Peptide Composition and Method of Use Thereof

Abstract

Provided are triple agonist peptides that are active against each of the GLP-1, glucagon, and GIP receptors. Also described are triple agonist analogs having one or more biotin moieties and / or fatty acid moieties conjugated thereto for improved bioavailability and pharmacokinetics. Compositions and formulations of these triple agonist peptides are particularly suitable for treating, reducing, and / or preventing one or more metabolic diseases, such as obesity, type 2 diabetes, or non-alcoholic fatty liver disease. Also described are compositions and methods of use thereof for treating, reducing, and / or preventing one or more neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Patent Application No. 63 / 346,605, filed on May 27, 2022, which is hereby incorporated by reference in its entirety.

[0002] Reference to a Sequence Listing The sequence listing submitted as a text file named "DDP_107_PCT_ST26.xml", created on March 25, 2023, and having a size of 287,384 bytes, is hereby incorporated by reference in accordance with 37 C.F.R. § 1.834(c)(1).

[0003] Field of the Invention The present invention generally relates to peptides and their analogs having activity against all of the glucagon, GLP - 1, and GIP receptors, and to the fields of their use.

Background Art

[0004] Background of the Invention Recently, due to economic development and medical progress, the elderly population has been increasing rapidly. Aging increases the risk of chronic diseases such as dementia, heart disease, type 2 diabetes, arthritis, and cancer. The mortality rates of heart disease and cerebrovascular disease, which are complications associated with obesity, are ranked first and second. Obesity has been suggested as a cause of various adult diseases such as type 2 diabetes and non - alcoholic fatty liver disease.

[0005] Obesity refers to a state in which fat is accumulated in an amount higher than normal. The most accurate method for evaluating obesity is to measure the amount of body fat. However, accurate measurement of body fat is costly, and therefore, it is evaluated using indirect methods. The most commonly used indirect methods are to measure the body mass index (BMI) and waist circumference. The World Health Organization (WHO) has published a classification based on data associating BMI with the risk of death, which is as follows: normal weight: 18.5 - 24.9 kg / m 2 , overweight: 25 - 29.9 kg / m 2 , and obesity: 30 kg / m 2 or higher.

[0006] The causes of obesity are known to be an energy imbalance resulting from excessive calorie intake and relatively decreased activity, and the resulting increase in body fat. However, since various risk factors, such as eating habits, lifestyle, age, race, genetic factors, etc., are involved in obesity, it is difficult to specify only one factor. Obese patients are mainly advised to control their weight through a healthier diet and physical activity, but if these methods are not effective, the patient may be treated with drug therapy or surgery. These often provide limited effectiveness.

[0007] Diabetes is classified into insulin-dependent diabetes (Type I diabetes), insulin-independent diabetes (Type II diabetes), and malnutrition-related true diabetes (MRDM). Type II diabetes, which accounts for more than 90% of diabetic patients, is a metabolic disease characterized by hyperglycemia and is reported to be caused by a decrease in insulin secretion from pancreatic beta cells or an increase in insulin resistance in peripheral tissues due to genetic, metabolic, and environmental factors. In this regard, as body fat increases, insulin sensitivity decreases. The accumulation of abdominal fat is particularly known to be related to glucose intolerance. Also, as obesity becomes more severe, insulin resistance also increases, and it is known that insulin resistance is closely correlated with obesity in patients suffering from Type II diabetes.

[0008] Non-alcoholic fatty liver disease (NAFLD) refers to a series of diseases including simple steatosis with excessive accumulation of fat in liver cells unrelated to alcohol consumption, hepatocyte injury (hepatocellular ballooning), inflammation, fibrosis, and in more advanced cases, cirrhosis, including non-alcoholic steatohepatitis (NASH). The prevalence of non-alcoholic fatty liver disease has increased rapidly worldwide along with the increasing prevalence of obesity. The prevalence of diabetes varies from country to country, but it accounts for about 20 - 30% of the total population in Western countries, and its incidence has reached about 16% in South Korea.

[0009] GLP-1 is a hormone secreted by the small intestine that is stimulated by food intake. GLP-1 promotes insulin secretion in the pancreas and inhibits glucagon secretion in a glucose-dependent manner, thus helping to lower blood glucose levels. In addition, GLP-1 acts as a satiety factor, exhibits digestive effects in the gastrointestinal tract, and reduces food intake by delaying the time for emptying the food digested in the gastrointestinal tract. Administration of GLP-1 to rats has been reported to have the effect of inhibiting food intake and reducing body weight, and these effects have been confirmed to occur equally in both normal and obese states, thus indicating the potential of GLP-1 as a drug for treating obesity.

[0010] GIP, one of the gastrointestinal hormones secreted by the stimulation of food intake, is a 42-amino acid hormone secreted by intestinal K cells like GLP-1. GIP promotes insulin secretion in the pancreas in a glucose-dependent manner and plays a role in helping to lower blood glucose levels, and it has been reported to show the effect of increasing the activation, anti-inflammation, etc. of GLP-1.

[0011] Glucagon is produced in the pancreas when blood glucose levels drop due to reasons such as drug therapy, disease, or deficiencies of hormones or enzymes. Glucagon sends a signal for glycogenolysis in the liver, induces the release of glucose, and increases blood glucose levels to normal levels. In addition to its effect of increasing blood glucose levels, glucagon suppresses appetite in animals and humans, activates hormone-sensitive lipase in adipocytes, promotes lipolysis and energy consumption, and thereby exhibits an anti-obesity effect.

[0012] Based on the effects of GLP-1 in controlling blood glucose levels and reducing body weight, GLP-1 has been developed as a therapeutic agent for treating diabetes and obesity. Exendin-4, which is prepared from Gila monster venom and has approximately 50% amino acid homology with GLP-1, is under development as a therapeutic agent for treating the same types of diseases. However, therapeutic agents containing GLP-1 and exendin-4 have been reported to exhibit side effects such as vomiting and nausea (Syed Y Y., Drugs, 2015 July; 75 (10): 1141-52).

[0013] For maximizing weight loss and as an alternative to GLP-1-based therapeutic materials, research has focused on dual agonists that bind to both GLP-1 receptors and glucagon receptors. These have been shown to be more effective in weight loss due to the activation of glucagon receptors compared to when GLP-1 is used alone (Jonathan W et al., Nat Chem Bio., 2009 October (5); 749-757). In research on triple agonists that simultaneously bind to GLP-1, GIP, and glucagon receptors, attempts have been made to increase resistance to dipeptidyl peptidase-IV (DPP-IV), which degrades gastrointestinal hormones and removes their activity, by substituting the amino acid sequence, and subsequently increase the half-life of the triple agonist by adding an acyl group to its specific region (Finan B et al., Nat Med., 2015 21 (1): 27-36). However, their effects of activating three different types of receptors were not significant, and none of the triple agonists showed various activity ratios against them.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0015] Therefore, it is an object of the present invention to provide compositions and methods for treating or preventing one or more symptoms of metabolic diseases including type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and / or obesity.

[0016] It is also an object of the present invention to provide compositions and methods suitable for activating GLP-1, GIP, and glucagon receptors to control blood glucose levels and reduce body weight without causing side effects.

[0017] It is a further object of the present invention to provide compositions and methods that provide effective glucose control with weight loss benefits and a favorable side effect profile.

[0018] It is a further object of the present invention to provide a therapeutic agent having an extended period of action.

Means for Solving the Problems

[0019] Summary of the Invention There is provided a triple agonist peptide that is active against each of the GLP-1, glucagon, and GIP receptors. Generally, the triple agonist peptide has the following polypeptide formula I: X 1 X 2 X 3 GTFTSDX 10 SX 12 X 13 LDX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 G X 30 X 31 SX 33 X 34 X 35 PP X 38 X 39 X 40 (SEQ ID NO: 131) (wherein X 1 is H or Y; X 2 is A or 2-aminoisobutyric acid (Aib); X 3 is Q or E; X 12 is R, W or K; X 13 is L or Y; X 19 is Q, A or T; X 21 is either D or L; X 22 is either F or R; X 23 is either V, G or D; X 25 is either W, Y or A; X 26 is either L or D; X 27 is either I, L, M, G or P; X 30 is either G or P; X 31 is either P or S; X 33 is either S or G; X 34 is either G or A; X 35 is either A or P; X 38 is either P or S; X 39 is absent, S, or C; X 40 is absent, C, or K; With optional amide modification at the C-terminus, X 10 , X 16 , X 17 , X 18 , X 20 , X 24 , and X 28 are each independently any one of the 20 amino acids excluding cysteine.) has. In some embodiments, X 17 , X 18 , X 20 are non-natural amino acids. In some embodiments, X 17 , X 18 , X 20 are non-natural amino acids, then X 17 , X 18 , X 20 are each independently one of the following: methoxinine, 2-aminoisobutyric acid, and alpha-methylarginine.

[0020] In other embodiments, X 10 is Y, W, K, F, H, S, L, A, E, M, Q, or D; X 16 is Y, Q, G, K, S, R, F, P, or A; X 17 is M, Y, Q, K, S, W, P, D, A, F, or methoxynine; X 18 is A, I, M, W, T, D, Y, or methoxynine; X 20 is R, Q, H, G, A, P, N, K, Aib, or alpha-methylarginine; X 24 is Q, D, K, L, N, W, or M; X 28 is N, E, G, D, H, or Q.

[0021] In a preferred embodiment, the triple agonist peptide has one of the amino acid sequences of SEQ ID NOs: 1 to 130.

[0022] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 134: YXQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 134) (wherein X is any one of the 20 amino acids).

[0023] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 134: YAibQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 135) and does not have the amino acid sequence of

[0024] Also described are triple agonist analogs having one or more biotin moieties and / or fatty acid moieties conjugated thereto for improved bioavailability and pharmacokinetics. In some embodiments, the one or more biotin moieties and / or one or more fatty acids, or derivatives thereof, are conjugated to an amino acid sequence of any one of SEQ ID NOs: 1-130 via one or more amino acid residues selected from the group consisting of cysteine and lysine. In other embodiments, one or more amino acid residues of cysteine and lysine are introduced into an amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to enable conjugation to one or more biotin moieties and / or one or more fatty acids, or derivatives thereof. In preferred embodiments, one or more amino acid residues of lysine at position 10, lysine at position 12, lysine at position 17, and one or more of the one or more C-terminal cysteine residues are introduced into an amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to enable conjugation to one or more biotin moieties and / or one or more fatty acids, or derivatives thereof. Exemplary biotin moieties suitable for conjugation are N-biotinoyl-N'-(6-maleimidocaproyl) hydrazide, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-CONH 2 , 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH 2 , propionate-N-hydroxysuccinimide ester-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH 2 and 3-maleimidopropionate-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH 2 .

