GIPR and GLP-1R co-agonistic polypeptide and application thereof

Abstract

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

Description

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

[0001] Reference to Related 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 contents of both of which are incorporated by reference in their entirety and for all purposes. TECHNICAL FIELD

[0003] The present application relates to the field of biopharmaceuticals, and more specifically, to GIPR and GLP-1R co-agonist polypeptides, or pharmaceutically acceptable salts, amides, or esters thereof, and uses of the aforementioned polypeptides, salts, amides, or esters. BACKGROUND

[0004] According to the American Diabetes Association’s classification and diagnostic criteria for diabetes in 2018, diabetes is mainly divided into the following types: the first type is type I diabetes, which is caused by the autoimmune system mistakenly attacking and destroying pancreatic beta cells, resulting in absolute lack of insulin; the second type is type II diabetes (T2D), which occurs in the context of insulin resistance, and the amount of insulin produced by beta cells gradually decreases; the third type is gestational diabetes (GDM), which is a diabetes first diagnosed during pregnancy; there are also other specific factors causing diabetes, such as monogenic diabetes syndrome, pancreatic exocrine diseases (such as cystic fibrosis and pancreatitis), and diabetes caused by drugs or chemicals (such as diabetes after using steroids to treat HIV / AIDS or organ transplantation). Among these types, T2D is the most common, previously known as adult-onset diabetes, accounting for more than 90% of all diabetes patients. Currently, an increasing number of children are diagnosed with T2D, which may be related to the rising rate of childhood obesity. As the most common metabolic disease, T2D has seen a steady increase in the number of patients over the past half century, and has shown a trend of spreading from the United States and Europe to Asia, the Pacific, and Africa. According to existing models, it is predicted that nearly 700 million people worldwide will be affected by this disease by 2045. Unlike type I diabetes caused by congenital insulin secretion deficiency, the occurrence and development of T2D are mainly related to insulin resistance.

[0005] Obesity is a complex disease caused by multiple factors, and excessive accumulation of fat in the body can have adverse effects on health. Currently, obesity is growing rapidly, forming a large-scale epidemic phenomenon, and there is no trend of slowing down in the short term. Obesity is a risk factor for non-communicable diseases such as diabetes and heart disease, and has a serious impact on people’s quality of life and life expectancy.

[0006] Obesity is also one of the major causes of T2D, and the progression from obesity to diabetes is generally as follows: obesity - impaired glucose tolerance - T2D - uncontrolled hyperglycemia - diabetic complications. Therefore, for the treatment of diabetes with obesity, in addition to reducing blood glucose, reducing body weight is also a factor that must be considered.

[0007] Glucagon-like peptide-1 receptor (GLP-1R) belongs to the B cluster of G protein-coupled receptors and is a member of the glucagon receptor subfamily. It has distinct structural features, including a long extracellular N-terminal region (ECD) of about 100-150 amino acids, seven transmembrane domains (TMDs), and a short intracellular C-terminal region (ICD). The extracellular region consists of two reverse beta sheets connected by a loop, contains six conserved cysteines forming three pairs of disulfide bonds, and has a flexible alpha helix.

[0008] GLP-1R is widely distributed in various tissues of the body, including the islets, stomach, small intestine, heart, kidney, lung, and brain. In islet beta cells, GLP-1R mainly promotes insulin secretion, enhances the regenerative capacity of beta cells, inhibits cell apoptosis, and reduces the release of glucagon. In tissues such as the gastrointestinal tract, GLP-1R slows down gastrointestinal motility, inhibits gastric juice secretion, delays gastric emptying, and enhances satiety through binding with its agonists. In the nervous system, GLP-1R agonists can penetrate the brain, protect neural cells from apoptosis by activating GLP-1R, and enhance learning and memory. In addition, activation of GLP-1R can also help to reduce body weight by controlling food intake. In terms of cardiovascular, studies have found that knocking out the GLP-1R gene in mice leads to decreased resting heart rate and increased left ventricular diastolic pressure. In addition, activation of GLP-1R can also reduce oxidative stress and inhibit cardiomyocyte apoptosis.

[0009] Glucose-dependent insulinotropic polypeptide receptor (GIPR) is also a G protein-coupled receptor with seven transmembrane domains. The N-terminal of GIPR contains a glycosylation sequence and a third cytoplasmic loop, while the C-terminal is rich in threonine and serine, 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, bone, lung, and spleen. In the islets, only alpha and beta cells express GIPR. GIPR activates G proteins to increase intracellular cAMP and Ca 2+GIPR signals can promote insulin secretion and reduce blood glucose levels after food intake. GIPR also affects fat cell storage and metabolism, 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 fat metabolism, and is essential for maintaining normal metabolic status.

[0010] In recent years, the importance of the GIPR-GIP pathway has been increasingly recognized. The GIPR signaling pathway is involved in various physiological processes, including islet proliferation, energy metabolism regulation, and control of intestinal function, and is closely related to lipid accumulation in adipose tissue, insulin resistance, etc., but the downstream signaling pathways activated by GIPR-GIP need further study. In adipocytes, GIPR-GIP promotes PKB phosphorylation through pathways such as activation of CREB, TORC2 or insulin, enhances lipoprotein lipase LPL activity, and promotes fat accumulation. GIP can also promote the expression of IL-6 in adipose tissue and induce insulin resistance. GIPR-GIP reduces caspase3 and bax gene activity through MAPK, Akt and FoxO1 pathways, and plays a role in promoting proliferation and anti-apoptosis. There are also reports that activation of the GIPR-GIP signaling pathway can promote the occurrence of leptin resistance in obesity. GIP inhibits the activation of leptin receptor signaling pathway molecules and promotes the expression of negative regulators of the leptin signaling pathway by activating the Epac / Rapl signaling pathway in the hypothalamus, inhibiting leptin-induced pro-opiomelanocortin neuron activation, thereby inducing central leptin resistance. In addition, GIPR-GIP upregulates TCF4 through the Akt signaling pathway to enhance GIPR expression, increase beta cell proliferation and insulin secretion function, and maintain blood glucose homeostasis.

[0011] In 2022, Eli Lilly launched the world's first GLP-1R / GIPR dual receptor agonist, tirzepatide. Tirzepatide is a GIPR and GLP-1R dual-target hypoglycemic weight-reducing polypeptide with better clinical effects. Its activation of GIPR activity is affected by the activation of GLP-1R activity.

[0012] Given the promising prospects of GLP-1R / GIPR dual receptor agonists, developing more such drugs is urgently needed in the art.

[0013] SUMMARY

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

[0015] R1X1X2GluX4X5X6X7X8X9X10 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 , 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 are independently any natural amino acid residue, non-natural amino acid residue, or absent; and / or R1is any peptide segment, or absent; and / or R2is -NH2, any peptide segment, or absent.

[0016] The second aspect of the present application relates to a pharmaceutical composition comprising a GIPR and GLP-1R co-agonistic polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide or ester thereof, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0017] The third aspect of the present application relates to a method of preventing, treating and / or ameliorating a metabolic disease or disorder, comprising administering to a patient or subject in need thereof 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 the present application relates to a method for reducing body weight, the method comprising administering to an individual in need thereof an effective amount of the polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition as described in the second aspect.

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

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

[0021] The seventh aspect of the present application relates to the use of the polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition as described in the second aspect, in the manufacture of a medicament for one or more of the following uses: (1) preventing, treating and / or alleviating a metabolic disease or disorder; (2) preventing, treating and / or alleviating diabetes; preferably, the diabetes is type II diabetes; (3) preventing, treating and / or alleviating obesity or overweight; (4) reducing body weight; (5) reducing food intake; (6) improving glycemic control (e.g. improving glucose tolerance).

[0022] BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 shows the chemical structural formula of one exemplary molecule DTM0001 of the present application.

[0024] Figure 2 shows the chemical structural formula of one exemplary molecule DTM0002 of the present application.

[0025] Figure 3 shows the chemical structural formula of one exemplary molecule DTM0003 of the present application.

[0026] Figures 4 and 5 show the chemical structural formula of exemplary molecules DTM0026 and DTM0028 of the present application.

[0027] DETAILED DESCRIPTION

[0028] The embodiments described below are to better illustrate the content of the present application, but are not intended to limit the content of the present application to only the embodiments. Non-essential modifications and adjustments to the embodiments can be made by those skilled in the art based on the above disclosure, and still fall within the scope of the present application.

[0029] While various embodiments of the application are described in detail herein, it is understood that the application is not limited to the specific methods, protocols, and reagents described herein as these can vary. It is also to 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 the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0030] The recitation of a range of values herein is merely intended to serve as a disclosing the value. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0031] Throughout this specification several documents are cited. Each of the documents cited herein, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., is hereby incorporated by reference in its entirety.

[0032] The term

[0033] Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps to the subject-matter defined by the expression comprising but in some embodiments can exclude such other member, integer or step or group of members, integers or steps as the subject-matter is specifically intended to include the stated member, integer or step or group of members, integers or steps.

[0034] Unless otherwise indicated herein, or otherwise apparent from context, the terms "a" and "an" and "the" and similar referents used in the description within the context of describing the application (especially in the context of the claims) are to be construed to cover both the singular as well as the plural unless otherwise indicated herein or otherwise apparent from context.

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

[0036] In the present application, the term "natural" when used in connection with amino acids refers to the 20 conventional 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 (lie or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin 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, pyrrolysine (PYL), and pyrroline-carboxy lysine (PCL).

[0037] Herein, all natural amino acids not designated as optically isomeric are understood to refer to the L-isomer, unless otherwise indicated.

[0038] As used herein, the term "non-natural" when used in connection with amino acids is meant to refer to an amino acid that is not naturally encoded or found in the genetic code of any organism. It can be, for example, a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, gamma-carboxyglutamate, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-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, t-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-methylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, iso-desmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, piperidic acid, and thioproline.

[0039] The term "amino acid residue" includes an amino acid from which a hydrogen atom has been removed from an amino group and / or a hydroxyl group has been removed from a carboxyl group and / or a hydrogen atom has been removed from a thiol group. Inaccurately, an amino acid residue can sometimes be referred to herein as an amino acid. Herein, each amino acid abbreviation in a sequence represents the corresponding amino acid residue.

[0040] Herein, the term "Aib" refers to 2-aminoisobutyric acid, which when forming a polypeptide means 2-aminoisobutyryl.

[0041] Herein, the term "AEEA" refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid, which when a group in a compound means 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl.

[0042] Herein, the term "γ-Glu" means γ-glutamyl.

[0043] The term "modification" of a peptide in the present application includes any change made to the peptide, such as a change in the length of the peptide, a change in the amino acid sequence, a change in the chemical structure, a co-translational modification or a post-translational modification of the peptide. In some cases, the peptides of the present application comprise one or more modified amino acid residues. The types of modifications are well known in the art. Modifications useful in the present application include amidation modification, fatty acid modification, methylation, myristoylation, PEG modification, fluorine element modification, biotin modification, fluorescent label modification, cyclization, carboxylation, acetylation modification, phosphorylation modification, glycosylation modification, or other known polypeptide modifications.

