the test molecule against human GLP-1R & GIPR activation. 50 (nM) is shown in Table 7.

[0301] Table 7 ECGs of GLP-1R and GIPR activation activities 50

[0302]

[0303] Results analysis:

[0304] The agonistic activity of the test molecule and control telpoeptide on human GLP-1 and GIP receptors was measured using EC50. 50 The value indicates that EC 50A smaller value indicates stronger receptor activation activity. In the assay system of Detection Buffer 1, the GLP-1R agonist activity of DTM0003 and DTM0026 was slightly lower than that of telpolide; the agonist activity of DTM0001, DTM0002, DTM0030, and DTM0028 was stronger than that of telpolide. In the assay system of Detection Buffer 1, the GIPR agonist activity of the six test molecules was lower than that of telpolide. In the assay system of Detection Buffer 2, the in vitro agonist activity of all test molecules and control telpolide for GIPR and GLP-1R was significantly weakened, and compared with telpolide, the weakening of in vitro activity of DTM0003, DTM0001, DTM0002, DTM0026, and DTM0028 was more pronounced in Detection Buffer 2. Detection Buffer 2 EC 50 / Detection buffer 1 EC 50 A higher T value indicates that the compound binds more readily to HSA (human serum albumin), which is expected to improve the drug's activity level in the body. 1 / 2 The extension of (half-life).

[0305] Example 5: Pharmacokinetics in cynomolgus monkeys

[0306] In this embodiment, the cynomolgus monkey was used as a model to test the in vivo pharmacokinetic properties of the six test molecules in Example 3, and telpolide (SEQ ID No: 100) was used as a control molecule.

[0307] 0.144 mg / kg of telpotetamide and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, and DTM0030 were subcutaneously injected in PBS phosphate buffer (pH 7.4) at a volume of 1 mL / kg. At 0, 1, 2, 6, 24, 48, 72, 120, 168, 240, and 336 hours post-administration, 0.5 mL of blood was collected via venous sampling from the extremities, placed in EDTA-K2 tubes, gently vortexed to mix, and centrifuged over 30 min in an ice-water bath (4°C, 2000 g, 10 min). The plasma was separated and frozen at –70°C for analysis.

[0308] LC-MS / MS detection conditions:

[0309] Sample pretreatment method: protein precipitation method

[0310] Chromatographic column: ACQUITY UPLC BEH C18 Column (1.7μm*2.1*50mm)

[0311] Mobile phase A: ultrapure water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Injection washing solution: methanol / acetonitrile / isopropanol / ultrapure water = (1:1:1:1, v / v / v / v)

[0312] Assay Method: An acetonitrile / 5mM ammonium acetate aqueous solution (7:3, v / v) was used as a stock solution to dilute the analyte, obtaining the required series of working solution concentrations. 5 μL of working solution (50, 100, 200, 500, 1000, 2000, 5000, 10000 ng / mL) was added to 45 μL of blank cynomolgus monkey plasma to achieve a total volume of 5-1000 ng / mL (5, 10, 20, 50, 100, 200, 500, 1000 ng / mL) calibration standard. Five quality control (QC) samples (10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 800 ng / mL) were prepared on the day of analysis in the same manner as the calibration standard. 50 μL of standard, 50 μL of QC sample, and 50 μL of sample were added to 200 μL of an internal standard mixture containing acetonitrile to precipitate the protein. The sample was then vortexed for 3 min. After centrifugation at 3200 g for 15 min at 4 °C, the supernatant was diluted with 5 mM ammonium acetate at a ratio of 1:1 (V / V), and 20 μL of the diluted supernatant was injected into an LC-MS / MS system (SCIEX 4500) for quantitative analysis.

[0313] result:

[0314] As shown in Table 8, the mean half-lives (T1 / 2) of the tested molecules DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, DTM0030 and telpolide in cynomolgus monkeys after subcutaneous injection (0.144 mg / kg) were 54.4, 122, 94.1, 50.1, 46.0, 69.1 and 65.9 hours, respectively, and the mean plasma exposure (AUC0-336h) were 98579, 369488, 220022, 109599, 129438, 127653 and 113088 h*ng / ml. The results showed that the pharmacokinetic properties of DTM0003, DTM0026, DTM0028 and DTM0030 were similar to those of telpolide, while DTM0001 and DTM0002 were superior to telpolide.

[0315]

[0316] Example 6: Effects on weight loss in diet-induced obese (DIO) monkeys

[0317] The in vivo weight-loss efficacy of DTM0001, DTM0002, DTM0026, and DTM0028 was further evaluated in a diet-induced obese (DIO) monkey (Shanghai WuXi AppTec) model, with telpolide (SEQ ID No:100) used as a control molecule.

[0318] In Study 1, male obese (DIO) cynomolgus monkeys aged 10–21 years, weighing 10–16 kg, were used. The animals were housed in temperature-controlled facilities (16–26°C) with a 12-hour light / dark cycle and free access to food and water. Nine DIO monkeys were randomly assigned to experimental groups (n = 3 / group) based on their weight, thus ensuring similar starting average weights for each group.

[0319] DTM0001, DTM0002 (dose range 60 nmol / kg), and telpolide (60 nmol / kg) were dissolved in excipients (100 mM phosphate buffer, pH 7.2–7.4) and administered via single subcutaneous injection. The onset date of administration was recorded as day 0. Body weight of the DIO monkeys in each group was measured on days 7 and 14. The absolute rate of change in body weight of the DIO monkeys was calculated using the following formula:

[0320]

[0321] Table 9 Percentage of body weight change in DIO monkeys (change in body weight from initial weight %)

[0322]

[0323] Note: *p<0.05, **p<0.01, ***p<0.001 are relative to the telpoeptide group (One-Way ANOVA, Dunnett's). Results are expressed as per group.