[0025] Exemplary fatty acids suitable for conjugation are C16-C22 fatty acids, optionally via one or more hydrophilic spacers such as γGlu or 8-amino-3,6-dioxaoctanoic acid. In some embodiments, fatty acids and their derivatives suitable for conjugation are C16-NHS, C16-MAL, C18-NHS, C18-MAL, C16-γGlu-NHS, C16-γGlu-MAL, C18-γGlu-NHS, C18-γGlu-MAL, C18-γGlu-OEG-NHS, C18-γGlu-OEG-MAL, C18-γGlu-2OEG-NHS, C18-γGlu-2OEG-MAL, C20-γGlu-2OEG-NHS, C20-γGlu-2OEG-MAL, C18-γGlu-2OEG-TFP, C18-γGlu-2OEG-NPC, and C20-γGlu-2OEG-NPC. Pharmaceutical formulations, triple agonist peptides or analogs thereof, and methods of using the same are also described.

[0026] A method for treating one or more diseases selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease in a subject in need thereof is provided. The method comprises administering a pharmaceutical formulation of an effective amount of a triple agonist peptide or an analog thereof to treat or alleviate one or more symptoms of the one or more diseases. In a preferred embodiment, the pharmaceutical formulation is administered in an amount effective to induce weight loss, reduce body fat, reduce food intake, improve glucose homeostasis, or a combination thereof in a normal or obese patient. In some embodiments, the subject has non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer. In the case of NAFLD, the pharmaceutical formulation is administered in an amount effective to inhibit or reduce the serum level of one or more of alanine aminotransferase, aspartic acid aminotransferase, triglyceride, gamma-glutamyltransferase, total cholesterol, low density lipoprotein, fasting blood glucose, or a combination thereof. In a preferred embodiment, the pharmaceutical formulation is administered in an amount effective to reduce one or more of steatosis, inflammation, hypertrophy, fibrosis, cirrhosis, or a combination thereof in a subject having NAFLD.

[0027] Typically, pharmaceutical formulations are administered via enteral and parenteral routes, such as oral or subcutaneous administration. In some embodiments, the pharmaceutical formulations are administered in the form of pills, capsules, tablets, solutions, and suspensions. In some embodiments, the pharmaceutical formulations are administered at intervals of once a month, once every two weeks, once a week, once every three days, once every two days, once a day, or twice a day. In other embodiments, the pharmaceutical formulations are administered to a subject once a week for a period of up to six months, or including the periods at both ends, for a period of 1 to 10 days, 1 to 10 weeks, 1 to 10 months, or 1 to 10 years. In some embodiments, the pharmaceutical formulations are administered to a human subject at a dose of 0.001 mg per kg of the subject's body weight to 10 mg per kg of the subject's body weight, including the doses at both ends. In a preferred embodiment, the pharmaceutical formulations are administered to a human subject at a dose of 0.01 mg per kg of the subject's body weight to 1 mg per kg of the subject's body weight, including the doses at both ends. In a further embodiment, the pharmaceutical formulations are administered to a human subject at a dose of 1.0 mg to 100 mg, including the doses at both ends.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0053] Detailed Description of the Invention I. Definitions The term "therapeutic agent" refers to an agent that can be administered to treat one or more symptoms of a disease or disorder. The term "preventive agent" generally refers to an agent that can be administered to prevent a disease or to prevent a particular condition.

[0054] As used herein, the term "pharmaceutically acceptable salt" refers to derivatives of the compounds defined herein, where the parent compound is modified by making its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral and organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. Such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, naphthalenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isethionate.

[0055] The terms "pharmaceutically acceptable" or "biocompatible" refer to compositions, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material involved in the carrying or transporting of any subject composition from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient.

[0056] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that produces a desired effect to a degree that represents a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount can vary depending on factors such as the disease or condition being treated, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation. In some embodiments, the term "effective amount" refers to an amount of a prophylactic or therapeutic agent that reduces or decreases the risk of developing a liver disease / disorder, or reduces or decreases one or more symptoms of a liver disease / disorder, e.g., to reduce inflammation in the liver. Additional desired results include reducing and / or inhibiting the serum levels of alanine aminotransferase (ALT), aspartic acid aminotransferase (AST), triglyceride (TG) and total cholesterol (TC), fat accumulation or steatosis, inflammation, hypertrophy, fibrosis, long-term pathological conditions, and mortality. The effective amount can be administered in one or more administrations.

[0057] The terms "inhibit" or "reduce" mean, in the context of inhibition, to reduce or decrease activity and amount. This can be a complete or partial inhibition or reduction of activity or amount. Inhibition or reduction can be compared to a control or standard level. The inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a sustained GLP-1r agonist can inhibit or reduce the activity and / or amount of activated microglia by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from the activity and / or amount of the same cells in equivalent tissue in a subject not receiving or treated with the sustained GLP-1r agonist. In some embodiments, inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ levels.

[0058] The term "treating" or "treatment" refers to the amelioration, alleviation or reduction of one or more symptoms of a disease, disorder, or condition in a person who may have a predisposition to, but who has not yet been diagnosed as having, the disease, disorder, and / or condition; reducing the symptoms of a disease; inhibiting a disease, disorder or condition, e.g., preventing its progression; and alleviating a disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition includes ameliorating at least one symptom of a particular disease or condition, even if it does not affect the underlying pathophysiology, e.g., treating a subject's pain by administration of such an agent even if an analgesic does not treat the cause of the pain. Desired effects of treatment include decreasing the rate of disease progression, ameliorating or alleviating the state of the disease, and regression, or improved prognosis. For example, if one or more symptoms associated with a liver disease / disorder are reduced and / or inhibited, including, but not limited to, an increase in transaminases including alanine transaminase (ALT) and aspartate transaminase (AST), reducing the growth of cancerous cells in the case of liver cancer, increasing the quality of life of those suffering from the disease, reducing the dosage of other drug therapies required to treat the disease, delaying the progression of the disease, and / or prolonging the survival of the individual, the individual is successfully "treated" if the disease is alleviated or eliminated. The term "ameliorate" refers to a decrease, suppression, attenuation, reduction, arrest, or stabilization of the development or progression of a disease.

[0059] The terms "prevent", "prevention" or "preventing" mean administering a composition or method to a subject or system that has a risk of, or a predisposition to, one or more symptoms caused by a disease or disorder, reducing the likelihood that a subject will develop one or more symptoms of a disease or disorder, or reducing the severity, duration, or time of onset of one or more symptoms of a disease or disorder.

[0060] The term "biodegradable" generally refers to materials that, under physiological conditions, can be broken down or eroded into smaller units and chemical species that can be metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and form.

[0061] The term "protein" or "polypeptide" or "peptide" refers to any chain of two or more natural or unnatural amino acids that, regardless of post-translational modifications (such as glycosylation and phosphorylation), constitutes all or part of a polypeptide or peptide, whether naturally occurring or not.

[0062] The terms "biotinylation" and "biotinylated" refer to the process and product of the covalent attachment of one or more biotin moieties or their derivatives to molecules and macromolecular structures such as therapeutic proteins.

[0063] The terms "lipidation" and "lipidated" refer to the process and product of the covalent attachment of one or more fatty acid moieties or their derivatives to molecules and macromolecular structures such as therapeutic proteins.

[0064] The term "PEGylation" refers to the process of both covalent and non-covalent attachment or fusion of polyethylene glycol (PEG) polymer chains to molecules and macromolecular structures such as drugs, therapeutic proteins, or vesicles.

[0065] The use of the term "about" is intended to describe values that exceed or are below the stated value by approximately + / - 10%, and in other embodiments, this value may be in the range of values that exceed or are below the stated value by approximately + / - 5%.

[0066] II. Compositions A composition is provided that includes an isolated peptide having activity against glucagon receptors, glucagon-like peptide 1 (GLP-1) receptors, and glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0067] A. Triple agonist peptide In some embodiments, the triple agonist peptide is the following polypeptide formula (I): X 1 X 2 X 3 GTFTSDX 10 SX 12 X 13 LDX 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 G X 30 X 31 SX 33 X 34 X 35 PP X 38 X 39 X 40 (SEQ ID NO: 131) (wherein X 1 is either H or Y; X 2 is either A or 2-aminoisobutyric acid (Aib); X 3 is either Q or E; X 12 is either R, W or K; X 13 is either L or Y; X 19 is either Q, A or T; X 21 is either D or L; X 22 is either F or R; X 23 is either V, G or D; X25 is W, Y or A; X 26 is L or D; X 27 is I, L, M, G or P; X 30 is G or P; X 31 is P or S; X 33 is S or G; X 34 is G or A; X 35 is A or P; X 38 is P or S; X 39 is absent, S or C; X 40 is absent, C or K; With an optional amide modification at the C-terminus, X 10 、X 16 、X 17 、X 18 、X 20 、X 24 、and X 28 has an amino acid sequence that is any one of the 20 amino acids excluding cysteine). In some embodiments, X 17 、X 18 、X 20 is a non-natural amino acid. In some embodiments, X 17 、X 18 、X 20 is a non-natural amino acid, X 17 、X 18 、X 20 is independently one of the following: methoxynine, 2-aminoisobutyric acid, and alpha-methylarginine.

[0068] In other embodiments, X 10 is Y, W, K, F, H, S, L, A, E, M, Q or D; X 16 is Y, Q, G, K, S, R, F, P or A; X 17 is M, Y, Q, K, S, W, P, D, A, F or methionine; X 18 is A, I, M, W, T, D, Y or methionine; X 20 is R, Q, H, G, A, P, N, K, Aib or alpha-methylarginine; X 24 is Q, D, K, L, N, W or M; X 28 is N, E, G, D, H or Q.

[0069] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 134: YXQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 134) (wherein X is any one of the 20 amino acids).

[0070] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 135: YAibQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 135)

[0071] In some embodiments, the polypeptide of formula (I) has the amino acid sequence of any one of SEQ ID NOs: 1-130.

[0072] 1. Modifications to the triple agonist peptide ​The direct use of native polypeptides as biopharmaceuticals is often limited by their very short systemic half-lives resulting from rapid metabolism, enzymatic degradation, and effective renal clearance for smaller proteins and peptides. Modifications to exenatide, such as semaglutide, liraglutide, and NLY01, have extended the half-life and pharmacokinetics of the active agent. Therefore, further modifications are being made to further improve oral bioavailability, stability, and / or pharmacokinetics.

[0073] In some embodiments, the triple agonist peptide has an amino acid sequence of any one of SEQ ID NOs: 1 to 130. In preferred embodiments, the triple agonist peptide is a modified triple agonist analog with one or more biotin moieties and / or one or more fatty acids, optionally with one or more spacers, to achieve the desired pharmacokinetics, stability, and bioavailability. In other embodiments, the triple agonist peptide is a modified triple agonist analog with one or more biotin moieties, one or more fatty acids, and / or one or more PEG moieties, optionally with one or more spacers, to achieve the desired pharmacokinetics, stability, and bioavailability. In further embodiments, the triple agonist peptides disclosed herein are modified by C-terminal amidation.

[0074] The selection of suitable functional groups for modification is based on the type of available reactive groups on the molecule to which the biotin moiety and / or fatty acid is coupled. Typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and C-terminal carboxylic acid can also be used for site-specific conjugation. In preferred embodiments, the reactive amino acids are lysine and cysteine.