[0044] As used herein, the terms "treat," "treating," or "treatment" with respect to a disease, disorder, or condition mean to alleviate or ameliorate the disease, disorder, or condition (i.e., to slow or arrest the development or progression of a disease, disorder, or condition or at least one clinical symptom thereof); or to alleviate or ameliorate at least one physical parameter or biomarker associated with the disease, disorder, or condition, including those that can not be discernible by the patient.

[0045] As used herein, the terms "prevent," "preventing," or "prevention" with respect to a disease, disorder, or condition mean prophylactic treatment of the disease, disorder, or condition; or delay in the onset or progression of the disease, disorder, or condition.

[0046] As used herein, a subject, patient, individual is "in need of" a treatment if such subject, patient, individual would benefit biologically, medically, or in quality of life from the treatment. In certain instances herein, the terms "subject," "patient," and "individual" are used interchangeably 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 effective amount described herein can, for example, activate GLP-1R and / or GIPR activity, ameliorate one or more symptoms, reduce one or more conditions, slow, lessen, or delay the progression of a disease, disorder, or condition, or prevent a disease, disorder, or condition.

[0048] Metabolic disease as described herein has the meaning described in the present application.

[0049] The term "body mass index" or "BMI" of a human patient or subject is defined as weight in kilograms divided by the square of height in meters, so the BMI has units of kg / m 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 persists, obesity results, i.e., body weight balance has two components, and abnormalities on either side (intake or expenditure) can lead to obesity. In this context, obesity is best viewed as any degree of excess adipose tissue that carries health risks. The distinction between normal and obese individuals can only be approximated, but the health risks associated with obesity can be a continuum with increasing adipose tissue. The term "obesity" refers to an excessive accumulation of adipose tissue in the body to such an extent that a metabolic disease affecting the whole body is formed. In certain instances herein, "obesity" and "obese" are used interchangeably.

[0051] The WHO definition of "overweight" is a condition in which an individual has a BMI greater than 25 kg / m 2 and less than 30 kg / m 2 . In certain instances, the terms "overweight" and "pre-obese" have the same meaning.

[0052] The WHO definition of "obesity" is a condition in which an individual has a BMI equal to or greater than 30 kg / m 2 . According to the WHO definition, the term obesity can be classified as follows: the term "class I obesity" is a condition in which the BMI is equal to or greater than 30 kg / m 2 but less than 35 kg / m 2 ; the term "class II obesity" is a condition in which the BMI is equal to or greater than 35 kg / m 2 but less than 40 kg / m2 obesity; the term "class III obesity" is a condition in which the BMI is equal to or greater than 40 kg / m 2 obesity.

[0053] In certain instances herein, the term "obese" refers to a condition in which a subject who is otherwise healthy has a body mass index (BMI) greater than or equal to 30 kg / m 2 obesity; the term "class III obesity" is a condition in which the BMI is equal to or greater than 40 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m

[0054] In Asian populations, increased risk associated with obesity occurs at lower body mass index (BMI). In Asian countries, "obese" refers to a condition in which a subject has a BMI greater than or equal to 25 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m 2 obese" is a subject who is otherwise healthy having a body mass index (BMI) greater than or equal to 30 kg / m

[0055] As used herein, the term "obese" is intended to encompass all of the above obesity definitions.

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

[0057] The term "weight loss" relates to the reduction of the body weight of a human and / or an animal. Weight loss can be in response to concerns about improving health, fitness level, and / or appearance. The terms "weight loss", "weight reduction", and "slimming" have the same meaning and are used interchangeably.

[0058] The term "diabetes mellitus" as used herein includes both insulin-dependent diabetes mellitus (IDDM, also known as Type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, also known as Type II diabetes), a metabolic disease. Type I diabetes or insulin-dependent diabetes mellitus is the result of an absolute deficiency of insulin, a hormone that regulates glucose utilization. Type II diabetes or diabetes independent of insulin (i.e., non-insulin-dependent diabetes mellitus) often occurs in the presence of normal or even elevated levels of insulin and appears to be the result of tissues failing to respond appropriately to insulin.

[0059] The term "improving glycemic control" includes, but is not limited to, improving glucose tolerance, reducing the level of fasting plasma glucose, postprandial plasma glucose, and / or glycosylated hemoglobin (HbAlc) in a patient or subject.

[0060] The term "HbAlc" or "glycosylated hemoglobin" as used herein is the product of the non-enzymatic reaction of hemoglobin in red blood cells with sugars (mainly glucose) in the serum. The non-enzymatic reaction that forms glycosylated hemoglobin is continuous, slow, and irreversible, so the amount of glycosylated hemoglobin is determined by the average blood glucose concentration over the past 8-12 weeks, not by the immediate glucose level. It is generally accepted that the concentration of glycosylated hemoglobin effectively reflects the average blood glucose level over the past 8-12 weeks. Glycosylated hemoglobin is composed of HbAla, HbAlb, and HbAlc, of which HbAlc accounts for about 70% and has a relatively stable structure. HbAlc is commonly used as a monitoring index for diabetes control in the clinic, and its concentration is expressed as a percentage of the total hemoglobin in adults.

[0061] As used herein, unless otherwise indicated, the term "generic sequence" or each amino acid in other polypeptide sequences is arranged in order from N-terminus to C-terminus.

[0062] In general, the present application provides GIPR and GLP-1R co-agonistic polypeptides or pharmaceutically acceptable salts, amides or esters thereof, combinations or compositions comprising the GIPR and GLP-1R co-agonistic polypeptides or pharmaceutically acceptable salts, amides or esters thereof, various uses of the GIPR and GLP-1R co-agonistic polypeptides or pharmaceutically acceptable salts, amides or esters thereof, combinations or compositions comprising the polypeptides or pharmaceutically acceptable salts, amides or esters thereof, including but not limited to preventing, treating and / or alleviating type II diabetes, obesity and overweight, etc.

[0063] Hereinafter, the description includes certain elements of the present application, which can be listed with specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce further embodiments. The differently described embodiments and exemplary embodiments should not be interpreted as limiting the present application to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or exemplary elements. In addition, unless the context indicates otherwise, it should be considered that the specification of the present application discloses any arrangement and combination of all described elements in the present application.

[0064] Specifically, the first aspect of the present application relates to a GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, the polypeptide having the following generic sequence of 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 the 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 are independently any natural amino acid residue, any unnatural amino acid residue, or absent; and / or said R1 is any peptide segment, or absent; and / or said R2 is -NH2, any peptide segment, or absent.

[0066] In some embodiments, the polypeptide has a general sequence as set forth in any one of SEQ ID Nos: 2-21. In the general sequence as set forth in any one of SEQ ID Nos: 2-21, the variables (e.g., R1, R2, X n , etc.) that are common to the general sequence as set forth in SEQ ID No: 1 can be referred to the definitions for such variables in the general sequence as set forth in SEQ ID No: 1, where not specified.

[0067] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has a general sequence as set forth 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 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 are independently any natural amino acid residue, non-natural amino acid residue, or absent.

[0069] In some embodiments, said X1is His, Phe, or Tyr; and / or said X2is Aib or Ala; and / or said X4is Gly or Ala; and / or said X5is Thr, Asn, or Ser; and / or said X6is Phe or His; and / or said X7is Thr or Val; and / or said X8is Thr or Ser; and / or said X9is Asp or Glu; and / or said X 10 is Tyr or Phe; and / or said X 11 is Thr or Ser; and / or said X 12 is lie or Asn; and / or said X 13 is Tyr, Gin, Ala, or Gly; and / or said X 15 is Asp, Glu, or Gin; and / or said X 16 is Glu or Lys; and / or said X 17 is Gin, Leu, or lie; and / or said X 18 is Ala, His, or Gly; and / or said X 19 is Gin or Asn; and / or said X 20 is Arg, Gin, or Lys; and / or said X 21 is Asp, Glu, or Ala; and / or said X 22 is Phe or Tyr; and / or said X 23 is lie or Val; and / or said X 24 is Gin, Lys, or Asn; and / or said X 25 is Trp or Tyr; and / or said X 26 is Leu or Ala; and / or said X 27 is Val or lie; and / or said X 33 is Ser or Lys.

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

[0071] In some embodiments, X2 is Aib or Ala. This site is a cleavage site for dipeptidyl peptidase-4 (DPP-4) for both GLP-1 and GIP. In some embodiments, X2 is Aib, substitution of X2 with Aib completely prevents degradation by DPP-4, and the Aib substitution has little effect on its activity to activate GLP-1R and GIPR.

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

[0073] In some embodiments, X5 is Thr, Asn, or Ser. In some embodiments, X5 is Thr, substitution of X5 with Thr significantly enhances its activity to activate GLP-1R and GIPR.

[0074] In some embodiments, X6 is Phe or His. In some embodiments, X6 is Phe, substitution of X6 with His slightly reduces its activity to activate GLP-1R and GIPR.

[0075] In some embodiments, X7 is Thr or Val. In some embodiments, X7 is not He, introduction of He to this site results in a decrease in its activity to activate GLP-1R. 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, substitution of X9 with Glu slightly reduces its activity to activate GLP-1R and GIPR.

[0078] In some embodiments, X 10 is Tyr or Phe. In some embodiments, X 10 is Tyr.

[0079] In some embodiments, X 11 is Thr or Ser. In some embodiments, X 11 is Ser.

[0080] In some embodiments, the X 12 is lie or Asn. In some embodiments, the X 12 is lie, X 12 The mutation of lie to Ala at position 8 can significantly reduce its insulinotropic effect. The use of lie at this position does not weaken its GLP-1R activity relative to Ala, but rather enhances its GIPR activity.

[0081] In some embodiments, the X 13 is Tyr, Gin, Ala or Gly. In some embodiments, the X 13 is Tyr, X 13 The substitution of Tyr with Gin slightly reduces its GLP-1R and GIPR activity.

[0082] In some embodiments, the X 15 is Asp, Glu or Gin. In some embodiments, the X 15 is Glu, X 15 The mutation of Glu to Ala or Gly at position 10 negatively affects its receptor activity.

[0083] In some embodiments, the X 16 is Glu or Lys. In some embodiments, the X 16 is Glu, X 16 The substitution of Glu increases its GLP-1R and GIPR affinity to some extent. In some embodiments, the X 16 is Lys, X 16 The substitution of Lys increases affinity and GLP-1 activity to some extent.

[0084] In some embodiments, the X 17 is Gin, Leu or lie. In some embodiments, the X 17 is lie, X 17 The lie at position 14 is important for GIPR activity. In some embodiments, the X 17 is Gin, Gin is important for GLP-1R activity. In some embodiments, the X 17 is lie.

[0085] In some embodiments, the X 18 is Ala, His or Gly. In some embodiments, the X 18 is Ala, X 18 The use of Ala at this position increases its GLP-1R and GIPR activity.