[0324] Mean ± standard deviation of the rate of change in body weight of 3 DIO monkeys.

[0325] In Study 2, male obese (DIO) cynomolgus monkeys aged 10–21 years, weighing 10–16 kg, were used. The animals were housed in temperature-controlled facilities (16–26°C) with a 12-hour light / dark cycle and free access to food and water. Nine DIO monkeys were randomly assigned to experimental groups (n = 3 / group) based on their weight, thus ensuring similar starting average weights for each group.

[0326] DTM0026, DTM0028 (dose range 30 nmol / kg), or telpolide (30 nmol / kg) were dissolved in excipients (100 mM phosphate buffer, pH 7.2–7.4). The first dosing was recorded as day 0, day 7, and day 21, with the second and third doses administered on these dates. The body weight of the DIO monkeys was measured twice weekly to observe changes in body weight in each group. The absolute rate of change in body weight of the DIO monkeys was calculated using the following formula:

[0327]

[0328] Table 10 Percentage of body weight change in DIO monkeys (change in body weight from initial weight %)

[0329]

[0330]

[0331] As shown in Tables 9 and 10, when administered subcutaneously at a single dose of 60 nmol / kg, DTM0001 and DTM0002 showed significantly greater effects in reducing the body weight of DIO monkeys compared to telpotetide. The body weight reduction rates of DTM0001, DTM0002, and telpotetide on day 14 were -9.17% ± 1.57%, -8.19% ± 3.03%, and -1.69% ± 1.80%, respectively, showing a significant difference compared to telpotetide. After multiple subcutaneous injections of 30 nmol / kg, the body weight reduction rates of DIO monkeys in the DTM0026, DTM0028, and telpotetide groups on day 28 were -7.65% ± 4.49%, -8.96% ± 3.75%, and -4.18% ± 6.96%, respectively. DTM0028 showed a stronger effect in reducing monkey body weight than telpotetide.

[0332] Example 7: Effect of a single subcutaneous administration on glucose tolerance in C57 mice

[0333] This embodiment evaluates the effect of a single subcutaneous administration of DTM0026 and DTM0028 on glucose tolerance in C57BL / 6JNifdc mice (Vitolliwa), using telpolide (SEQ ID No:100) as a control molecule.

[0334] In this study, male C57BL / 6JNifdc mice, aged 6-8 weeks and weighing 23-26g, were used. The animals were housed in a temperature-controlled facility (20-24℃) with a 12-hour light / dark cycle and free access to food and water. After one week of acclimatization, the mice were randomly assigned to experimental groups (n=6 / group) based on their weight. Therefore, each group had a similar starting average weight, with an average weight of 24.6-24.7g on the day of group assignment.

[0335] DTM0026, DTM0028 (30 nmol / kg), and telpolide (30 nmol / kg) were dissolved in excipients (100 mM phosphate buffer, pH 7.2–7.4) and administered as a single subcutaneous injection. Mice in each group were fasted until all blood collection points were reached after administration. Approximately 12 hours post-administration, glucose was injected intraperitoneally at a dose of 2 g / kg. Blood samples were collected via tail tip sampling (10 μL whole blood + 90 μL physiological saline) at 0 min before glucose administration and at 15, 30, 60, and 120 min after glucose administration. The samples were incubated on ice for 30 min, centrifuged at 3000 rpm for 10 min at 4°C, and serum was collected. Blood glucose levels were measured using a Solarbiol blood glucose assay kit. A standard curve was plotted linearly using a method pre-set on the microplate reader (Infinite E PLEX), and blood glucose levels were calculated based on the standard curve. The blood glucose concentration curves for each group of mice from 0 to 120 min were plotted using Graphpad Prism 8.0.2 software, and the area under the curve (AUC) was calculated using the formula. (0-120min) Statistical comparisons between groups were performed using one-way ANOVA followed by Dunnett's multiple comparison test. The AUC formula is as follows:

[0336]

[0337] n is the number of sampling points, Gi is the blood glucose value at time ti, and ti+1-ti is the width between ti cells.

[0338] Table 11 Blood glucose levels at various time points in C57 mice

[0339]

[0340] Note: Results are expressed as the mean ± standard deviation of blood glucose levels at each time point for each of the six mice in each group.

[0341] Table 12 AUC of C57 mice in each group (0-120min) blood sugar level

[0342]

[0343] Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the control group (One-Way ANOVA, Dunnett's), results table

[0344] The AUC of 6 mice in each group is shown. (0-120min) The mean ± standard deviation.

[0345] As shown in Tables 11 and 12, the area under the blood glucose time curve (AUC) for groups DTM0028 and DTM0026 is as follows: (0-120min) The glucose tolerance levels (AUC) were 141.53 ± 15.40 mg / dL*h and 155.82 ± 38.40 mg / dL*h, respectively. Compared with the blank control group (excipient), glucose administration significantly reduced blood glucose levels in mice, improving glucose tolerance. Compared with the same dose of telpolide, DTM0028 and DTM0026 AUC were... (0-120min) Slightly lower.

Claims

1. A GIPR and GLP-1R co-stimulatory polypeptide, the structure of which is shown in SEQ ID NO:

101.

2. A pharmaceutical composition comprising the GIPR and GLP-1R co-stimulatory peptide of claim 1, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

3. Use of the GIPR and GLP-1R co-activating peptide of claim 1 in the preparation of a medicament for one or more of the following uses: (1) Treatment and / or relief of type 2 diabetes; (2) Treatment and / or relief of obesity or overweight; (3) Lose weight.

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