[0075] In a preferred embodiment, one or more biotin moieties and / or one or more fatty acids, or derivatives thereof, are conjugated to any one of the amino acid sequences of SEQ ID NOs: 1 to 130 via one or more of the amino acid residues of lysine at position 10, lysine at position 12, lysine at position 17, lysine at position 20, lysine at position 24, and cysteine or lysine residues inserted at one or more C-termini. In other embodiments, one or more cysteine and lysine residues are introduced via substitution or insertion into any one of the amino acid sequences of SEQ ID NOs: 1 to 130 to facilitate conjugation to biotin moieties and / or fatty acids, or derivatives thereof. In certain embodiments, the triple agonist peptide has any one of the amino acid sequences of SEQ ID NOs: 1 to 130.

[0076] a. Biotinylation Biotin modification of exendin derivatives has been previously described, for example, in International Publication Nos. WO2009107900A1, WO2020242268A1, and WO2021107519A1.

[0077] Korean Patent Registration No. 10-0864584 describes that exendin-4 in which biotin is modified at the lysine residue of exendin-4 can be orally administered and has improved bioavailability in the intestine. However, in this case, there are problems in that biotin is conjugated at various lysine positions of exendin-4 to form various isomers, thereby reducing the reaction rate and yield, and biotin is conjugated at the position of the N-terminal lysine which is the active site of exendin-4, inhibiting the activity of exendin-4.

[0078] Therefore, in a preferred embodiment, one or more biotin moieties are conjugated to amino acids at suitable positions (e.g., cysteine or lysine) to provide excellent oral bioavailability without inhibiting the activity of the triple agonist peptide.

[0079] In some embodiments, the triple agonist peptide has improved in vivo bioavailability compared to the same triple agonist peptide that does not contain one or more biotin moieties conjugated thereto. In a preferred embodiment, the biotin-conjugated triple agonist peptide retains most of the activity of the same triple agonist peptide that does not contain one or more biotin moieties conjugated thereto.

[0080] In some embodiments, the biotin moiety conjugated to one or more amino acid residues (e.g., cysteine or lysine) of the triple agonist peptide is represented by the following general formula A. [General formula A] [Chemical formula] (In the formula, X is a functional group capable of conjugating to a polypeptide, Y is a spacer, Z is a linking unit, B is the following chemical formula A-1 [Chemical formula A-1] [Chemical formula] (T is a terminal group, m is an integer from 1 to 10, n is an integer from 1 to 10, p is an integer of 0 or 1) is represented by).

[0081] In some embodiments, when n = 0, Y can be directly linked to B or T.

[0082] In some embodiments, the biotinylated polypeptide is a peptide having any one of the amino acid sequences of SEQ ID NOs: 1-130. Alternatively, one or more cysteine or lysine residues are inserted internally at any position within any one of the amino acid sequences of SEQ ID NOs: 1-130 to facilitate conjugation to biotin.

[0083] In some embodiments, the biotin moiety is conjugated to the triple agonist peptide via one or more additional cysteine or lysine residues added to the C-terminus of the polypeptide. In one embodiment, the biotin moiety is conjugated to the triple agonist peptide via one additional cysteine residue added to the C-terminus of any one of the amino acid sequences of SEQ ID NOs: 1-130. In another embodiment, the biotin moiety is conjugated to the triple agonist polypeptide via one additional lysine residue added to the C-terminus of any one of the amino acid sequences of SEQ ID NOs: 1-130. In some embodiments, the amino acid at the second position of SEQ ID NOs: 1-18 is replaced with 2-aminoisobutyric acid (Aib). In some embodiments, the biotin moiety is conjugated to the triple agonist polypeptide via one or more internal lysine residues, such as lysine at position 10, lysine at position 12, lysine at position 17, lysine at position 20, and / or lysine at position 24, of any one of SEQ ID NOs: 1-130.

[0084] In general formula A representing the biotin moiety, X is a functional group capable of conjugating with the cysteine of the polypeptide. Without limitation, for example, the functional group may be a maleimide, amine, succinimide, N-hydroxysuccinimide, aldehyde or carboxyl group, more specifically, it may be a maleimide.

[0085] In one embodiment, when the functional group X in general formula A is conjugated to the cysteine or lysine of the polypeptide, its structure may be maintained, or removed, or modified.

[0086] In General Formula A, Y may be a spacer and may have a structure that is cleavable in the body. Without limitation, for example, Y is a directly bonded, substituted or unsubstituted alkylene, where the alkylene may include at least one of -O-, -C(=O)NR-, -C(=O)O- or -C(=O)-, -NR-, and -NOR-, and R may be hydrogen, and substituted or unsubstituted alkyl or aryl.

[0087] In one embodiment, the spacer may include a structure represented by the following formula.

Chemical formula

[0088] In some embodiments, in General Formula A, Z is a bonding unit capable of bonding to B, for example, without limitation, it may include an amino acid, polypeptide, alkyleneamine, or polyamideamine structure.

[0089] Without limitation, for example, the amino acid may be lysine, 5-hydroxylysine, 4-oxallysine, 4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexenoic acid, cis-4-dehydrolysine, 6-N-methyllysine, diminopimelic acid, ornithine, 3-methyornithine, α-methyornithine, citrulline or homocitrulline, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, ornithine, proline, serine, or threonine.

[0090] When n is 0, B may be directly bonded to Y (spacer).

[0091] In some embodiments, in General Formula A, T is a terminal group and, without limitation, is, for example, hydrogen or NH 2 and may be such.

[0092] When p is 0, B can be terminal.

[0093] In one embodiment, in the above General Formula A, "m" may be an integer from 1 to 10, specifically, may also be an integer from 1 to 8, from 1 to 5, and from 1 to 4.

[0094] In one embodiment, the biotin moiety is represented by the following General Formula 1A: [General Formula 1A] [Chemical Formula] (wherein Lys is lysine, T is hydrogen or NH 2 and q is an integer from 1 to 5, r is 0 or an integer from 1 to 3, B, n, m, and p are as defined in the above General Formula A) and may be represented by.

[0095] In one embodiment, the biotin moiety is represented by the following General Formula 2A or 3A: [General Formula 2A] [Chemical Formula] (wherein Lys is lysine, T is hydrogen or NH 2 and R 3 is hydrogen or -SO 3 - and q is 0 or an integer from 1 to 4, B, n, m, and p are as defined in the above General Formula A) [General Formula 3A] [Chemical Formula] (wherein, R 1 is a direct bond or NH, R 3 is hydrogen or -SO 3 -, B and m are as defined in the general formula A above). It may be represented by

[0096] In one embodiment, the biotin moiety may be represented by the following structures I to III. Structure I.

Chemical formula

Chemical formula

Chemical formula

[0097] Exemplary biotin derivatives are shown in Tables 1 and 2 below.

[0098] Table 1. Examples of biotin derivatives.

Table 1

[0099] Table 2. Examples of biotin derivatives.

Table 2-1

Table 2-2

[0100] For B39 and B40, desthiobiotin, an analog of biotin shown in Structure IV, is used. Structure IV. Desthiobiotin [Chem.]

[0101] In some embodiments, the NHS-desthiobiotin of the biotin analog shown in Structure V is used for conjugation. Structure V. NHS-desthiobiotin [Chem.] b. Lipidation

[0102] Lipidated peptides have increased lipophilicity, increased in vivo half-life (allowing once-daily oral administration), and reduced variability in steady-state pharmacokinetics. In some embodiments, additional amino acids are added to the C-terminus of the triple agonist peptide to allow conjugation of one or more fatty acid molecules having increased linker stability. In preferred embodiments, the amino acid is either cysteine or lysine.

[0103] In some embodiments, the fatty acid moiety is conjugated to the triple agonist peptide via one or more additional cysteine or lysine residues added to the C-terminus of the polypeptide. In one embodiment, the fatty acid moiety is conjugated to the triple agonist peptide via one additional cysteine residue added to the C-terminus of the amino acid sequence of SEQ ID NOs: 1-130. In some embodiments, the amino acid at the second position of SEQ ID NOs: 1-18 is replaced with 2-aminoisobutyric acid (Aib). In some embodiments, the fatty acid moiety is conjugated to the triple agonist polypeptide via one or more internal lysine residues of any of SEQ ID NOs: 1-130, such as lysine at position 10, lysine at position 12, lysine at position 17, and / or lysine at position 20, lysine at position 24.

[0104] The first lipidated biopharmaceutical to receive regulatory approval was insulin detemir in 2004. Insulin detemir, a basal insulin for the treatment of diabetes, contains desB30 human insulin conjugated to myristic acid (C14) through the Nε-amine of LysB29.

[0105] Current lipidated biopharmaceuticals have a hydrophilic spacer, typically γGlu and / or OEG (8-amino-3,6-dioxaoctanoic acid), between the lipid and peptide moieties to increase parameters such as albumin affinity, potency, water solubility, and oligomerization. An example is liraglutide, a once-daily glucagon-like peptide 1 (GLP-1) analog marketed for the treatment of diabetes and obesity. The liraglutide sequence is identical to that of native GLP-1 except for a Lys34Arg substitution, which enables selective palmitoylation through the γGlu spacer at the Nε of Lys26 (Lau J.; et al., J. Med. Chem. 2015, 58 (18), 7370-7380). Due to albumin binding and slow absorption, liraglutide has a significantly extended half-life (11-15 hours, s.c.) compared to native GLP-1 (1-1.5 hours, s.c.). From the dietary fatty acids used in insulin detemir and liraglutide, the preferred fatty acids for lipidation have progressed to non-dietary dicarboxylic acid fatty acids used in insulin degludec, a once-daily basal insulin, and semaglutide, a once-weekly GLP-1 analog. Insulin degludec is lipidated at LysB29 with palmitic diacid spaced by γGlu. The peptide backbone of semaglutide is similar to that of liraglutide except for the substitution of alanine 8 with 2-aminoisobutyric acid (Aib) to reduce degradation by dipeptidyl peptidase IV (DPP-4) (Lau, J. et al., Journal of Medicinal Chemistry (2015), 58 (18), 7370-7380). Semaglutide is lipidated at Lys26 with octadecanedioic acid through a spacer containing γGlu and two OEG units, which induces an albumin affinity 5.6-fold higher than that of liraglutide.High albumin affinity and DPP-4 resistance confer a half-life of approximately one week in humans (s.c.) to semaglutide (van Witteloostuijn, S. B.; Pedersen, S. L.; Jensen, K. J. ChemMedChem 2016, 11, 1-23). Impressively, this extended half-life is obtained without reducing the efficacy of the GLP-1 receptor compared to the native ligand. Recently, lipidation has also been shown as a viable strategy for extending the half-life of larger proteins, as demonstrated by somatropin, a human growth hormone administered once a week. The lipidation of somatropin involves a rather long spacer region and a non-carboxylic acid fatty acid with a tetrazole head.

[0106] Therefore, in some embodiments, the triple agonist peptide is preferably conjugated to one or more of the fatty acid chains using one or more hydrophilic spacers, such as γGlu or OEG (8-amino-3,6-dioxaoctanoic acid), between the lipid and the peptide. Exemplary fatty acids can include dietary fatty acids, such as those used in insulin detemir and liraglutide, and preferred fatty acids for lipidation are non-dietary dicarboxylic acid fatty acids, such as those used in insulin degludec and semaglutide.