[0086] In some embodiments, the X 19 is Gin or Asn. In some embodiments, the X 19 is Gin. In some embodiments, the X 19 is Asn, X 19 Substitution of Asn for Gin slightly reduces its activity in activating GLP-1R and GIPR.

[0087] In some embodiments, the X 20 is Arg, Gin, or Lys. In some embodiments, the X 20 is Arg, X 20 Arg improves its activity in activating GLP-1R.

[0088] In some embodiments, the X 21 is Asp, Glu, or Ala. In some embodiments, the X 21 is Glu. In some embodiments, the X 21 is Asp, which can form a hydrogen bond with Arg on GIPR, which is important for maintaining GIP activity.

[0089] In some embodiments, the X 22 is Phe or Tyr. In some embodiments, the X 22 is Phe, X 22 Substitution of Tyr for Phe significantly reduces its activity in activating GIPR target.

[0090] In some embodiments, the X 23 is He or Val. In some embodiments, the X 23 is He.

[0091] In some embodiments, the X 24 is Gin, Lys, or Asn. In some embodiments, the X 24 is Gin, X 24 Gin can improve its activity in activating GLP-1R.

[0092] In some embodiments, the X 25 is Trp or Tyr. In some embodiments, the X 25 is Trp.

[0093] In some embodiments, the X 26 is Leu or Ala. In some embodiments, the X 26 is Leu. In some embodiments, the X 26 is not His, X26 Substitution of His for Leu significantly reduces its activity in activating GLP-1R and GIPR.

[0094] In some embodiments, the X 27 is Val or lie. In some embodiments, the X 27 is Val.

[0095] In some embodiments, the X 33 is Ser or Lys. In some embodiments, the X 33 is Ser.

[0096] In some embodiments, the X5is Thr; and / or the X 10 is Tyr; and / or the X 22 is Phe; and / or the X 26 is Leu.

[0097] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has the general sequence set forth 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 the 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 any natural amino acid residue, non-natural amino acid residue, absent, or has the foregoing definition; and R1, R2have the foregoing definitions.

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

[0100] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has the general sequence set forth 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 any natural amino acid residue, non-natural amino acid residue, absent, or has the foregoing definition; and R1, R2have the foregoing definitions.

[0102] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has the general sequence set forth 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 LeuX 27 AlaGlyGlyProSerX 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 GlnX25 X 26 X 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2(SEQ ID No:6), wherein the 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 are independently any natural amino acid residue, non-natural amino acid residue or absent or have the foregoing definitions; the R1, R2have the foregoing definitions.

[0107] In some embodiments, the X 12 is lie; and / or the X 15 is Glu; and / or the X 16 is Lys; and / or the X 18 is Ala; and / or the X 20 is Arg; and / or the X 24 is Gin.

[0108] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has the general 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 the X1, X2, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 13 , X 17 , X 19 , X21 , X 22 , X 23 , X 25 , X 26 , X 27 and X 33 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; and R1, R2have the foregoing definitions.

[0110] In some embodiments, the X 12 is lie; and / or the X 15 is Glu; and / or the X 16 is Glu; and / or the X 18 is Ala; and / or the X 20 is Lys; and / or the X 24 is Gin.

[0111] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptide has the general sequence set forth 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 the 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 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; and R1, R2have the foregoing definitions.

[0113] In some embodiments, the X 12is Ile; and / or said X 15 is Glu; and / or said X 16 is Glu; and / or said X 18 is Ala; and / or said X 20 is Arg; and / or said X 24 is Lys.

[0114] In some embodiments, the GIPR and GLP-1R co-agonist polypeptide has the general sequence of 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 said 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 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; said R1, R2have the foregoing definitions.

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

[0117] R1X1X2GluX4X5X6X7X8X9X 10 X 11 IleX 13 LeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX25 X 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] R1X1X2GluGlyX5PheX7X8AspX 10 X 11 IleTyrLeuGluGluX 17 AlaX 19 ArgX 21 X 22 X 23 GlnX 25 X 26 X 27 AlaGlyGlyProSerX 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, said X1is Tyr; and / or said X2is Aib; and / or said X7is Thr; and / or said X8is Ser; and / or said X 11 is Ser; and / or said X 17 is Gln; and / or said X 19 is Gln and / or said X 21 is Glu; and / or said X 23 is lie; and / or said X 25 is Trp; and / or said X 27 is Val.

[0125] In some embodiments, said GIPR and GLP-1R co-agonistic polypeptide has the general sequence of 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 said 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 are independently any natural amino acid residue, non-natural amino acid residue or absent or have the aforementioned definitions; said R1, R2have the aforementioned definitions.

[0127] In some embodiments, said GIPR and GLP-1R co-agonistic polypeptide has the general sequence of SEQ ID No: 15:

[0128] R1TyrAibGluX4ThrX6ThrSerX9TyrSerX 12 X 13 LeuX15 X 16 GlnX 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] R1TyrAibGluX4X5X6ThrSerX9X 10 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 polypeptide 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 13Leu GIu GIu GIn Ala GIn Arg GIu Phe lie GIn Trp Leu VaI Ala GIy GIy Pro Ser X 33 Gly Ala Pro Pro Pro Ser R2 (SEQ ID No: 19), wherein the X4, X6, X9, X 13 and X 33 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; the R1, R2have the foregoing definitions.

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

[0138] R1 Tyr Aib GIu GIy X5 Phe Thr Ser Asp X 10 Ser lie Tyr Leu GIu GIu GIn Ala GIn Arg GIu X 22 Ile GIn Trp X 26 VaI Ala GIy GIy Pro Ser X 33 Gly Ala Pro Pro Pro Ser R2 (SEQ ID No: 20), wherein the X5, X 10 , X 22 , X 26 and X 33 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; the R1, R2have the foregoing definitions.

[0139] In some embodiments, the GIPR and GLP-1R co-agonist polypeptide has the general sequence set forth in SEQ ID No: 21:

[0140] R1 Tyr Aib GIu GIy Thr Phe Thr Ser Asp Tyr Ser lie Tyr Leu GIu GIu GIn Ala GIn Arg GIu Phe lie GIn Trp Leu VaI Ala GIy GIy Pro Ser X 33 Gly Ala Pro Pro Pro Ser R2 (SEQ ID No: 21), wherein the X 33 are independently any natural amino acid residue, non-natural amino acid residue or absent, or have the foregoing definitions; the R1, R2have the foregoing definitions. In further embodiments, the X 33 is Lys.

[0141] In some embodiments of the general sequences of SEQ ID Nos: 1-21, the X 16 , X 20 , X 24 , and / or X 33 is Lys. In further embodiments, the Lys further comprises a fatty acid modification.

[0142] In some embodiments, the N-terminal, C-terminal, and / or amino acid residues within the polypeptide sequence of the polypeptide further comprise a chemical modification. In some embodiments, the chemical modification comprises one or more of amidation modification, fatty acid modification, methylation, myristoylation, PEG modification, fluorine element modification, biotin modification, fluorescent label modification, cyclization, carboxylation, acetylation modification, phosphorylation modification, glycosylation modification. In some embodiments, the chemical modification comprises an amidation modification and / or a fatty acid modification. In some embodiments, the chemical modification is an amidation modification and a fatty acid modification.

[0143] In some embodiments, the chemical modification occurs at the C-terminal. In some embodiments, the chemical modification of the C-terminal comprises 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 an amino acid residue within the polypeptide sequence. In some embodiments, the chemical modification comprises or is a fatty acid modification. In some embodiments, one or more Lys residues in the polypeptide comprise a fatty acid modification.

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

[0146] In some embodiments, the fatty acid modification occurs at one or more amino acid residues that are X 16 , X 20 , X 24 , or X 33 . In some embodiments, the fatty acid modification occurs at one that is X 16 , X 20 , X 24 , or X 33 . In some embodiments, the fatty acid modification occurs at a lysine residue that is X 16 , X 20 , X 24 , or X 33 .

[0147] In some embodiments, the fatty acid modification has the structure of -AEEA a -(y-Glu) b -CO-(CH2) n -COOH, where a, b, n are natural numbers. In some embodiments, 12

[0148] In some embodiments, the fatty acid modification has the structure of -AEEA n -COOH, where n is a natural number, and 12 n -COOH, where n is a natural number, n is 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the fatty acid modification has the structure of -AEEA 16 -COOH. In some embodiments, the fatty acid modification has the structure of -AEEA 18 -COOH.

[0149] In some embodiments, the fatty acid modification is linked to the epsilon-amino group of the Lys residue. In some embodiments, R1and / or R2in the general sequence is absent. In some embodiments, R1is absent. In some embodiments, R2is absent. In some embodiments, both R1and R2are absent.

[0150] In some embodiments, the GIPR and GLP-1R co-agonist polypeptides involved in the present application have a sequence set forth in any one of SEQ ID Nos. 22-41, 43-73, 76, 78-80, 82-86, 88-98, 101-131, or the sequence of the polypeptide consists of the sequence set forth in any one of SEQ ID Nos. 22-41, 43-73, 76, 78-80, 82-86, 88-98, 101-131.

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

[0152] Table 1A

[0153] In some embodiments, the GIPR and GLP-1R co-agonist polypeptides of the present application comprise or have a sequence that is identical to the sequences shown in Table 1A except that the A (Ala) at position 2 is substituted with Aib. In some other embodiments, the sequences comprising the A (Ala) at position 2 substituted with Aib are shown in Table IB.

[0154] Table IB

[0155] In some embodiments, the pharmaceutically acceptable amides of the GIPR and GLP-1R co- agonist polypeptides of the present application are formed by C-terminal amidation of the GIPR and GLP-1R co-agonist polypeptides of the present application, such as any one of the sequences shown in any one of SEQ ID Nos. 76, 78-80, 82-86, 88-98 in Table 3 below. In some embodiments, the pharmaceutically acceptable amides of the GIPR and GLP-1R co-agonist polypeptides of the present application, the GIPR and GLP-1R co-agonist polypeptide sequence is selected from a sequence identical to a sequence in Table 1A or a sequence identical to a sequence shown in Table 1A except that the A (Ala) at position 2 is substituted with Aib, and is C-terminally amidated.

[0156] In some embodiments, the GIPR and GLP-1R co-agonist polypeptides of the present application comprise or consist of a modified sequence selected from Table 2.

[0157] Table 2

[0158] In some embodiments, the pharmaceutically acceptable amides of the GIPR and GLP-1R co- agonist polypeptides of the present application are formed by C-terminal amidation of the GIPR and GLP-1R co-agonist polypeptides of the present application, such as any one of the sequences shown in any one of SEQ ID Nos. 101-121 and 128-130. In some embodiments, the amidation modification is a primary amide modification.

[0159] The foregoing {K(AEEA-AEEA-y-Glu-17-carboxyheptadecanoyl)} indicates that the lysine residue at this site is chemically modified by conjugation with -AEEA-AEEA-y-Glu-CO-(CH2) 16 -COOH to the epsilon-amino group of the lysine side chain. The foregoing {K(AEEA-AEEA-y-Glu-19-carboxynonadecanoyl)} indicates that the lysine residue at this site is chemically modified by conjugation with -AEEA-AEEA-y-Glu-CO-(CH2) 18-COOH is chemically modified by conjugation to the epsilon-amino group of the lysine side chain.

[0160] Figures 1 to 5 show the chemical structure of five exemplary molecules of the present application.

[0161] In some embodiments, the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, described herein comprise or have a sequence that is a variant of a sequence selected from Table 2, wherein the variant is substituted with an amino acid residue other than -AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl and -AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl. In some embodiments, the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, described herein comprise or have a sequence that is a variant of a sequence selected from Table 2, wherein the variant is substituted with an amino acid residue other than arginine. In some embodiments, the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, described herein comprise or have a sequence selected from Table 2, wherein -AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl or -AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl is substituted with -R(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl) or -R(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl). In some embodiments, the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, described herein comprise or have a sequence that is a variant of a sequence selected from Table 2, wherein the variant is substituted with -K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl). In some embodiments, the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, described herein comprise or have a sequence selected from Table 2, wherein -AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl is substituted with -K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl).In some embodiments, the GIPR and GLP-1R co-agonist polypeptide comprises or has a sequence that is a variant of a sequence selected from Table 2, the variant having {K(AEEA-AEEA-γ-Glu-19-carboxynonadecanoyl)} replaced with {K(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)} relative to the sequence selected from Table 2.

[0162] In some embodiments, the GIPR and GLP-1R co-agonist polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, involved in the present application comprises or has a sequence that is a variant of a sequence selected from Table 2, the variant having other fatty acid modification replacing the fatty acid modification used relative to the sequence selected from Table 2. In some embodiments, the other fatty acid modification is HOOC(CH2) n CO-, wherein 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 modification can take the form of COOH(CH2) 14～20 CO-γ-Glu-AEEA-AEEA-.

[0163] In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-agonist polypeptide involved in the present application is formed by C-terminal amidation of the GIPR and GLP-1R co-agonist polypeptide of the present application. In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-agonist polypeptide involved in the present application comprises or consists of C-terminal amidation of a sequence selected from Table 2. In some embodiments, the pharmaceutically acceptable amide of the GIPR and GLP-1R co-agonist polypeptide involved in the present application comprises or consists of C-terminal amidation of a sequence in Table 2 to a C-terminal primary amide.

[0164] In some embodiments, the GIPR and GLP-1R coagonist polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, involved in the present application, the GIPR and GLP-1R coagonist polypeptide comprises or has a sequence that is a variant of a sequence selected from Table 2, which is further modified at the N-terminal 1st amino acid and / or 2nd amino acid relative to the sequence selected from Table 2 to reduce the susceptibility to DPP-4 cleavage. More specifically, in some embodiments, the N-terminal 1st amino acid and / or 2nd amino acid is replaced with a DPP-4 resistant amino acid. In some embodiments, the N-terminal 1st amino acid and / or 2nd amino acid of the sequence in Table 2 is replaced with an amino acid selected from D-serine, D-alanine, glycine, N-methyl serine and ε-aminobutyric acid.

[0165] In some embodiments, the GIPR and GLP-1R coagonist polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, involved in the present application, the GIPR and GLP-1R coagonist polypeptide comprises or has a sequence that is a variant of a sequence selected from Table 2, which is further modified at the N-terminal 1st amino acid and / or 2nd amino acid relative to the sequence selected from Table 2 to reduce the susceptibility to DPP-4 cleavage. More specifically, in some embodiments, the N-terminal 1st amino acid and / or 2nd amino acid is replaced with a DPP-4 resistant amino acid. In some embodiments, the N-terminal 1st amino acid and / or 2nd amino acid of the sequence in Table 2 is replaced with an amino acid selected from D-serine, D-alanine, glycine, N-methyl serine and ε-aminobutyric acid.

[0166] In some embodiments, the GIPR and GLP-1R coagonist polypeptide involved in the present application has the general sequence shown in SEQ ID No: 132:

[0167] 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.

[0168] 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.

[0169] In some embodiments, the GIPR and GLP-1R co-excitation peptide involved in this application has the general formula sequence shown in SEQ ID No:133:

[0170] 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 is Val or lie; X 33 is Ser or Lys.

[0171] In some embodiments of the general sequence shown in SEQ ID No: 133, X 16 , X 20 , X 24 and / or X 33 is Lys.

[0172] In some embodiments of the general sequence shown in SEQ ID No: 133, the Lys comprises a fatty acid modification -(AEEA) a -(y-Glu) b -CO-(CH2) n -COOH, wherein a, b, n are natural numbers, and 12n<20. In some embodiments, the n is 16 or 18, the a is 2, and the b is 1.

[0173] In some embodiments of the general sequence shown in SEQ ID No: 133, the polypeptide has a sequence, or consists of a sequence, of any one of SEQ ID Nos. 47-51, 61, 62, 65, 66, 71, 73, 131, 101-105, 116, 117, 120, 121, 128, 129, 130, 122-127.

[0174] In some embodiments, the GIPR and GLP-1R co-agonistic polypeptides of the present application have a general sequence shown in SEQ ID No: 134:

[0175] Tyr X2Glu Gly Thr Phe Thr Ser Asp Tyr Ser lie Tyr Leu Glu X 16 Gln Ala Gln Arg X 21 Phe lie X 24 Trp Leu Val Ala Gly Gly Pro Ser X 33 Gly Ala Pro Pro Pro Ser R2(SEQ ID No: 134), wherein X2is Ala or Aib; X 16 is Glu or Lys; X 21 is Glu or Asp; X 24 is Gin, Lys or Asn; X 33is Ser or Lys.

[0176] In some embodiments of the general sequence set forth in SEQ ID No: 134, X 16 , X 24 , and / or X 33 is Lys.

[0177] In some embodiments of the general sequence set forth in SEQ ID No: 133, the Lys comprises a fatty acid side chain modification -(AEEA) a -(y-Glu) b -CO-(CH2) n -COOH, wherein a, b, n are natural numbers, and 12n<20. In some embodiments, the n is 16 or 18, the a is 2, and the b is 1.

[0178] In some embodiments of the general sequence set forth in SEQ ID No: 133, the polypeptide has the sequence set forth in any one of SEQ ID Nos. 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 set forth in any one of SEQ ID Nos. 38, 95, 47-49, 71, 73, 131, 101, 102, 103, 128, 129, 130, 122-127.

[0179] The present application also relates to a polypeptide composition comprising the GIPR and GLP-1R co-agonistic polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof. In some embodiments, the polypeptide composition comprises two or more of the GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof.

[0180] The second aspect of the present application relates to a pharmaceutical composition comprising the GIPR and GLP-1R co-agonistic polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide, or ester thereof, and a pharmaceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the pharmaceutical composition comprises the polypeptide composition described above.

[0181] In some embodiments, the pharmaceutical composition is used for one or more of the following: (1) preventing, treating, and / or ameliorating a metabolic disease or disorder; (2) preventing, treating, and / or ameliorating diabetes; preferably, the diabetes is Type II diabetes; (3) preventing, treating, and / or ameliorating obesity or overweight; (4) reducing body weight; (5) reducing food intake; (6) improving glycemic control (e.g., improving glucose tolerance). In some embodiments, the metabolic disease or disorder is diabetes. In some embodiments, the diabetes is Type II diabetes. In some embodiments, the metabolic disease or disorder is Type II diabetes.

[0182] A third aspect of the present application relates to a method of preventing, treating, and / or ameliorating a metabolic disease or disorder, the method comprising administering to a patient or subject in need thereof 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.

[0183] In some embodiments, the metabolic disease or disorder is diabetes. In some embodiments, the diabetes is Type II diabetes. In some embodiments, the metabolic disease or disorder is Type II diabetes.

[0184] In some embodiments, the metabolic disease or disorder is obesity or overweight.

[0185] A fourth aspect of the present application relates to a method of reducing body weight, the method comprising administering to an individual in need thereof 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 an obese patient or an overweight individual. In some embodiments, the individual is a diabetic patient, e.g., a Type II diabetic patient. In some embodiments, the individual is an obese patient or an overweight individual, and is a diabetic patient, e.g., a Type II diabetic patient.

[0186] A fifth aspect of the present application relates to a method of reducing food intake, the method comprising administering to an individual in need thereof 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 an obese patient or an overweight individual. In some embodiments, the individual is a diabetic patient, e.g., a Type II diabetic patient. In some embodiments, the individual is an obese patient or an overweight individual, and is a diabetic patient, e.g., a Type II diabetic patient.

[0187] The sixth aspect of the present application relates to a method for improving glycemic control (e.g., improving glucose tolerance), comprising administering to a subject in need thereof an effective amount of the polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition as described in the second aspect. In some embodiments, the subject is an obese patient or an overweight individual. In some embodiments, the subject is a diabetic patient, e.g., a type II diabetes patient. In some embodiments, the subject is an obese patient or an overweight individual, and is a diabetic patient, e.g., a type II diabetes patient.

[0188] The seventh aspect of the present application relates to the use of the polypeptide as described in the first aspect, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition as described in the second aspect, in the manufacture of a medicament for one or more of the following uses: (1) preventing, treating and / or alleviating a metabolic disease or disorder; (2) preventing, treating and / or alleviating diabetes; preferably, the diabetes is type II diabetes; (3) preventing, treating and / or alleviating obesity or overweight; (4) reducing body weight; (5) reducing food intake; (6) improving glycemic control (e.g., improving glucose tolerance).

[0189] The following supplementary descriptions are provided to supplement the above first to seventh aspects. In the absence of conflicts, the following supplementary descriptions apply to each of the technical solutions described in the first to seventh aspects.

[0190] GIPR and GLP-1R co-agonistic polypeptides, or pharmaceutically acceptable salts, amides or esters thereof

[0191] The present application provides general structures and examples of various GIPR and GLP-1R co-agonistic polypeptides, or pharmaceutically acceptable salts, amides or esters thereof. One of the disclosed or expected benefits is that the activation of GLP-1R is enhanced while maintaining the activation activity of GIPR, which is beneficial to enhance the effect in reducing blood glucose level, and further enhance the effect in reducing body weight.

[0192] The GIPR and GLP-1R co-agonistic polypeptides provided by the present application can be synthesized by methods known in the art.

[0193] The GIPR and GLP-1R co-agonistic polypeptides of the present application can be in the form of a pharmaceutically acceptable salt, amide or ester.