[0107] Exemplary fatty acid derivatives are shown in Table 3 below. Table 3. Examples of Fatty Acid Derivatives

Table 3

[0108] In a preferred embodiment, the triple agonist peptide is lipidated and / or biotinylated. In certain embodiments, the triple agonist peptide has an amino acid sequence of any one of SEQ ID NOs: 1-130 and is modified at one or more sites listed in Table 23 (A, B, C, D, E, and F) using one or more biotin derivatives listed in Tables 1 and 2, and / or one or more fatty acid derivatives listed in Table 3.

[0109] c. Pegylation In some embodiments, the triple agonist peptide is modified with polyethylene glycol, polyethylimine, or derivatives thereof. In a preferred embodiment, the triple agonist peptide is modified by pegylation at a site similar to the biotinylated and lipidated sites. The modification can alter pharmacokinetics, pharmacodynamics, stability, and bioavailability.

[0110] Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. The structure of PEG is (note the repeating element in parentheses): H-(O-CH 2 -CH 2 ) nIt is -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. PEG, PEO, or POE refers to oligomers or polymers of ethylene oxide. The three names are chemically synonymous, but historically, PEG is preferred in the biomedical field, while PEO is more prevalent in the field of polymer chemistry. Since different applications require different polymer chain lengths, PEG tends to refer to oligomers and polymers with a molecular mass below 20,000 g / mol, PEO refers to polymers with a molecular mass above 20,000 g / mol, and POE refers to polymers of any molecular mass. PEG and PEO are liquids or low-melting solids depending on their molecular weight. PEG is prepared by the polymerization of ethylene oxide and is commercially available over a wide range of molecular weights from 300 g / mol to 10,000,000 g / mol. PEGs and PEOs with different molecular weights are used in different applications and have different physical properties (e.g., viscosity) due to the effect of chain length, but their chemical properties are almost the same. Different forms of PEG are also available depending on the initiator used in the polymerization process 2, and the most common initiator is monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG. Lower molecular weight PEGs are also available as purer oligomers, referred to as monodisperse, uniform, or discrete. Very high purity PEG has recently been shown to be crystalline, enabling the determination of crystal structure by X-ray diffraction. Since the purification and separation of pure oligomers are difficult, the price of this type of quality is often 10 to 10,000 times the price of polydisperse PEG.

[0111] PEGs with different geometries are also available. Branched PEGs have 3 - 10 PEG chains emanating from a central core group. Star PEGs have 10 - 100 PEG chains emanating from a central core group. Comb PEGs have multiple PEG chains typically grafted onto a polymer backbone. The numbers often included in the name of the PEG indicate its average molecular weight (e.g., a PEG with n = 9 will have an average molecular weight of approximately 400 Daltons and will likely be labeled PEG400). Most PEGs contain molecules with a distribution of molecular weights (i.e., they are polydisperse). The size distribution can be statistically characterized by its weight average molecular weight (Mw) and its number average molecular weight (Mn), the ratio of which (Mw / Mn) is called the polydispersity index. MW and Mn can be measured by mass spectrometry.

[0112] In some embodiments, the polyethylene glycol or their derivatives are in a linear or branched form, and for the branched form, preferably, a dimeric or trimeric form may be used, more preferably, a trimeric form may be used. Specifically, the polyethylene glycol derivative is, for example, methoxypolyethylene glycol succinimidyl propionate, methoxypolyethylene glycol N - hydroxysuccinimide, methoxypolyethylene glycol propionaldehyde, methoxypolyethylene glycol maleimide, or a multi - branched form of these derivatives. Preferably, the polyethylene glycol derivative is linear methoxypolyethylene glycol maleimide, branched - form methoxypolyethylene glycol maleimide or trimeric methoxypolyethylene glycol maleimide, more preferably, trimeric methoxypolyethylene glycol maleimide.

[0113] PEG is a particularly attractive polymer for conjugation. Specific features of the PEG moiety relevant to pharmaceutical applications are water solubility, high mobility in solution, lack of toxicity and low immunogenicity, rapid clearance from the body, and altered distribution in the body.

[0114] PEGylation (also often called pegylation) refers to the process of both covalent and non-covalent attachment or fusion of polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as drugs, therapeutic proteins or vesicles, which results in what is then described as PEGylation (pegylation). PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. Covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity) and can increase the hydrodynamic size (size in solution) of the agent, which extends its circulation time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

[0115] PEGylation can improve the safety and efficacy of many therapeutic agents, such as peptides, proteins, and antibody fragments. This results in alterations in physicochemical properties, including changes in conformation, electrostatic binding, hydrophobicity, etc. These physical and chemical changes increase the systemic retention of the therapeutic agent. It can also affect the binding affinity of the therapeutic agent moiety to cell receptors and can alter absorption and distribution patterns.

[0116] PEGylation increases the molecular weight, protection of metabolic sites and inhibition of immunogenic sites, increased in vivo half-life and stability, as well as reduced immunogenicity. Furthermore, the renal excretion of peptides and proteins conjugated to PEG is reduced due to the increase in the molecular weight of the peptides and proteins by PEG, and as a result, PEGylation has the advantage of increasing the effect both pharmacokinetically and pharmacodynamically.

[0117] In some embodiments, the triple agonist peptide is modified with polyethylene glycol, polyethylimine, or derivatives thereof. In a preferred embodiment, the triple agonist peptide is modified by PEGylation at a site similar to the biotinylation and lipidation sites. After the triple agonist peptide is PEGylated with polyethylene glycol or derivatives thereof are prepared, the molecular structure of the analog can be confirmed by comparison between a mass spectrometer, liquid chromatography, X-ray diffraction analysis, optical rotation analysis, and the calculated and measured values of representative elements constituting the PEGylated triple agonist peptide.

[0118] In some embodiments, the PEG or derivatives thereof are conjugated to the triple agonist peptide via one or more additional cysteine or lysine residues added to the C-terminus of the polypeptide. In one embodiment, the PEG or derivatives thereof are conjugated to the triple agonist peptide via one additional cysteine residue added to the C-terminus of the amino acid sequence of SEQ ID NOs: 1-53. In some embodiments, the amino acid at the second position of SEQ ID NOs: 1-53 is replaced with 2-aminoisobutyric acid (Aib). In some embodiments, the PEG or derivatives thereof are conjugated to the triple agonist polypeptide via one or more internal lysine residues of any of SEQ ID NOs: 1-64, such as lysine at position 10, lysine at position 12, lysine at position 17, lysine at position 20, and / or lysine at position 24.

[0119] B. Pharmaceutical Formulations In some embodiments, the triple agonist peptide or an analog thereof is formulated using one or more pharmaceutical additives, adjuvants, or fillers. For example, in some embodiments, the triple agonist peptide or an analog thereof is formulated into a pharmaceutical formulation for administration to a subject. The composition containing the triple agonist peptide or an analog thereof may be formulated in a conventional manner using one or more physiologically acceptable carriers, including additives and adjuvants that facilitate the processing of the active compound into a pharmaceutically useful preparation.

[0120] Suitable formulations depend on the selected route of administration. In preferred embodiments, the composition is formulated for parenteral delivery. In preferred embodiments, the composition is formulated for subcutaneous delivery. In some embodiments, the composition is formulated for intravenous injection. Typically, the composition is formulated in sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition can be lyophilized and stored in a single-use vial for rehydration immediately prior to use. Other means for rehydration and administration are known to those skilled in the art.

[0121] The pharmaceutical formulation contains the triple agonist peptide or an analog thereof in combination with one or more pharmaceutically acceptable additives. Representative additives include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable additives are preferably selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0122] Generally, pharmaceutically acceptable salts can be prepared by reacting the free acid or base form of the drug with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of the drug derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by the reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). A list of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 20 thIt can be found on p. 704 of the 2000 ed., Lippincott Williams & Wilkins, Baltimore, MD. Examples of ophthalmic drugs that may be administered in pharmaceutically acceptable salt forms include timolol maleate, brimonidine tartrate, and diclofenac sodium.

[0123] 1. Dosage unit The composition is preferably formulated into dosage unit forms for ease of administration and uniformity of dosage. The phrase "dosage unit form" refers to physically discrete units suitable for the patient to be treated. However, it will be understood that the total daily dosage of the composition will be determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose can initially be estimated in cell culture assays or animal models, usually in any of mice, rats, rabbits, dogs, or pigs. Animal models are also used to achieve the desired concentration range and route of administration. Such information should then be useful for determining useful dosages and routes of administration in humans.

[0124] 2. Formulations for administration In some embodiments, the composition of the triple agonist peptide or its analog is formulated into a pharmaceutically acceptable formulation for administration via a particular route. In some embodiments, the composition is administered locally, for example, by direct injection into the site to be treated. In some embodiments, the composition is injected, topically applied, or otherwise administered directly to the vasculature in or adjacent to the vascular tissue at the site of injury, surgery, or implantation. For example, in some embodiments, the composition is topically applied to vascular tissue exposed during a surgical procedure. Typically, local administration causes an increased localized concentration of the composition, which is higher than that achievable by systemic administration.

[0125] Pharmaceutical compositions formulated for parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection) administration and enteral routes of administration are described.

[0126] a. Enteral administration In some embodiments, the triple agonist peptide or an analog thereof is administered orally. For oral administration, suitable formulations include tablets, pellets, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, troches, etc. These formulations can include, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), slip modifiers (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). Tablets may also contain binders, such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and may also contain disintegrants, such as starch, agar, alginic acid or its sodium salt, or boiling mixture, and / or, if necessary, absorbents, colorants, flavoring agents, and sweetening agents.

[0127] In a preferred embodiment, one or more absorption or permeation enhancers are used for oral formulations. Exemplary permeation enhancers include bile acids, cholic acid, deoxycholic acid, glycocholic acid, glycochonodeoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, Labrasol® (caprylocaprylyl polyoxyl-8 glyceride), SNAC (sodium N-(8-[2-hydroxybenzoyl]amino)caprylate), propyl gallate, and their salt forms.

[0128] b. Parenteral administration In some embodiments, the triple agonist peptide or an analog thereof is formulated into a pharmaceutically acceptable formulation for parenteral administration. The terms "parenteral administration" and "administered parenterally" are terms recognized in the art and include modes of administration other than enteral and topical administration, e.g., injections, including but not limited to intravenous (i.v.), intramuscular (i.m.), intraperitoneal (i.p.), subcutaneous (s.c.) injections and infusions. The sustained GLP-1r agonist can be administered parenterally, e.g., by intravenous, intraperitoneal, or subcutaneous routes.

[0129] For liquid formulations, the pharmaceutically acceptable carrier may be, for example, aqueous and non-aqueous solutions, suspensions, emulsions, or oils. Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Examples of non-aqueous solvents are injectable organic esters such as propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrin, emulsions or suspensions including saline and buffered media. The sustained triple agonist can also be administered in an emulsion, e.g., an oil-in-water emulsion. Examples of oils are of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0130] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile suspensions which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, and suspending, solubilizing, thickening, stabilizing and preserving agents. Examples of intravenous vehicles include fluid replenishers and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0131] Injectable pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0132] III. Methods of Preparation A. Methods of Preparing the Triple Agonist Peptide Analogue The triple agonist can be prepared and modified via various chemical reaction steps. Typically, methods for modifying the triple agonist include biotinylation and / or lipidation. In some embodiments, biotinylation and lipidation can be applied during peptide synthesis (assembly of amino acids by solid-phase peptide synthesis) using biotin-conjugated amino acids and lipid-conjugated amino acids.