[0194] The salt can be a basic salt, an acid salt, or a neutral salt. The basic salt produces hydroxyl ions in water, and the acid salt produces hydronium ions. The synthesis of the salt of the co-agonistic polypeptide of the present application can be achieved by introducing a corresponding counter ion into a polypeptide solution. For example, if the polypeptide molecule contains a protonatable amino acid residue, forming a negatively charged anion group, a cation (such as a metal cation Na + , K+ or a proton, H + ) to form a polypeptide cationic salt. Conversely, if the polypeptide molecule contains amino acid residues that can be deprotonated to form positively charged cationic groups, then the polypeptide can be reacted with the addition of anions such as CI - , SO4 2- , etc. to form a polypeptide anionic salt. These counterion groups can be located within the peptide portion and / or in the side chains of the co-agonist polypeptides of the present application.

[0195] Non-limiting examples of anionic groups of the co-agonist polypeptides of the present application include free carboxyl groups in the side chains (if any) and in the peptide portion. The peptide portion typically includes a free carboxylic acid at the C-terminus, and it can also include free carboxyl groups on internal acidic amino acid residues, such as Asp and Glu. Non-limiting examples of cationic groups of the peptide portion include a free amino group at the N-terminus (if any) and any free amino groups on internal basic amino acid residues, such as His, Arg, and Lys.

[0196] Esters of the co-agonist polypeptides of the present application can be formed, for example, by reaction of a free carboxylic acid group with an alcohol or phenol, resulting in substitution of at least one hydroxyl group with an alkoxy or aryloxy group. The formation of an ester can involve the free carboxyl group at the C-terminus of the peptide, and / or any free carboxyl groups of the side chains.

[0197] Amides of the co-agonist polypeptides of the present application can be formed, for example, by reaction of a free carboxylic acid group with an amine or substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid. The formation of an amide can involve the free carboxyl group at the C-terminus of the peptide, any free carboxyl groups of the side chains, the free amino group at the N-terminus of the peptide, and / or any free or substituted peptide amino groups in the peptide and / or side chains.

[0198] In some embodiments, the amino acid residues in the GIPR and GLP-1R co-agonist polypeptides, or a pharmaceutically acceptable salt, amide or ester thereof, include chemical modifications. In some embodiments, the chemical modifications include amidation modifications, fatty acid modifications, methylation, myristoylation, PEG modifications, fluorine element modifications, biotin modifications, fluorescent label modifications, cyclization, carboxylation, acetylation modifications, phosphorylation modifications, glycosylation modifications, and other modifications known in the art (see, e.g., U.S. 5856298; U.S. 2003-0120045, U.S. 2004-0063917, U.S. 2005-0220800, U.S. 2005-0107591, U.S. 2006-0035322, and U.S. 2006-0073563; and WO 200181405) or combinations thereof. Methods for the foregoing chemical modifications can be achieved using methods conventional in the art.

[0199] In some embodiments, the amino acid residues in the GIPR and GLP-1R co-agonist polypeptides described herein, or pharmaceutically acceptable salts, amides, or esters thereof, include amidation modifications.

[0200] In some embodiments, the amino acid residues in the GIPR and GLP-1R co- agonist polypeptides described herein, or pharmaceutically acceptable salts, amides, or esters thereof, include fatty acid modifications.

[0201] In some embodiments, the chemical modification comprises or is a fatty acid modification within the polypeptide sequence. In some embodiments, the fatty acid modification is attached to the epsilon-amino group of a Lys residue. In some embodiments, the fatty acid modification has the structure of -AEEA a -(y-Glu) b -CO-(CH2) n -COOH, where a, b, n are natural numbers, 12 < n < 20; a is 1, 2, or 3; and b is 1 or 2. Herein, “-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. Herein, “(y-Glu)2” refers to -y-Glu-y-Glu.

[0202] In some embodiments, the fatty acid modification has the structure of -AEEA- AEEA-y-Glu-CO-(CH2) 16 -COOH (i.e., -AEEA-AEEA-y-Glu-17-carboxyheptadecanoyl). In some embodiments, the lysine residue is chemically modified by conjugation of -AEEA-AEEA-y-Glu-CO-(CH2) 16 -COOH to the epsilon-amino group of the lysine side chain.

[0203] In some embodiments, the fatty acid modification has the structure of -AEEA- AEEA-y-Glu-CO-(CH2) 18 -COOH (i.e., -AEEA-AEEA-y-Glu-19-carboxynonadecanoyl). In some embodiments, the lysine residue is chemically modified by conjugation of -AEEA-AEEA-y-Glu-CO-(CH2) 18 -COOH to the epsilon-amino group of the lysine side chain.

[0204] Pharmaceutical compositions

[0205] The present application provides pharmaceutical compositions comprising the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0206] The pharmaceutical compositions of the present 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, in certain exemplary embodiments, does not interact with the action of the active agents of the pharmaceutical composition.

[0207] As used herein, the term "carrier" refers to an organic or inorganic component of natural or synthetic nature, in which the active ingredient is combined in order to facilitate, enhance or achieve application. According to the present application, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject.

[0208] As used herein, the term "excipient" is intended to include all substances that can be present in a pharmaceutical composition and are not active ingredients.

[0209] In some embodiments, the compositions can be formulated in a unit dosage form suitable for administration into the body of a patient, particularly can be formulated in a form for polypeptide drug administration, and can be administered by oral administration route or parenteral administration route such as intradermal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, intrapulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal route, but not limited to.

[0210] The administration dose and frequency of the pharmaceutical compositions of the present application are determined according to the type of drug as an active ingredient and various related factors such as the disease to be treated, the administration route, the age, sex and body weight of the patient, and the severity of the disease.

[0211] Metabolic disease or disorder

[0212] The present application provides a method for preventing, treating and / or alleviating a metabolic disease or disorder, the method comprising administering to a patient or subject in need thereof an effective amount of the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof or a pharmaceutical composition, polypeptide composition comprising the same. The GIPR and GLP-1R co-agonistic polypeptide of the present application activates both GIPR and GLP-1R, two enteroincretin receptors involved in blood glucose control, through a dual mechanism of action, which can achieve the effects of lowering blood glucose and reducing weight.

[0213] In some embodiments, the metabolic disease or disorder includes, but is not limited to, diabetes. In some embodiments, the diabetes is type II diabetes.

[0214] In some embodiments, the metabolic disease or disorder includes, but is not limited to, obesity or overweight.

[0215] Type II diabetes treatment / prevention and glycemic control

[0216] The present application provides a method of improving glycemic control, comprising administering to an individual in need thereof an effective amount of a preceding GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition.

[0217] In some embodiments, the term "glycemic control" means maintaining or reducing the individual's HbAlc level. "Hemoglobin Alc" or "HbAlc" means glycated hemoglobin, which is formed when hemoglobin is joined with glucose in the blood, and the level thereof. HbAlc level is a commonly used indicator of glycemic control in diabetic individuals, and a reduced HbAlc level generally indicates improved glycemic control.

[0218] In some embodiments, improving glycemic control includes reducing the patient's or subject's Hb1Ac level to below 7%, below 6.5%, or below 6.0%.

[0219] In some embodiments, improving glycemic control means improving the individual's glucose tolerance.

[0220] The present application provides a method of preventing, treating and / or ameliorating type II diabetes, comprising administering to a patient or subject in need thereof an effective amount of a preceding GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition, which is particularly effective for treating type II diabetes.

[0221] In some embodiments, the method of preventing, treating and / or ameliorating type II diabetes further comprises administering one or more additional drugs for treating type II diabetes.

[0222] In some embodiments, the subject diagnosed with type II diabetes in the present application is a type II diabetes patient determined by the usual clinical diagnostic criteria.

[0223] In some embodiments, the subject diagnosed with type II diabetes in the present application has a fasting blood glucose level higher than 125 mg / dL.

[0224] In some embodiments, the subject diagnosed with type II diabetes in the present application has a blood glucose level after glucose tolerance test higher than 199 mg / dL.

[0225] In some embodiments, the subject diagnosed with diabetes mellitus type II in the present application can have an HbAlC level greater than 6.4%.

[0226] In some embodiments, the diabetes is obesity-induced diabetes. Obesity-induced diabetes refers to diabetes that has symptoms of obesity that cause diabetes, particularly diabetes type II, or symptoms of obesity in a patient with diabetes type II. About 80% to 90% of patients with diabetes type II have symptoms of obesity, and these patients are characterized by insulin resistance. Proper exercise, diet, and medication therapy can prevent and alleviate obesity-induced diabetes. In the present application, in some cases, obesity-induced diabetes is caused by obesity.

[0227] Treatment / prevention of overweight and obesity and weight loss

[0228] The present application provides methods of preventing, treating, or alleviating overweight, the methods comprising administering to an individual in need thereof an effective amount of the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition.

[0229] In some embodiments, the overweight patient or subject has a BMI greater than 25 kg / m 2 and less than 30 kg / m 2 .

[0230] In some embodiments, the overweight patient or subject has a BMI greater than 25 kg / m 2 to less than 27 kg / m 2 and has at least one comorbidity.

[0231] In some embodiments, the overweight patient or subject is of Asian descent and has a BMI greater than 23 kg / m 2 to less than 25 kg / m 2 .

[0232] The present application provides methods of preventing, treating, or alleviating obesity, the methods comprising administering to an individual in need thereof an effective amount of the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition.

[0233] In some embodiments, the patient or subject with obesity has a BMI equal to or greater than 30 kg / m 2 .

[0234] In some embodiments, the patient or subject with obesity has a BMI greater than or equal to 27 kg / m 2 and has at least one comorbidity.

[0235] In some embodiments, the patient or subject suffering from obesity is of Asian descent, has a condition of BMI greater than or equal to 25 kg / m2, and has at least one co-morbidity. 2 of BMI greater than or equal to 25 kg / m2, and has at least one co-morbidity.

[0236] The present application provides a method of reducing body weight, comprising administering to an individual in need thereof an effective amount of the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition.

[0237] The present application provides a method of reducing food intake, comprising administering to an individual in need thereof an effective amount of the aforementioned GIPR and GLP-1R co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition. Prevention, treatment, alleviation of obesity, overweight or body weight loss can be achieved by reducing food intake.

[0238] Herein, the term "reducing food intake" refers to a spontaneous reduction in the amount of food intake under the condition of obtaining sufficient food. The GIPR and GLP-1R co-agonistic polypeptide of the present application, after binding to GLP-1R, can produce the effects of slowing down gastrointestinal peristalsis, inhibiting gastric juice secretion, delaying gastric emptying, and enhancing satiety, and can help reduce body weight by reducing food intake.

[0239] In one embodiment, the GIPR and GLP-1R co-agonistic polypeptide of the present application, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition is used for treating overweight, obesity and / or eating disorders with one or more of the following clinical outcomes as a treatment target: reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety.

[0240] In one embodiment, the GIPR and GLP-1R co-agonistic polypeptide of the present application, or a pharmaceutically acceptable salt, amide or ester thereof, or a pharmaceutical composition comprising the same, polypeptide composition can also be combined with one or more additional pharmacologically active substances. EXAMPLE

[0241] Example 1: General synthetic route of GIPR and GLP-1R co-agonistic polypeptide

[0242] The synthesis of GIPR and GLP-1R co-agonistic polypeptide was completed in a polypeptide synthesizer S890 using Fmoc / t-Bu strategy.

[0243] The lys residue and its side chain containing fatty acid side chain were synthesized using Fmoc-Lys(AEEA) a -(y-Glu) b -CO-(CH2)n -COOH (wherein a, b, n are determined by the side chain required) (purchased from Chengdu Pukang Biotechnology Co., Ltd.);

[0244] lys without fatty acid side chain used Fmoc-Lys(Boc)-OH as raw material (purchased from Chengdu Kolon Chemical Co., Ltd.);

[0245] Other amino acid residues used standard side chain protected amino acid residue raw material (purchased from Chengdu Kolon Chemical Co., Ltd.);

[0246] Resin with C-terminal amide: Rink Amide MBHA Resin, 1% DVB (divinylbenzene), 100-200 mesh, substitution degree 0.4-0.5 mmol / g (purchased from Xi'an Lanxiao New Material Science and Technology Co., Ltd.).

[0247] Resin with C-terminal carboxylic acid: 2-Chlorotrityl Chloride Resin (dichloride resin), 1% DVB, 100-200 mesh, substitution degree 0.4-0.5 mmol / g (purchased from Xi'an Lanxiao New Material Science and Technology Co., Ltd.).

[0248] The general preparation process is as follows:

[0249] (1) Connection of resin with C-terminal amide and C-terminal first amino acid: 20% piperidine / DMF was added to the reactor containing Rink Amide MBHA Resin, and reacted for 20 minutes. Then the reaction solution was filtered off, and the resin was washed with DMF for 6 times. C-terminal first amino acid mixture was added to the resin after deprotection, and the condensation reaction was carried out for 3h. The molar ratio of C-terminal first amino acid mixture to resin was resin:amino acid:diisopropyl carbodiimide:2-hydroxymethyl acetic acid ethyl ester=1:3:3:3. After the reaction was completed, filtration was carried out, and the resin was washed with DMF for 6 times.

[0250] (2) Connection of resin with C-terminal carboxylic acid and C-terminal first amino acid: C-terminal first amino acid mixture was added to the reactor containing 2-Chlorotrityl Chloride Resin, and reacted for 2h. The molar ratio of C-terminal first amino acid mixture to resin was resin:amino acid:diisopropyl ethylamine=1:1:6. After the reaction was completed, filtration was carried out, and the resin was washed with DMF for 2 times. Then 10% methanol / DMF was added, and reacted for 0.5h. After the reaction was completed, filtration was carried out, and the resin was washed with DMF for 6 times.

[0251] (3) Deprotection: 20% piperidine / DMF was added to the reactor containing resin, and reacted for 20 minutes. Then the reaction solution was filtered off, and the resin was washed with DMF for 6 times.

[0252] (4) Condensation: add the amino acid mixture to the resin after deprotection, and condense for 3 h. The molar ratio of the amino acid mixture to the resin is resin: amino acid: diisopropyl carbodiimide: 2-hydroximoyl cyanacetic acid ethyl ester = 1:3:3:3. After the reaction is completed, filter, and wash the resin with DMF 6 times.

[0253] According to the sequence of the polypeptide from the C-terminal to the N-terminal, repeat steps (3) and (4) in order to connect each amino acid or polypeptide fragment, and complete the synthesis of the whole polypeptide. Then wash the resin with methanol 3 times, and dry under vacuum to obtain the polypeptide resin with the C-terminal as amide or carboxylic acid.

[0254] (5) Cleavage: add the cleavage solution (trifluoroacetic acid: water: triisopropylsilyl, 95:2.5:2.5 v / v) 10 times the mass of the polypeptide resin to the obtained polypeptide resin, and react for 3 h, filter, add 10 times the volume of methyl tert-butyl ether to the filtrate to precipitate the polypeptide, and filter to obtain the polypeptide precipitate. After drying under vacuum, obtain the crude polypeptide.

[0255] (6) Purification: dissolve the crude polypeptide in 10% acetonitrile aqueous solution, adjust the pH to 8-9 by adding ammonia, and separate and purify by reverse phase high performance liquid chromatography (RP-HPLC). The chromatographic column used for RP-HPLC is 21x 250 mm, Kromasil C8, 10 μm; the mobile phase used is 100% acetonitrile and 0.1% TFA / water buffer system; 0-60 min, gradient is 20-50% acetonitrile, collect the components with purity >95%, and freeze-dry to obtain the freeze-dried powder.

[0256] (7) Analysis and detection: the purity of the polypeptide freeze-dried powder is 98.5% detected by analytical HPLC (Agilent, 1260 Infinity II), and the molecular weight is consistent with the theoretical calculation value determined by LC-MS (Thermo, OE240).

[0257] Example 2: Detection of GLP-1R / GIPR agonistic activity of the test molecule

[0258] Functional activity was determined in HEK-293 clonal cell lines expressing GIPR and luciferase or GLP-1R and luciferase, respectively. In a 100 μl assay volume, the cell lines expressing the respective receptors were treated with different concentrations of the respective polypeptide working solutions in DMEM medium (Gibco Cat# 12800) supplemented with 1% fetal bovine serum (FBS) (Gibco Cat# 10099141C). After 4 h incubation at 37 °C, cells were lysed using Glo lysis buffer (Promega, Cat# E2661) and Stead-Glo Luciferase buffer (Promega, Cat# E2520) substrate was added. Luciferase catalyzes the oxidation of the substrate in the presence of ATP, which results in bioluminescence. The luminescence was read on a microplate reader.

[0259] The luminescence values were fitted using GraphPad Prism 8 software (Ver 8.3.0) with the X-axis as the concentration of the test molecule (nM) and the Y-axis as the luminescence value. The data were fitted according to 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. Hill slope is the Hill coefficient, which describes the shape of the curve, i.e. the sensitivity of Y to changes in X). The EC 50 values obtained for the luciferase reporter cell lines treated with the different test molecules are shown in Tables 3 and 4.

[0260] Candidate test molecules with more optimal agonistic activity at the GLP-1R end and comparable or more optimal agonistic activity at the GIPR end compared to the corresponding tirzepatide control (backbone peptide or modified peptide) are considered to have a better prospect.

[0261] Table 3 GIPR / GLP-1R agonistic activity EC 50 Note: The lower the EC 50 value, the higher the activity; “*” indicates the control; “-NH2” indicates C-terminal amidation of the polypeptide; “-” in the results indicates no activity; Tir-0* and Tir-Aib* indicate tirzepatide backbone derivatives

[0262] Table 4 GIPR / GLP-1R agonistic activity EC 50 Note: The lower the EC 50The lower the value, the higher the activity; "*" indicates the control group; "-NH2" indicates the C-terminal amidation of the polypeptide;

[0263] Example 3: Effects of GIPR and GLP-1R co-agonistic polypeptides on body weight loss in diet-induced obese (DIO) mice

[0264] According to the test results of GIPR / GLP-1R agonistic activity of Example 2, DTM0001 to DTM0003 were selected for further in vivo test, and tirzepatide (SEQ ID No: 100) was used as a control molecule.

[0265] Diet induced obesity (DIO) mice (C57 / B16 mice purchased from Vivotecnia were induced to be DIO mice by high-fat diet) were used to evaluate the in vivo weight loss efficacy of DTM0001 to DTM0003.

[0266] In this study, 24-week-old male diet-induced obese (DIO) C57 / B16 mice were used. The mice were housed in a facility with a temperature controlled at 20-24°C, a 12-hour light / dark cycle, and the mice had free access to food and water. After 3 weeks of acclimation, the mice were randomly assigned to each test group (n=9 / group, 3 mice per cage) according to body weight, so that each group had a similar average starting body weight, and the average body weight of the mice in each group on the day of grouping was 46g.

[0267] Excipient control (PBS, 100mM phosphate buffer, pH 7.2-7.4), DTM0001 to DTM0003 (dose 30nmol / kg) or tirzepatide (30nmol / kg) were dissolved in excipient and administered to DIO mice with free feeding by SC (subcutaneous) injection, once every three days for 15 days. SC injection was performed on days 1, 4, 7, 10 and 13. The body weight of the mice was measured daily throughout the study. The absolute change rate of the body weight of the mice was calculated by the following formula:

[0268] Statistical comparisons between groups were made using One-Way ANOVA followed by Dunnett's multiple comparison test. The results showed that DTM0001 to DTM0003 all had significant weight loss efficacy, among which DTM0003 had a better body weight change rate than tirzepatide on the 7th day of administration (p<0.05), and the body weight reduction rates of DTM0003, DTM0001, DTM0002 and tirzepatide in DIO mice on the 15th day were -25.76±6.18, -23.73±2.09, -23.60±3.08 and -24.94±4.12, respectively.

[0269] Table 5 Percentage of body weight change in DIO mice (% change from initial body weight) Note: ***p<0.001 vs PBS, # p<0.05 vs tirzepatide. Results are expressed as mean ± standard deviation of the rate of body weight change for 9 mice per group.

[0270] Example 4: In vitro agonistic activity test of hGLP-1R and hGIPR

[0271] Based on the test results of Examples 2 and 3, the superior properties possessed by DTM0001 to DTM0003 were confirmed. In combination with the analysis of the structural commonalities 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 agonistic activity tests of hGLP-1R and hGIPR, with tirzepatide (SEQ ID No: 100) used as a control molecule.

[0272] Table 6

[0273] The in vitro agonistic activity of the above six test molecules on human GLP-1 receptor (hGLP-1R) was evaluated by the cAMP detection method in HEK293T cells expressing hGLP-1R; the in vitro agonistic activity of the above six test molecules on human GIP receptor was evaluated in HEK293 cells expressing hGIPR receptor.

[0274] Test method:

[0275] 1. Cell recovery and preparation:

[0276] The stable GLP-1R or GIPR cell strain was recovered after centrifugation after being added to Hank's balanced salt solution, the supernatant was discarded, and the cells were resuspended with experimental buffer (detection buffer 1 (5 mM hydroxyethylpiperazine ethanesulfonic acid, 0.5 mM 3-isobutyl-1-methylxanthine, 1*Hank's balanced salt solution, 0.1% casein) or 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)), and the viable cell density was adjusted to 1 x 10 5 / mL.