[0133] 1. Biotinylation and Lipidation Biotinylation of exendin derivatives has been previously described, for example, in International Publication Nos. WO2009107900A1, WO2020242268A1, and WO2021107519A1.

[0134] In some embodiments, biotinylation and lipidation are performed simultaneously. In one embodiment, the method comprises dissolving a peptide, C18-γGlu-2OEG-MAL (F12), and biotin-N-hydroxysuccinimide ester (B1-NHS, B38) in a 0.3% triethylamine (TEA) (v / v) solution containing dimethyl sulfoxide (DMSO). In one embodiment, the peptide and F12 are mixed at a 1:1 volume ratio. An exemplary concentration of the peptide is 5 mg / mL and the molar ratio is 1:2 (peptide:lipid). In one embodiment, the mixture is reacted at 25 °C for 10 minutes with gentle shaking. In another embodiment, B38 is added at a 1:0.2 volume ratio and the molar ratio is 1:3 (peptide:biotin). In one embodiment, the mixture is reacted at 25 °C for 60 minutes with gentle shaking.

[0135] In another embodiment, the method comprises dissolving a peptide, biotin-maleimide (B1-MAL, B1), and C18-γGlu-2OEG-NPC (F16) in a 0.3% TEA (v / v) solution containing DMSO. In one embodiment, the peptide and B1 are mixed at a 1:1 volume ratio. An exemplary concentration of the peptide is 5 mg / mL and the molar ratio is 1:2 (peptide:biotin). In one embodiment, the mixture is reacted at 25°C for 10 minutes with gentle shaking, then F16 is added at a 1:0.2 volume ratio and the molar ratio is 1:2 (peptide:lipid). In one embodiment, the mixture is reacted at 25°C for 90 minutes with gentle shaking. The lipidated and biotinylated peptide can be purified by Prep-LC and the eluate can be collected into individual fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated at 45°C for 40 minutes using a centrifugal evaporator. The solvent can be changed to water by ultrafiltration. The purified sample can be analyzed by reverse-phase HPLC for purity check. In one embodiment, the sample is lyophilized at -88°C for 18 hours and then stored at -20°C.

[0136] 2. Lipidation In one embodiment, the method comprises dissolving a peptide and C18-γGlu-2OEG-MAL (F12) in a 0.3% TEA (v / v) solution containing DMSO; and mixing the respective solutions at a 1:1 volume ratio. In one embodiment, the concentration of the peptide is 5 mg / mL and the molar ratio is 1:2 (peptide:lipid). The mixture is then reacted at 25°C for 30 minutes with gentle shaking. In one embodiment, the lipidated peptide is purified by Prep-LC and the eluate is collected into individual fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated at 45°C for 40 minutes using a centrifugal evaporator. The solvent can be changed to water by ultrafiltration. The purified sample can then be analyzed by reverse-phase HPLC for purity check. In one embodiment, the sample is lyophilized at -88°C for 18 hours and then stored at -20°C.

[0137] IV. Method of Use A pharmaceutical formulation containing one or more of the triple agonist peptides or analogs thereof described herein can be administered to a subject in need thereof to treat or prevent one or more diseases.

[0138] A. Method of Treatment A method of using a triple agonist peptide to treat or prevent one or more metabolic diseases is described. The method is preferably effective in treating or preventing one or more metabolic diseases, such as dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, and type 2 true diabetes (T2DM) with minimal side effects.

[0139] In a preferred embodiment, the triple agonist peptide or an analog thereof is administered in an amount and dosing regimen effective to prevent, inhibit, or reduce one or more symptoms associated with one or more metabolic diseases in a subject, such as dyslipidemia, fatty liver disease, metabolic syndrome, NAFLD, obesity, and T2DM. In a preferred embodiment, the disease is fatty liver disease, obesity, NAFLD, or T2DM.

[0140] The triple agonist peptide or an analog thereof is administered to the subject in one or more doses at one or more time points after the starting dose. The amount of the composition administered to the subject is selected to deliver an effective amount to reduce, prevent, or otherwise alleviate one or more clinical or molecular symptoms of the disease or disorder being treated as compared to a subject treated with an agonist that primarily targets a single receptor, such as GLP-1, glucagon, or a GIP receptor. The composition and method are also suitable for prophylactic use.

[0141] The method is also suitable for treating or preventing one or more neurodegenerative diseases, such as Alzheimer's disease (AD) or Parkinson's disease. In some embodiments, the method comprises administering to an individual in need thereof an effective amount of a triple agonist peptide or an analog thereof.

[0142] B. Conditions to be Treated Compositions and formulations of these triple agonist peptides are effective for reducing or preventing one or more symptoms of metabolic or neurological diseases with minimal off-target toxicity or side effects.

[0143] In some embodiments, the subject to be treated is a human. In some embodiments, the subject to be treated is a pediatric or infant subject. All methods can include the step of identifying and selecting a subject in need of treatment or a subject who would benefit from administration with the described composition.

[0144] 1. Obesity and Type 2 Diabetes Mellitus Obesity is a medical condition in which excess body fat has accumulated to the extent that it can have harmful effects on health, leading to a reduction in life expectancy and / or an increase in health problems. The body mass index (BMI), a measure comparing weight and height, defines people as overweight (pre-obese or overweight) when their BMI is 25 - 30 kg / m 2 and obese when it is higher than 30 kg / m 2 or more.

[0145] Obesity increases the risk of many physical and mental disorders. Excess body weight is associated with various diseases, particularly cardiovascular disease, type 2 diabetes mellitus, obstructive sleep apnea, certain types of cancer, and osteoarthritis. These diseases are either directly caused by obesity or indirectly related through mechanisms that share common causes such as poor diet and / or a sedentary lifestyle. One of the strongest associations is with type 2 diabetes. Excess body fat underlies 64% of cases of diabetes in men and 77% of cases in women. An increase in body fat alters the body's response to insulin and potentially leads to insulin resistance.

[0146] A method for treating and / or preventing one or more symptoms of obesity and / or T2DM comprises administering to a subject in need thereof an effective amount of a composition for treating and / or alleviating one or more symptoms associated with obesity and / or T2DM. In some embodiments, the composition or a pharmaceutical formulation thereof is administered in an amount effective to induce weight loss, reduce body fat, reduce food intake, improve glucose homeostasis, prevent weight gain, and / or prevent an increase in the obesity index in normal or obese patients, or a combination thereof.

[0147] In some embodiments, the pharmaceutical formulation is administered to a patient suffering from obesity, an obesity-related disease or disorder, diabetes, insulin resistance syndrome, non-alcoholic steatohepatitis, cardiovascular disease, or metabolic syndrome.

[0148] In some embodiments, the pharmaceutical formulation is administered to normalize blood glucose, and the formulation is preferably administered in an amount effective to lower the blood glucose level to less than about 180 mg / dL. The formulation can be co-administered with other anti-diabetic therapies, if necessary, to improve glucose homeostasis.

[0149] The pharmaceutical formulation can also be administered to a patient suffering from a disease or disorder that predisposes the patient to obesity or becoming obese.

[0150] 2. Non-alcoholic fatty liver disease (NAFLD) In preferred embodiments, the methods and compositions are used to treat or prevent non-alcoholic steatohepatitis, liver fibrosis associated with non-alcoholic steatohepatitis, and primary biliary cholangitis.

[0151] In some embodiments, the composition is used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD represents the clinicopathological spectrum of diseases (steatosis) mainly manifested as excessive accumulation of fat in hepatocytes. NAFLD encompasses the entire spectrum of diseases ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which can lead to life-threatening cirrhosis and its most severe form, hepatocellular carcinoma. This is considered to be the hepatic manifestation of the metabolic syndrome, and other pathologies include obesity, insulin resistance, hypertension, and hyperlipidemia. Histologically, NASH is characterized by hepatic steatosis and signs of lobular inflammation with hypertrophic degeneration of hepatocytes. The estimated prevalence of NASH is much lower than that of NAFLD and ranges from 3 to 5%. Twenty percent of patients with NASH have been reported to develop cirrhosis, and 30 to 40% of patients with NASH cirrhosis experience liver-related death.

[0152] NAFLD is broadly classified into two phenotypes, namely: non-alcoholic fatty liver (NAFL) represented by isolated steatosis, and non-alcoholic steatohepatitis (NASH), a more aggressive subtype characterized by cell injury, inflammatory cell infiltration, and hepatocyte hypertrophy, which can further progress to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In some embodiments, the composition is used in an effective amount to treat and ameliorate one or more symptoms of non-alcoholic steatohepatitis (NASH).

[0153] NAFLD is closely associated with metabolic syndrome, including obesity, type II diabetes, dyslipidemia, etc., based on insulin resistance. In fact, many prediabetic and type II diabetic patients have been shown to present with non-alcoholic fatty liver / non-alcoholic steatohepatitis, and it is known that the rate of progression to cirrhosis and liver cancer (i.e., hepatocellular carcinoma) is high in these patients. On the other hand, the prevalence of diabetes in patients with non-alcoholic fatty liver disease is high, which is evident in patients with non-alcoholic steatohepatitis.

[0154] Non-alcoholic fatty liver disease may include one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer.

[0155] In some embodiments, the composition is administered in an amount effective to prevent the conversion of NAFLD to NASH and to improve the pathophysiology of the disease.

[0156] A method for treating and / or preventing one or more symptoms of NAFLD or NASH typically involves administering to a subject in need thereof an effective amount of a composition for treating and / or alleviating one or more symptoms associated with NAFLD or NASH.

[0157] In some embodiments, the composition is administered in an amount effective to inhibit or reduce the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and total cholesterol (TC), fat accumulation or steatosis, inflammation, hypertrophy, fibrosis, long-term pathological conditions, and mortality.

[0158] 3. Neurological and neurodegenerative diseases The compositions and their formulations can be used to treat one or more neurological and neurodegenerative diseases. The compositions and methods are particularly suitable for treating one or more neurological or neurodegenerative diseases associated with the activation of microglia and / or astrocytes. In some embodiments, the disease or disorder is selected from, but not limited to, neuropathy (e.g., Alzheimer's disease (AD), Parkinson's disease (PD)). In one embodiment, the composition is used to treat Alzheimer's disease (AD) or Parkinson's disease.