[0277] 2. Compound dilution and addition:

[0278] Test peptides and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, DTM0030 were diluted to working concentration (2x starting concentration) with Assay Buffer 1 (5 mM Hepes, 0.5 mM 3-isobutyl-1-methylxanthine, 1* Hank's Balanced Salt Solution, 0.1% Casein) and Assay Buffer 2 (5 mM Hepes, 0.5 mM 3-isobutyl-1-methylxanthine, 1* Hank's Balanced Salt Solution, 0.1% Casein, 1% Human Serum Albumin) respectively. The diluted compounds were pipetted into Echo Qualified 384-well plates in a volume of 40 μL, and 5 μL of the 2x test molecules and test peptides diluted in gradient were added to the reaction plates (Optiplate-384) using a Bravo V11 automated liquid handling platform. In GLP-1R cAMP assay, the starting concentrations of test peptides and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, DTM0030 in Assay Buffer 1 reaction system were 100, 1000, 20, 20, 1000, 20 and 20 nM respectively; and in Assay Buffer 2 reaction system were all 10000 nM. In GIPR cAMP assay, the starting concentrations of test peptides and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, DTM0030 in Assay Buffer 1 reaction system were 200, 10000, 500, 500, 10000, 200 and 200 nM respectively; and in Assay Buffer 2 reaction system were 10000, 30000, 10000, 10000, 30000, 10000 and 10000 nM respectively, all with 4-fold serial dilution, 10 points, double replicates. High control groups were 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) respectively, and low control group was Assay Buffer. cAMP standard (Revvity, 62AM4PEJ) was prepared with a starting concentration of 800 nM, serially diluted 4-fold for 10 points, and added to the reaction plates (Optiplate-384) in a volume of 10 μL per well.

[0279] 3. cAMP detection:

[0280] Add 10 μL of cell-containing assay buffer 1 or assay buffer 2 to the reaction plate and incubate at room temperature for 30 min. Add 10 μL of cAMP detection solution (Revvity, 62AM4PEJ) to the corresponding wells of the reaction plate and incubate at room temperature for 1 hour in the dark. Read the ratio of emission light at 665 nm to 615 nm in the EnVision microplate reader.

[0281] 4. Data analysis:

[0282] a) Calculate the actual cAMP level (nM) of each sample well by the cAMP standard curve

[0283] b) The formula for calculating the activity % of the sample well:

[0284] Activity % = (average cAMP level of sample well - average cAMP level of low control group) / (average cAMP level of high control group - average cAMP level of sample well) * 100%

[0285] c) Calculate the EC by fitting the "log (agonist) vs. response - Variable slope" model in GraphPad Prism 5.0 50 .

[0286] The EC of the test molecules for the activation activity of human GLP-1R & GIPR was determined in assay buffer 1 and assay buffer 2 as shown in Table 7. 50

[0287] Table 7 EC for the activation activity of GLP-1R and GIPR 50

[0288] Results analysis:

[0289] The agonistic activity of the test molecules and the control exendin-4 on human GLP-1 and GIP receptors is expressed as EC 50 values, and the EC 50 ​The smaller the value, the stronger the activity of the activated receptor. In the detection buffer 1 test system, the GLP-1R agonistic activity of DTM0003 and DTM0026 was slightly lower than that of tirzepatide; the agonistic activity of DTM0001, DTM0002, DTM0030 and DTM0028 was stronger than that of tirzepatide. In the detection buffer 1 test system, the GIPR agonistic activity of the six test molecules was lower than that of tirzepatide. In the detection buffer 2 test system, the in vitro agonistic activity of GIPR and GLP-1R of all test molecules and the control tirzepatide was significantly weakened, and compared with tirzepatide, DTM0003, DTM0001, DTM0002, DTM0026 and DTM0028 were more strongly weakened in in vitro activity in the detection buffer 2. 50 / Detection buffer 1 EC 50 The higher, the easier the compound binds to HSA (human serum albumin), and it is expected to achieve the prolongation of the drug T 1 / 2 (half-life) in the human body.

[0290] Example 5: Pharmacokinetics in Cynomolgus monkeys

[0291] In this example, Cynomolgus monkeys were used as a model to test the in vivo pharmacokinetic properties of the six test molecules in Example 3, and tirzepatide (SEQ ID No: 100) was used as a control molecule.

[0292] Tirzepatide and DTM0003, DTM0001, DTM0002, DTM0026, DTM0028 and DTM0030 were subcutaneously injected at a dose of 0.144 mg / kg in a volume of 1 mL / kg in PBS phosphate buffer (pH 7.4). Blood was taken 0, 1, 2, 6, 24, 48, 72, 120, 168, 240 and 336 hours after administration 0.5 mL from the limbs, placed in EDTA-K2 test tubes, gently shaken to mix, placed in an ice water bath, and centrifuged (4°C, 2000g, 10 min) within 30 min, the plasma was separated and frozen in a -70°C refrigerator for testing.

[0293] LC-MS / MS detection conditions:

[0294] Sample pretreatment method: protein precipitation method

[0295] Chromatographic column: ACQUITY UPLC BEH C18 Column (1.7 μm*2.1*50 mm)

[0296] Mobile phase A: ultrapure water containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid; needle washing liquid: methanol / acetonitrile / isopropanol / ultrapure water = (1:1:1:1, v / v / v / v)

[0297] Method of determination: The working solution series concentrations were obtained by using acetonitrile / 5 mM ammonium acetate aqueous solution (7:3, v / v) as the dilution of the analyte stock solution. 5 μL working solution (50, 100, 200, 500, 1000, 2000, 5000, 10000 ng / mL) was added to 45 μL blank cynomolgus monkey plasma to achieve a total volume of 50 μL of 5-1000 ng / mL (5, 10, 20, 50, 100, 200, 500, 1000 ng / mL) calibration standards. Five quality control (QC) samples (10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 800 ng / ml) were prepared in the same way as the calibration standards on the day of analysis. 50 μL of standard, 50 μL of QC sample 50 μL of sample were added to 200 μL of acetonitrile containing internal standard mixture to precipitate the protein. Then the samples were 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 then 20 μL of the diluted supernatant was injected into the LC-MS / MS system (SCIEX 4500) for quantitative analysis.

[0298] Results:

[0299] As shown in Table 8, the average half-life (T1 / 2) of test molecules DTM0003, DTM0001, DTM0002, DTM0026, DTM0028, DTM0030 and tirzepatide administered by subcutaneous injection (0.144 mg / kg) in cynomolgus monkeys were 54.4, 122, 94.1, 50.1, 46.0, 69.1 and 65.9 hours, respectively, and the average plasma exposure (AUC0-336h) were 98579, 369488, 220022, 109599, 129438, 127653, 113088 h*ng / ml. The results showed that the pharmacokinetic properties of DTM0003, DTM0026, DTM0028 and DTM0030 were similar to tirzepatide, and DTM0001 and DTM0002 were superior to tirzepatide.

[0300] Example 6: Effect on weight loss in diet-induced obese (DIO) monkeys

[0301] The in vivo weight loss efficacy of DTM0001, DTM0002, DTM0026, DTM0028 was further evaluated in diet-induced obese (DIO) monkey (Shanghai Viva) model, and tirzepatide (SEQ ID No: 100) was used as control molecule.

[0302] In study one, 10-21 year old male obese (DIO) cynomolgus monkeys with body weight of 10-16 kg were used. Animals were housed individually in a temperature-controlled (16-26 °C) facility with a 12-hour light / dark cycle and free access to food and water. Nine DIO monkeys were randomly assigned to test groups (n=3 / group) according to body weight, so that each group had similar starting mean body weight.

[0303] DTM0001, DTM0002 (dose range 60 nmol / kg) and tirzepatide (60 nmol / kg) were dissolved in excipient (100 mM phosphate buffer, pH 7.2-7.4) and administered by single subcutaneous injection. The starting date of administration was recorded as day 0, and the body weight of DIO monkeys in each group was detected on day 7, 14. The absolute change rate of DIO monkey body weight was calculated by the following formula:

[0304] Table 9 Percentage of body weight change in DIO monkeys (% change from starting body weight) Note: *p<0.05, **p<0.01, ***p<0.001 vs. tirzepatide group (One-Way ANOVA, Dunnett's), results are expressed as mean ± standard deviation of body weight change rate of 3 DIO monkeys in each group.

[0305] In study two, 10-21 year old male obese (DIO) cynomolgus monkeys with body weight of 10-16 kg were used. Animals were housed individually in a temperature-controlled (16-26 °C) facility with a 12-hour light / dark cycle and free access to food and water. Nine DIO monkeys were randomly assigned to test groups (n=3 / group) according to body weight, so that each group had similar starting mean body weight.

[0306] DTM0026, DTM0028 (dose range 30 nmol / kg) or tirzepatide (30 nmol / kg) were dissolved in excipient (100 mM phosphate buffer, pH 7.2-7.4). The starting date of first administration was recorded as day 0, and the second and third administrations were performed on day 7, 21. The body weight of DIO monkeys was detected twice a week, and the body weight change of DIO monkeys in each group was observed. The absolute change rate of DIO monkey body weight was calculated by the following formula:

[0307] Table 10 Percentage of body weight change in DIO monkeys (% change from starting body weight)

[0308] As shown in the results of Table 9 and Table 10: compared with tirzepatide, DTM0001, DTM0002 reduced the body weight of DIO monkeys more obviously at 60 nmol / kg single subcutaneous administration, the body weight reduction rates of DTM0001, DTM0002 and tirzepatide in DIO monkeys on day 14 were -9.17% ± 1.57% and -8.19% ± 3.03% and -1.69% ± 1.80% respectively, and there was a significant difference compared with tirzepatide. After multiple subcutaneous injection administration at 30 nmol / kg, the body weight reduction rates of DTM0026, DTM0028 and tirzepatide groups in DIO monkeys on day 28 were -7.65% ± 4.49%, -8.96% ± 3.75%, -4.18% ± 6.96% respectively. DTM0028 reduced the body weight of monkeys more effectively than tirzepatide.

[0309] Example 7: Effect of single subcutaneous administration on glucose tolerance of C57 mice

[0310] This example evaluates the effect of single subcutaneous administration of DTM0026 and DTM0028 on glucose tolerance of C57BL / 6JNifdc mice (Vital River) and uses tirzepatide (SEQ ID No: 100) as a control molecule.

[0311] In this study, 6-8 week old male C57BL / 6JNifdc mice weighing 23-26 g were used, and the animals were housed in a temperature-controlled (20-24°C) facility with a 12-hour light / dark cycle and free access to food and water. After one week of acclimatization to the facility, the mice were randomly assigned to the test groups (n = 6 / group) according to body weight. Therefore, each group had a similar average starting weight, and the average weight of the mice in each group on the day of grouping was 24.6-24.7 g.

[0312] 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:

[0313] 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.

[0314] Table 11 Blood glucose levels at different time points in C57 mice 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.

[0315] Table 12 AUC of C57 mice in each group (0-120min) blood sugar level Note: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 relative to the control group (One-Way ANOVA, Dunnett's), results are expressed as AUC of 6 mice per group. (0-120min) The mean ± standard deviation.

[0316] 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-lR co-agonist polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, having the general sequence of SEQ ID No: 1: 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 Ala GlyGlyProSerX 33 GlyAlaProProProSerR2(SEQ ID No: 1), wherein, The X1, X2, X4, X5, X6, X7, X8, X9, X 10 X 11 X 12 X 13 X 15 X 16 X 17 X 19 X 18 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 and X 33 Independently constitutes any natural amino acid residue, non-natural amino acid residue, or is absent; and / or R1is any peptide segment, or is absent; and / or R2is -NH2, any peptide segment, or is absent.

2. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to claim 1, wherein X1is His, Phe, or Tyr; and / or X2is Aib or Ala; and / or X4is Gly or Ala; and / or X5is Thr, Asn or Ser; and / or X6is Phe or His; and / or X7is Thr or Val; and / or X8is Thr or Ser; and / or X9is Asp or Glu; and / or said X 10 is Tyr or Phe; and / or said X 11 is Thr or Ser; and / or said X 12 is He or Asn; and / or said X 13 is Tyr, Gin, Ala or Gly; and / or said X 15 is Asp, Glu or Gin; and / or said X 16 is Glu or Lys; and / or said X 17 is Gin, Leu or He; and / or said X 18 is Ala, His or Gly; and / or said X 19 is Gin or Asn; and / or said X 20 is Arg, Gin or Lys; and / or said X 21 is Asp, Glu or Ala; and / or said X 22 is Phe or Tyr; and / or said X 23 is He or Val; and / or said X 24 is Gin, Lys or Asn; and / or said X 25 is Trp or Tyr; and / or said X 26 is Leu or Ala; and / or said X 27 is Val or lie; and / or Said X 33 is Ser or Lys.

3. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to claim 1 or 2, wherein X5is Thr; and / or said X 10 is Tyr; and / or said X 22 is Phe; and / or The X 26 is Leu.

4. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-3, wherein X4is Gly; and / or X6is Phe; and / or X9is Asp; and / or The X 13 is Tyr.

5. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-4, wherein said X is 12 is He; and / or The X 15 For Glu; and / or said X 16 is Glu or Lys; and / or said X 18 is Ala; and / or said X 20 is Arg or Lys; and / or The X 24 is Gin or Lys.

6. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-5, wherein X1is Tyr; and / or X2is Aib; and / or X7is Thr; and / or X8is Ser; and / or The X 11 For Ser; and / or said X 17 is Gin; and / or said X 21 is Glu or Asp; and / or said X is 23 is He; and / or said X is 25 Trp; and / or said X 27 is Val; and / or Said X 33 is Ser or Lys.

7. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-6, wherein the polypeptide has a general sequence as set forth in any one of SEQ ID Nos: 2-21; preferably, the polypeptide has a sequence as set forth in, or consisting of, any one of SEQ ID Nos. 22-41, 43-73, 76, 78-80, 82-86, 88-98, 101-131.

8. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-7, wherein the N-terminal, C-terminal and / or amino acid residues within the polypeptide sequence of the polypeptide further comprise a chemical modification; preferably, the chemical modification comprises one or more of amidation modification, fatty acid modification, methylation, myristoylation, PEG modification, fluorine element modification, biotin modification, fluorescent label modification, cyclization, carboxylation, acetylation modification, phosphorylation modification, glycosylation modification; further preferably, the chemical modification comprises amidation modification and / or fatty acid modification.

9. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to claim 8, wherein the chemical modification occurs at the C-terminal; preferably, the chemical modification of the C-terminal comprises amidation modification; further preferably, the amidation modification is primary amide modification.

10. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to claim 8 or 9, wherein the chemical modification occurs at an amino acid residue within the polypeptide sequence; preferably, the chemical modification comprises a fatty acid modification; further preferably, one or more Lys residues in the polypeptide comprise a fatty acid modification.

11. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, of any one of claims 1-10, wherein the X 16 , X 20 , X 24 and / or X 33 is Lys; preferably, the Lys further comprises a fatty acid modification.

12. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, of any one of claims 8-11, wherein the fatty acid modification has the structure of -(AEEA) a -(y-Glu) b -CO-(CH2) n -COOH, wherein, a, b, n are natural numbers and 12 < n < 20; preferably, n is 16 or 18, a is 2 and b is 1.

13. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 8-12, wherein the fatty acid modification is attached to the epsilon-amino group of one or more Lys residues in the polypeptide.

14. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 1-13, wherein R1and / or R2in the general sequence is absent.

15. The polypeptide according to claim 1 or 2, or a pharmaceutically acceptable salt, amide, or ester thereof, wherein the polypeptide has the general formula sequence shown in SEQ ID No: 132: X1AlaGluGlyThrPheThrSerAspTyrSerIleX 13 LeuX 15 X 16 X 17 AlaGlnX 20 X 21 PheX 23 X 24 TrpLeuX 27 AlaGlyGlyProSerSerGlyAlaProProProSerR2 (SEQ ID No: 132), where, X1is 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 He or Gin; X 20 selected from Lys or Arg; X 21 selected from Ala, Glu or Asp; X 23 selected from Val or He; X 24 selected from Gin or Asn; X 27 is selected from Val or He.

16. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to claim 15, wherein the polypeptide has a sequence as set forth in any one of SEQ ID Nos. 22, 24, 38-41, 76, 79, 95-98, or the sequence of the polypeptide consists of a sequence as set forth in any one of SEQ ID Nos. 22, 24, 38-41, 76, 79, 95-98.

17. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of claim 1 or 2, wherein the polypeptide has the general sequence set forth in SEQ ID No: 133: X1X2GluGlyThrPheThrSerAspTyrSerIleTyrLeuX 15 X 16 GlnAlaGlnX 20 X 21 PheX 23 X 24 TrpLeuX 27 AlaGlyGlyProSerX 33 GlyAlaProProProSerR2(SEQ ID No: 133), wherein, X1is Tyr, Phe or His; X2is Ala or Aib; X 15 is Glu or Asp; X 16 is Glu or Lys; X 20 is Arg or Lys; X 21 is Glu, Ala or Asp; X 23 is He or Val; X 24 is Gin, Lys or Asn; X 27 is Val or He; X 33 is Ser or Lys.

18. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of claim 17, wherein X 16 , X 20 , X 24 , and / or X 33 is Lys.

19. The polypeptide of claim 18, or a pharmaceutically acceptable salt, amide, or ester thereof, wherein the Lys comprises a fatty acid modification -(AEEA) a -(y-Glu) b -CO-(CH2) n -COOH, wherein, a, b, n are natural numbers and 12 < n < 20; preferably, n is 16 or 18, a is 2 and b is 1.

20. The polypeptide, or a pharmaceutically acceptable salt, amide or ester thereof, according to any one of claims 17-19, wherein the polypeptide has a sequence as set forth in any one of SEQ ID Nos. 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 a sequence as set forth in any one of SEQ ID Nos. 47-51, 61, 62, 65, 66, 71, 73, 131, 101-105, 116, 117, 120, 121, 128, 129, 130, 122-127.

21. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of claim 1 or 2, wherein the polypeptide has the general sequence set forth in SEQ ID No: 134: Tyr X2 Glu Gly Thr Phe Thr Ser Asp Tyr Ser lie Tyr Leu Glu X 16 Gln Ala Gln Arg X 21 Phe lie X 24 Trp Leu Val Ala Gly Gly Pro Ser X 33 Gly Ala Pro Pro Pro Ser R2 (SEQ ID No: 134), wherein, X2is Ala or Aib; X 16 is Glu or Lys; X 21 is Glu or Asp; X 24 is Gin, Lys or Asn; X 33 is Ser or Lys.

22. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of claim 21, wherein X 16 , X 24 , and / or X 33 is Lys.

23. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of claim 22, wherein the Lys comprises a fatty acid modification -(AEEA)a-(y-Glu)b-CO-(CH2)n-COOH, wherein, a, b, n are natural numbers and 12 < n < 20; preferably, n is 16 or 18, a is 2 and b is 1.

24. The polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of any one of claims 21-23, wherein the polypeptide has a sequence set forth in any one of SEQ ID Nos. 38, 95, 47-49, 71, 73, 131, 101, 102, 103, 128, 129, 130, 122-127, or the sequence of the polypeptide consists of a sequence set forth in any one of SEQ ID Nos. 38, 95, 47-49, 71, 73, 131, 101, 102, 103, 128, 129, 130, 122-127.

25. A pharmaceutical composition comprising the GIPR and GLP-lR co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of any one of claims 1-24, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

26. The pharmaceutical composition of claim 25, for one or more of the following uses: (1) preventing, treating, and / or ameliorating a metabolic disease or disorder; (2) preventing, treating, and / or ameliorating diabetes; preferably, the diabetes is Type II diabetes; (3) preventing, treating, and / or ameliorating obesity or excess weight; (4) reducing body weight; (5) reducing food intake; (6) improving glycemic control (e.g., improving glucose tolerance).

27. The pharmaceutical composition of claim 26, wherein the metabolic disease or disorder is diabetes; preferably, the diabetes is Type II diabetes.

28. The pharmaceutical composition of claim 26, wherein the metabolic disease or disorder is obesity or excess weight.

29. A method of preventing, treating, and / or ameliorating a metabolic disease or disorder, wherein the method comprises administering to a patient or subject in need thereof an effective amount of the GIPR and GLP-lR co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of any one of claims 1-24 or the composition of claim 25.

30. The method of claim 29, wherein the metabolic disease or disorder is diabetes; preferably, the diabetes is Type II diabetes.

31. The method of claim 29, wherein the metabolic disease or disorder is obesity or excess weight.

32. A method of reducing body weight, wherein the method comprises administering to an individual in need thereof an effective amount of the GIPR and GLP-lR co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of any one of claims 1-24 or the pharmaceutical composition of claim 25.

33. A method of reducing food intake, wherein the method comprises administering to an individual in need thereof an effective amount of the GIPR and GLP-lR co-agonistic polypeptide, or a pharmaceutically acceptable salt, amide, or ester thereof, of any one of claims 1-24 or the pharmaceutical composition of claim 25.

34. A method of improving glycemic control (e.g., improving glucose tolerance), wherein the method comprises administering to an individual in need thereof an effective amount of the GIPR and GLP-1R co-agonist polypeptide of any one of claims 1-24, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition of claim 25.

35. The method of any one of claims 32-34, wherein the individual is an obese individual or an overweight individual.

36. The method of any one of claims 32-34, wherein the individual is a diabetic, e.g., a type II diabetic.

37. The method of any one of claims 32-34, wherein the individual is an obese individual or an overweight individual and is a diabetic, e.g., a type II diabetic.

38. Use of the GIPR and GLP-1R co-agonist polypeptide of any one of claims 1-24, or a pharmaceutically acceptable salt, amide or ester thereof, or the pharmaceutical composition of claim 25, in the manufacture of a medicament for one or more of the following uses: (1) preventing, treating and / or ameliorating a metabolic disease or disorder; (2) preventing, treating and / or ameliorating diabetes; preferably, the diabetes is type II diabetes; (3) preventing, treating and / or ameliorating obesity or being overweight; (4) reducing body weight; (5) reducing food intake; (6) improving glycemic control (e.g., improving glucose tolerance).

39. The use of claim 38, wherein the metabolic disease or disorder is diabetes; preferably, the diabetes is type II diabetes.

40. The use of claim 38, wherein the metabolic disease or disorder is obesity or being overweight.

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