[0159] Neurodegenerative diseases are chronic progressive disorders of the nervous system that affect nerve and behavioral functions and result in distinct histopathological and clinical syndromes (Hardy H, et al., Science. 1998;282:1075-9). Abnormal proteins that are resistant to the cell's degradation mechanisms accumulate within the cell. The pattern of neuron loss is selective in the sense that certain groups are affected while others remain intact. In many cases, there is no clear inciting event for the disease. Diseases classically described as neurodegenerative are Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0160] Neuroinflammation mediated by activated microglia and astrocytes is a major hallmark of a variety of neuropathies that makes it a potential therapeutic target. Multiple scientific reports suggest that reducing early neuroinflammation by targeting these cells can delay disease onset, which in turn can provide a longer therapeutic window for treatment (Dommergues, MA et al., Neuroscience 2003, 121, 619; Perry, VH et al., Nat Rev Neurol 2010, 6, 193; Kannan, S et al., Sci. Transl. Med. 2012, 4, 130ra46; and Block, ML et al., Nat Rev Neurosci 2007, 8, 57). Delivery of therapeutic agents across the blood-brain barrier is a difficult challenge. Neuroinflammation causes disruption of the blood-brain barrier (BBB). The impaired BBB in neuroinflammatory disorders can be utilized to transport drug-loaded nanoparticles across the brain (Stolp, HB et al., Cardiovascular Psychiatry and Neurology 2011, 2011, 10; and Ahishali, B et al., International Journal of Neuroscience 2005, 115, 151).

[0161] The compositions and methods can also be used for the treatment of neurological diseases or disorders or neurodegenerative diseases or disorders, or central nervous system disorders. In preferred embodiments, the compositions and methods are effective to treat and / or reduce neuroinflammation associated with neurological diseases or disorders or neurodegenerative diseases or disorders, or central nervous system disorders. The methods typically comprise administering to a subject an effective amount of the composition for increasing cognition or reducing cognitive decline, increasing cognitive function or reducing cognitive dysfunction, increasing memory or reducing memory decline, increasing the ability or capacity to learn or reducing the decline in the ability or capacity to learn, or combinations thereof.

[0162] Neurodegeneration refers to the progressive loss of the structure or function of neurons, including neuron death. For example, the compositions and methods can be used to treat subjects having a disease or disorder such as Parkinson's disease (PD) and PD-related disorders, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and other dementias, prion diseases such as Creutzfeldt-Jakob disease, corticobasal degeneration, frontotemporal dementia, HIV-related cognitive impairment, mild cognitive impairment, motor neuron disease (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedrich's ataxia, Lewy body disease, Alpers' disease, Batten disease, cerebro-oculo-facio-skeletal syndrome, corticobasal degeneration, Gerstmann-Straussler-Scheinker disease, Kuru, Leigh disease, monomelic amyotrophy, multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis (MS), neurodegeneration with brain iron accumulation, oculoclonus-myoclonus, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, vascular dementia, progressive multifocal leukoencephalopathy, Lewy body dementia (DLB), Rett syndrome, hydrocephalus, Wernicke-Korsakoff syndrome, post-encephalitic dementia, cancer and chemotherapy-related cognitive impairment and dementia, as well as depression-induced dementia and pseudodementia. In a preferred embodiment, the disease or disorder is Alzheimer's disease (AD) or Parkinson's disease.

[0163] Criteria for evaluating the improvement of specific neurological factors include methods for evaluating cognitive skills, motor skills, memory ability, etc., as well as methods for evaluating physical changes in selected areas of the central nervous system, such as magnetic resonance imaging (MRI) and computed tomography (CT), or other imaging methods. Such evaluation methods are well-known in the fields of medicine, neurology, psychology, etc., and can be appropriately selected for diagnosing the state of specific neurological dysfunctions. One or more tests of the selected assessment or evaluation are administered prior to the start of the administration of the composition to evaluate the changes in Alzheimer's disease or related neurological changes. After this initial evaluation, a treatment method for the administration of the composition is initiated and continued at various time intervals. At selected time intervals after the initial evaluation of the dysfunction of neurological deficits, the same evaluation or tests of the evaluation are used again to re-evaluate the changes or improvements in the selected neurological criteria.

[0164] The individual is preferably an adult human, more preferably a human older than 30 years of age, and has some amount of loss of nerve function as a result of Alzheimer's disease or dementia. Generally, nerve loss means any nerve loss at the cellular level and includes the loss of neurites, nerve organization, or neural networks.

[0165] In other embodiments, the method includes selecting a subject who may benefit from treatment with the composition.

[0166] Compositions and formulations are suitable for reducing or preventing one or more pathological processes associated with the development and progression of PD. Accordingly, provided are methods for the treatment, reduction, and prevention of pathological processes associated with PD, the methods comprising administering to an individual suffering from PD a composition in an effective amount and dosing regimen effective to reduce microglial activation, abnormal accumulation of alpha-synuclein protein, reduction of neurofibrillary changes in the brain, and / or improvement of tremors, rigidity, bradykinesia, and gait difficulties. Provided are methods for reducing, preventing, or reversing the motor dysfunction in an individual suffering from PD. The methods comprise administering to a subject in need thereof an effective amount of a composition comprising one or more persistent GLP-1r agonists. In a preferred embodiment, the method comprises administering to the subject an effective amount of a composition comprising one or more triple agonist peptides having an amino acid sequence of any one of SEQ ID NOs: 1-64, or a pharmaceutically acceptable salt thereof.

[0167] C. Dosage and Effective Amount Dosage and dosing regimen depend on the severity of the disorder and / or the method of administration and can be determined by one of ordinary skill in the art. A therapeutically effective amount of a triple agonist peptide, or a pharmaceutical formulation thereof, used in the treatment of fatty liver disease, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, and / or type 2 diabetes mellitus (T2DM) is typically sufficient to reduce or alleviate one or more symptoms associated with the disease or disorder.

[0168] Preferably, the composition does not target the activity or amount of healthy cells that are not within and not associated with the diseased or target tissue, nor otherwise modulate it, or does so at a reduced level compared to the target cells. In this way, by-products and other side effects associated with the composition are reduced.

[0169] The actual effective amount can vary according to factors including the particular agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder. In some embodiments, the dosage of the triple agonist analog, or a pharmaceutical formulation thereof, can be about 0.01 to about 100 mg / kg body weight, about 0.01 mg / kg to about 10 mg / kg, and about 0.05 mg to about 5 mg / kg body weight. In other embodiments, the dosage is the absolute amount of the triple agonist analog, or a pharmaceutical formulation thereof, for a single administration to a subject, e.g., about 0.1 mg to a maximum of about 100 mg. For example, in some embodiments, the dosage of the triple agonist analog, or a pharmaceutical formulation thereof, is 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg, or more than 10 mg, e.g., 20 mg, 30 mg, 40 mg, 50 mg, or 100 mg. In an exemplary embodiment, the dosage of the triple agonist analog is 5 mg and is administered once a week. Generally, for intravenous injection or infusion, the dosage may be lower than for oral administration.

[0170] Generally, the timing and frequency of administration are adjusted to balance the effectiveness of a given treatment schedule with the side effects of a given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple administrations, e.g., hourly, daily, weekly, monthly, or annually.

[0171] The triple agonist analog, or a pharmaceutical formulation thereof, can be administered daily, biweekly, weekly, every two weeks, monthly, or less frequently, in an amount that provides a therapeutically effective increase in the blood level of the therapeutic agent. When the administration is by a route other than the oral route, the composition may be delivered over a period longer than 1 hour, e.g., 3 to 10 hours, to produce a therapeutically effective dosage within a 24-hour period. Alternatively, the composition can be formulated for controlled release, where the composition is administered as a single dose that is repeated on a once-a-week or less frequent regimen.

[0172] The dosage can be changed and can be administered once daily or more frequently, once a day, twice a week, once a week, once every two weeks, once a month, or less frequently. Guidance can be found in the literature regarding the appropriate dosage for a given class of pharmaceutical product. The optimal dosing schedule can be calculated from measurements of drug accumulation in the body of the subject or patient. One of ordinary skill in the art can readily determine the optimal dosage, dosing methodology, and repetition rate. The optimal dosage can vary depending on the relative potency of the individual pharmaceutical composition and is generally an EC that has been found to be effective in in vitro and in vivo animal models. 50 It can be estimated based on.

[0173] In some embodiments, the composition is administered to the subject for 1 to 20 years, for example, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20 years. If necessary, the composition is administered for 10 years. In one embodiment, the effect of the treatment lasts for at least 1 year.

[0174] In a preferred embodiment, the triple agonist analog is orally administered in an amount of about 1 mg to about 100 mg, preferably about 5 mg to about 50 mg, including the amounts at both ends. In some embodiments, the triple agonist peptide or its analog is orally administered once a week, once every three days, once every two days, once a day, or twice a day.

[0175] In other preferred embodiments, the triple agonist analog is administered parenterally, such as subcutaneously, at a concentration of about 0.1 mg / mL to about 10 mg / mL, preferably about 1 mg / mL to about 5 mg / mL, including the concentrations at both ends. In some embodiments, the triple agonist analog is administered parenterally once a week, once every three days, once every two days, or once a day.

[0176] In some embodiments, the regimen includes one or more cycles of a course of therapy followed by a drug-free (e.g., no drug) period of rest. The drug-free period of rest can be 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0177] In some embodiments, the amount of the triple agonist analog administered to the subject changes over time after the starting dose. Thus, in some embodiments, the amount of the triple agonist analog administered to the subject changes over time after the starting dose.

[0178] D. Combination Therapies and Procedures The composition can be administered alone or in combination with one or more conventional therapies. Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder, or condition. The additional therapeutic, prophylactic, or diagnostic agents can have the same or different mechanisms of action. In some embodiments, the combination results in an additive effect on the treatment of the disease or condition. In some embodiments, the combination results in an effect that exceeds the additive effect on the treatment of the disease or condition.

[0179] E. Controls The therapeutic outcome of a triple agonist analog, or a pharmaceutical formulation thereof, can be compared to a control or reference. The terms "control" or "reference" refer to a standard for comparison. The terms "sample" and "subject that has changed compared to the control" are understood to have levels that are statistically different from those of a normal, untreated, or control sample. Examples of control samples include cells in culture, one or more laboratory test animals, or one or more human subjects. Methods for selecting and testing control samples are within the ability of those skilled in the art. An analyte can be a naturally occurring substance (e.g., an antibody, a protein) characteristically expressed or produced by a cell or an organ, or a substance produced by a receptor construct (e.g., β-galactosidase or luciferase). Depending on the method used for detection, the amount of change and the measured values can vary. Determination of statistical significance, e.g., the number of standard deviations from the mean that constitutes a positive result, is within the ability of those skilled in the art. Suitable controls are known in the art and include, for example, untreated subjects or cells, or the same individual prior to treatment.

[0180] V. Kit The composition can be packaged into a kit. The kit can include a composition containing one or more of the triple agonist peptides or their analogs, either as a single-dose or multiple-dose, or a pharmaceutical formulation thereof, and instructions for administering the composition. In a preferred embodiment, the triple agonist peptide or its analog has an amino acid sequence of any one of SEQ ID NOs: 1 to 130. Specifically, the instructions direct the administration of an effective amount of the composition to an individual having the specified symptom, disease, defect, or dysfunction. The composition can be formulated as described above with respect to a particular method of treatment and can be packaged in any conventional manner.

[0181] The present invention will be further understood by reference to the following non-limiting examples.

Examples

[0182] (Example 1) Screening for Tri-Agonists Using Phage Display Method Screening Using Phage Display A custom peptide library was constructed using phage display. Peptides that bind to the human GLP-1 receptor, human glucagon receptor, and human GIP receptor were identified using phage display technology. Eighteen unique peptide sequences were identified from the phage display. The library was screened against the target using the trimer codon. The trimer codon technology allows for the creation of a random region with each amino acid position randomized by a defined amino acid composition, as well as variations in the length of the region. Also, this method produces less bias. Lead peptide sequences were identified after 3 - 4 rounds of biopanning. Biopanning is an affinity selection technique for selecting peptides that bind to a given target.

[0183] cAMP Screening for GLP-1R, GCGR, GIPR To determine the activity of the tri-agonist in a CHO-K1 cell line expressing the GLP-1 receptor (GLP-1R), glucagon receptor (GCGR), and glucose-dependent insulinotropic polypeptide receptor (GIPR), the cell line was purchased from Eurofins and the HITHUNTER® cAMP assay (DiscoverX) for small molecules was used.

[0184] Cells were seeded into 96-well plates at 7×10 3 cells / well using cell plating reagent, and the cells were incubated overnight at 37 °C and 5% CO 2 . To perform the cAMP detection assay, the cell plating reagent was replaced with cAMP assay buffer. Then each well was treated with diluted peptide and incubated for 30 minutes at 37 °C in a CO 2 incubator.

[0185] After incubation, the cAMP antibody reagent and the working cAMP detection solution were added to all wells. The plate was incubated for 1 hour at room temperature in the dark. Then, cAMP solution A was added and the plate was incubated for 3 hours at room temperature in the dark. Thereafter, luminescence was measured using a microplate reader device.

[0186] Results Using phage display technology, peptides that bind to the human GLP-1 receptor, the human glucagon receptor, and the human GIP receptor were identified. Phage display technology has been previously described, for example, in U.S. Patent No. 10,493,125.

[0187] For the phage library, a library of peptides was designed based on the GLP-1, glucagon, and GIP sequences. A custom peptide library was constructed by maintaining the same hot spot residues but changing other residues as shown in FIG. 1 and in polypeptide formula II. Amino acid positions 10, 16, 17, 18, 20, 24, and 28 were randomized by trimer incorporation of 19 codons excluding cysteine. Trimer codon technology allows for the generation of random regions with each AA position randomized by a defined AA composition, as well as variations in the length of the regions. Also, this is the best method that produces the least bias. Lead peptide sequences were generated after 3 - 4 rounds of biopanning. Biopanning is an affinity selection technique that selects peptides that can bind to a given target. First round of cell panning: GLP-1R→GCGR→GIPR; and second round of cell panning: GLP-1R→GCGR→GIPR.

[0188] The backbone for the triple agonist is the following polypeptide formula II: HX 2 QGTFTSDX 10 SX 12 YLDX 16 X 17 X 18 AX20 DFVX 24 WLX 27 X 28 GGPSSGAPPPSX 40 (wherein X 2 is A or 2-aminoisobutyric acid (Aib); X 12 is K or W; X 27 is L, M, I, G or P; X 40 is absent, C, or K) having the amino acid sequence of X(10, 16, 17, 18, 20, 24, or 28) is randomized by incorporation of 19-codon trimers excluding cysteine.

[0189] The cAMP assay was performed on the first and second rounds of enriched phage pools against three target cell lines and two control cell lines. Each enriched pool was tested for its activation against all target and control cell lines. Phage from the unscreened library and helper phage M13KO7 were also used as control phage. GLP-1R+ cells were activated from the first 2-P enriched pool to the second 3-P enriched pool, with the first 3-P enriched pool showing the highest activation effect. GCGR+ cells were also activated from the first 2-P enriched pool to the second 3-P enriched pool, with the first 3-P enriched pool showing the highest activation effect. GIPR+ cells were activated from the first 3-P enriched pool to the second 3-P enriched pool, with the second 2-P enriched pool showing the highest activation effect. There was no obvious activation of the six enriched phage pools against the control cells, nor obvious activation of the control phage against all target and control cells (Figure 2).

[0190] Based on the data from the cAMP assay, a second 2-P enriched pool was selected to perform a monoclonal phage activation test using a cAMP assay kit. Validation of the monoclonal phage cAMP activity assay was performed on the second 2-P phage. Ninety clones from the second 2-P phage were tested, and 29 clones were identified as being able to specifically activate all three target cells (GLP-1R+, GCGR+ and GIPR+). After sequencing, 18 unique sequences with activity against all three receptors GLP-1R, GCGR and GIPR were identified. The amino acid sequences of the 18 unique peptides are listed in Table 4. A C-terminal cysteine was introduced into each of these 18 peptides to allow incorporation of modifications such as lipidation and / or biotinylation. The sequences are listed in Table 5.

[0191] Table 4. Peptide sequences of 18 candidate peptides

Table 4

[0192] Table 5. Peptide sequences of 18 candidate peptides with C-terminal cysteine

Table 5

[0193] The 18 individual peptides were rescreened at four different concentrations against three target cell lines, GLP-1R+ cells, GCGR+ cells, GIPR+ cells, using a cAMP assay (Figures 3A - 3C).

[0194] (Example 2) Screening for candidates suitable for modification with fatty acids Method Lipid modification (Type A) The peptide and C18-γGlu-2OEG-MAL (F12) were dissolved in a 0.3% TEA (v / v) solution containing DMSO. The respective solutions were mixed in a 1:1 volume ratio. The concentration of the peptide was 5 mg / mL and the molar ratio was 1:2 (peptide:lipid). The mixture was reacted at 25 °C for 30 minutes while gently shaking. The lipidated peptide was purified by Prep-LC and the eluate was collected into individual fractions. The ACN contained in the fractionated solution was evaporated at 45 °C for 40 minutes using a centrifugal evaporator. The solvent was changed to water by ultrafiltration. The purified sample was analyzed by reverse-phase HPLC for purity check. The sample was lyophilized at -88 °C for 18 hours and then stored at -20 °C.

[0195] Lipid and biotin modification (type B) The peptide, C18-γGlu-2OEG-MAL (F12), and biotin-N-hydroxysuccinimide ester (B1-NHS, B38) were dissolved in a 0.3% TEA (v / v) solution containing DMSO. First, the peptide and F12 were mixed in a 1:1 volume ratio. The concentration of the peptide was 5 mg / mL and the molar ratio was 1:2 (peptide:lipid). The mixture was reacted at 25 °C for 10 minutes while gently shaking. Then, B38 was added in a 1:0.2 volume ratio and the molar ratio was 1:3 (peptide:biotin). The mixture was reacted at 25 °C for 60 minutes while gently shaking. The lipidated and biotinylated peptide was purified by Prep-LC and the eluate was collected into individual fractions. The ACN contained in the fractionated solution was evaporated at 45 °C for 40 minutes using a centrifugal evaporator. The solvent was changed to water by ultrafiltration. The purified sample was analyzed by reverse-phase HPLC for purity check. The sample was lyophilized at -88 °C for 18 hours and then stored at -20 °C.

[0196] Lipid and biotin modification (type C) The peptide, biotin-maleimide (B1-MAL, B1) and C18-γGlu-2OEG-NPC (F16) were dissolved in a 0.3% TEA (v / v) solution containing DMSO. First, the peptide and B1-MAL were mixed at a volume ratio of 1:1. The concentration of the peptide was 5 mg / mL and the molar ratio was 1:2 (peptide:biotin). The mixture was reacted at 25 °C for 10 minutes with gentle shaking. Then, C18-NPC was added at a volume ratio of 1:0.2 and the molar ratio was 1:2 (peptide:lipid). The mixture was reacted at 25 °C for 90 minutes with gentle shaking. The lipidated and biotinylated peptide was purified by Prep-LC and the eluate was collected into individual fractions. The ACN contained in the fractionated solution was evaporated at 45 °C for 40 minutes using a centrifugal evaporator. The solvent was changed to water by ultrafiltration. The purified sample was analyzed by reverse-phase HPLC for purity check. The sample was lyophilized at -88 °C for 18 hours and then stored at -20 °C.

[0197] Results Based on the data from the cAMP assays of 18 individual peptides, peptide numbers 5, 12, 18, and 57 were selected for full screening. Peptide numbers 5, 12, 18, and 57 were screened using the cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, to determine their EC 50 The relative ratios of the EC 50 of each of their native ligands to that of the triple agonist peptide are shown in Tables 6 - 8. Table 6. Percentage change in EC 50 for GLP-1 at human GLP-1R [Table 6]

[0198] Table 7. Percentage change in EC 50 for GCG at human GCGR [Table 7]

[0199] Table 8. Percentage change in EC 50 for GIP in human GIPR [Table 8]

[0200] The direct use of native polypeptides as biopharmaceuticals is often limited by their very short systemic half-lives, which result from rapid metabolism, enzymatic degradation, and effective renal clearance for smaller proteins and peptides. Modifications such as biotinylation and lipidation are introduced to improve the stability, bioavailability, and in vivo absorption of these peptides. Exemplary conjugations of a biotin moiety, a fatty acid moiety, or both a biotin and a fatty acid moiety to the triple agonist peptide are shown in FIGS. 4A - 4C. For ease of reference, these modifications are referred to as Type A (lipid at Cys40), Type B (lipid at Cys40 and biotin at Lys12), and Type C (lipid at Lys12 and biotin at Cys40).

[0201] Subsequently, the peptides of Table 5 were modified with lipid molecules conjugated via cysteine added to the respective C-terminus of the peptide (i.e., the Type A modification shown in FIG. 4A). Lipidated versions (Type A modification) of these peptides were also synthesized, resulting in peptide numbers 3A, 5A, 12A, 18A, 19A, 25A, 28A, 32A, 44A, 57A, 58A, 60A, 62A, 67A, 68A, 70A, 76A, and 77A. These lipidated peptides were also screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells (FIGS. 5A - 5C).

[0202] The lipidated peptides 5A, 12A, 18A, and 32A were screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, to determine their EC 50 values. The relative ratios of the EC 50 of each of their native ligands to that of the triple agonist peptide are shown below:

[0203] Table 9. Percentage change in EC 50 for GLP-1 at the human GLP-1R [Table 9]

[0204] Table 10. Percentage change in EC 50 for GCG at the human GCGR [Table 10]

[0205] Table 11. Percentage change in EC 50 for GIP at the human GIPR [Table 11]

[0206] Based on the cAMP assay results from peptides (5, 12, 18, 57) and lipidated peptides (5A, 12A, 18A, 32A), peptides number 5 and 12 were selected for further study. Glucagon and GIP activities were reduced by lipidation.

[0207] Peptide number 5 was further studied using all three types of modifications, types A, B, and C (Figures 4A - 4C). The modified peptides 5A, 5B, and 5C were screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, to determine their EC 50was determined. The EC of peptide number 5 and its modified forms, as well as their respective native ligands (STD) 50 are summarized in Table 12. The EC of each triple agonist peptide 50 for its respective native ligand (STD peptide) 50 is summarized in Table 13. The relative ratio of the EC of each triple agonist peptide 50 to the EC of the GLP-1 peptide 50 is summarized in Table 14. The data suggest that lipidation at Cys40 reduced glucagon and GIP activities. Type C (lipidation at Lys12) showed better activity than type B.

[0208] Table 12. EC of peptide number 5 and its modified forms 50

Table 12

[0209] Table 13. Ratio of EC of STD peptide to triple agonist peptide 50

Table 13

[0210] Table 14. Ratio of EC of GLP-1 peptide to triple agonist peptide 50

Table 14

[0211] (Example 3) In vivo efficacy study on weight loss in normal mice Method ​​To confirm the therapeutic effect of the peptide in vivo, the polypeptide was administered to mice and the change in body weight was measured. First, normal C57BL / 6 mice were administered the polypeptide subcutaneously at doses of 20 nmol / kg or 30 nmol / kg on days 0, 1, and 2 (once a day, QD) or days 0 and 3 (every other day, Q2D), respectively. The peptide was delivered with 0.02% polysorbate 80 (PS80) in PBS, and the dosing volume was 10 mL / kg. Body weight was monitored QD at 6 hours. The detailed treatment doses and regimens are summarized in Table 15.

[0212] The following amino acid sequence having a C-terminal cysteine that is pegylated (PEG MW 50 kDa): HAibQGT FTSDY SKYLD EQAAK EFVQW LMNTC (SEQ ID NO: 132) The DD01 peptide having the sequence is used as a positive control. Semaglutide, a GLP-1 receptor agonist manufactured by Novo Nordisk, is also used as a positive control.

[0213] Table 15. Treatment doses and regimens.

Table 15

[0214] Results The change in body weight was measured in all treatment groups (Figures 6A and 6B). Excellent body weight loss (BWL) was observed in the daily dosing of 5A. 5A showed similar BWL at a lower dose than DD01. 12A showed efficacy similar to semaglutide.

[0215] (Example 4) Peptide Design and Screening for Increased GIP Activity Methods Peptide numbers 5 and 12 were used for further peptide design to increase activity against GIPR. Peptide numbers 5 and 12, and their derivatives are listed in Table 16. A C-terminal cysteine was introduced into each of these peptides to allow incorporation of modifications such as lipidation and / or biotinylation. Lipidated peptides via conjugation to the C-terminal cysteine were also synthesized. These are peptide numbers 5-1A (SEQ ID NO: 50), 5-2A (SEQ ID NO: 51), 5-3A (SEQ ID NO: 52), 5-4A (SEQ ID NO: 53), 5-5A (SEQ ID NO: 54), 5-6A (SEQ ID NO: 55), 12-1A (SEQ ID NO: 56), 12-2A (SEQ ID NO: 57), 12-3A (SEQ ID NO: 58), 12-4A (SEQ ID NO: 59), 12-5A (SEQ ID NO: 60), 12-6A (SEQ ID NO: 61), and 12-7A (SEQ ID NO: 62).

[0216] Table 16. Peptide sequences derived from peptide numbers 5 and 12

Table 16

[0217] Results Peptide numbers 5-1, 5-2, 5-3, 5-4, 5-5, 5-6, 12-1, 12-2, 12-3, 12-4, 12-5, 12-6, and 12-7 were screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells (Figures 7A - 7C).

[0218] Lipidated versions (type A modification) of these peptides were also synthesized, yielding peptide numbers 5-1A, 5-2A, 5-3A, 5-4A, 5-5A, 5-6A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A, 12-6A, and 12-7A (SEQ ID NOs: 50 - 62). These lipidated peptides were also screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells (Figures 8A - 8C and 9A - 9C).

[0219] Peptide numbers 5A, 5-1A, 5-2A, and 5-5A were further screened using a cAMP assay in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, to determine their EC 50 which was determined. The EC 50 values of peptide numbers 5A, 5-1A, 5-2A, 5-5A, and their respective native ligands (STD) are summarized in Table 17. The EC 50 values of each triple agonist peptide relative to the EC 50 of its respective native ligand (STD peptide) are summarized in Table 18. The EC 50 values of each triple agonist peptide relative to the EC 50 of the GLP-1 peptide are summarized in Table 19.

[0220] Table 17. EC values of peptide numbers 5A, 5-1A, 5-2A, 5-5A 50

Table 17

[0221] Table 18. Ratio of EC of STD peptide to triple agonist peptide 50

Table 18

[0222] Table 19. Ratio of EC of GLP-1 peptide to triple agonist peptide 50

Table 19

[0223] (Example 5) In vivo efficacy study on weight loss in normal mice Method ​​To confirm the therapeutic effect of the peptides in obesity, diabetes, or NASH, the polypeptide was administered to mice to measure changes in food intake, blood glucose, and body weight. First, normal C57BL / 6 mice were administered the polypeptide subcutaneously at doses of 20 nmol / kg or 30 nmol / kg on days 0, 1, and 2 (once a day, QD) or days 0 and 3 (every other day, Q2D), respectively. In addition, an NASH animal model was prepared by feeding normal C57BL / 6 mice (approximately 6 weeks old) a high-fat, high-fructose, high-cholesterol diet for approximately 21 weeks to increase the body weight of the mice to approximately 50 g on average. Subsequently, the polypeptide was administered by subcutaneous injection once every other day for 2 weeks at a dose of 20 nmol / kg. Body weight and food intake were measured once every other day at a given time point. After a 2-week treatment period, the mice were sacrificed, and then liver weight, liver triglyceride, and NASH-related biomarkers were measured.

[0224] Peptides 5A, 5-1A, 5-2A, 5-3A, 5-4A, and 5-5A were administered at 20 nmol / kg, s.c., QD as shown in Table 20. DD01 administered at 40 nmol / kg, s.c., Q2D as a positive control was delivered with 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. Body weight was monitored QD for 4 days.

[0225] Table 20. Treatment doses and regimens.

Table 20

[0226] Peptides 5A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A, and 12-7A were administered at 20 nmol / kg, s.c., QD as summarized in Table 21. The peptides were delivered with 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. Food intake, blood glucose, and body weight changes were monitored QD.

[0227] Table 21. Treatment doses and regimens.

Table 21

[0228] Results The therapeutic effects of the peptides were evaluated in mice. Body weights were monitored over a 4-day period in mice treated with peptides 5A, 5-1A, 5-2A, 5-3A, 5-4A, and 5-5A (Figures 10A - 10B). Excellent weight loss effects were observed in the 5A and 5-1A treatment groups. Based on the body weight measurements, the treatment efficacy was most pronounced in the groups treated with peptides 5A and 5-1A, followed by 5-2A and 5-5A, then 5-4A, and the lowest effect was observed in 5-3A.

[0229] Food intake, blood glucose, and body weight changes were monitored over a 3-day period in mice treated with peptides 5A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A, and 12-7A. (Figures 11A - 11D).

[0230] (Example 6) Peptide Screening for Improved Sites for Lipidation Method Peptides 5, 5-1, 5-2, 5-3, 5-4, and 5-5 were synthesized with lipidation at new positions to further improve the activity of these peptides when lipidated. Three additional sequences were also synthesized as peptide numbers 5-6D, 5-7D, and 5-3F. The sequences with lipidation at the indicated residues (K * ) without lipidation at Cys40 are in Table 22. The fatty acid derivative used for conjugation here is 2OEG-γGlu-C18. The preferred sites and residues for lipidating these peptides are summarized in Table 23.

[0231] Table 22. Peptide Sequences

Table 22

[0232] Table 23. Residues and Positions Suitable for Modification [Table 23]

[0233] Results Secondary peptide screening was designed to screen for peptides with increased activity against GIPR using peptide numbers 5 and 12 as lead sequences without a decrease in their glucagon and GLP-1 activities. This was achieved at the peptide level, although it was lipidated at the C-terminus. Therefore, tertiary screening was designed to maintain the potency of its peptide derivatives based on peptide number 5 from secondary screening but vary the positions for lipidation for improved activity.

[0234] Peptide numbers 5A, 5-1A, 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E, and 5-1F were screened using the cAMP assay at five different concentrations of 0.3 nM, 1 nM, 10 nM, 100 nM, and 300 nM, or 1000 nM, in three target cell lines, GLP-1R+ cells, GCGR+ cells, GIPR+ cells (Figures 12A - 12C). The relative ratio of the EC 50 of each of the peptides used in the cAMP assay to the EC 50 of its respective native ligand is summarized in Table 24. The relative ratio of the EC 50 of each of the peptides used in the cAMP assay to the EC 50 of the GLP-1 peptide is summarized in Table 25. The EC 50 of each of its respective non-lipidated peptides to the EC 50The relative ratios are summarized in Table 26. Based on the cAMP assay, the activity of these peptides was maintained against GLP-1R when lipidated with K10, K12, K17, or C40, the activity was maintained against GCGR when lipidated with K17, and the activity was most decreased when lipidated with K12.

[0235] Table 24. EC of native ligands against the triple agonist peptide 50 Ratio

Table 24

[0236] Table 25. EC of GLP-1 against the triple agonist peptide 50 Ratio

Table 25

[0237] Table 26. EC of the candidate triple agonist peptide against its respective non-lipidated form 50 Ratio

Table 26

[0238] (Example 7) In vivo efficacy study on weight loss in normal mice Method Peptide numbers 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E, and 5-1F were administered at 20 nmol / kg, s.c., QD as shown in Table 27. The peptides were delivered with 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. Food intake, blood glucose, and body weight changes were monitored QD.

[0239] The following amino acid sequences: Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH 2 ) 18 -CO 2 H)AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH2 (SEQ ID NO: 133) EL.EX.14, a triple agonist from published PCT application WO2019 / 125938 having the above sequence, was used as a control.

[0240] Table 27. Treatment dosages and regimens. [Table 27]

[0241] Results Food intake, blood glucose, and body weight changes were monitored over a 4-day period in mice treated with peptides 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E, and 5-1F (Figures 13A - 13D).

[0242] The peptides showed a significant body weight loss effect.

[0243] Table 28. Body weight loss after 48 or 72 hours in normal mice [Table 28]

[0244] (Example 8) In vivo efficacy study in the mAMLN mouse model Methods Peptides 5-1A, 5D, 5-2D, 5-1D, 5-2D, DD01, and EL.EX.14 were administered at 20 nmol / kg, s.c., Q2D for 2 weeks as shown in Table 29. The peptides were delivered with 0.02% PS80 in PBS (DD01 only: F1). Food intake, body weight change, blood glucose (0 h, 4 h, 1 day, Q6D, end); serum chemistry (end); liver TG (end); inflammatory markers (end) were monitored Q2D. Animals were fasted for at least 4 h prior to blood glucose measurement. The control group received vehicle without peptide.

[0245] Table 29. Treatment doses and regimens.

Table 29

[0246] Results Non-alcoholic steatohepatitis (NASH) is a obesity-related liver disease with a significantly unmet medical need. Various dietary obesity animal models of NASH have been used in preclinical studies, target discovery, and drug development. A high trans-fat-containing amylin liver NASH (AMLN) diet high in fat, fructose, and cholesterol has been widely used in ob / ob mice and C57BL / 6J mice to reliably induce metabolic and liver histopathological changes that reproduce the prominent features of NASH.

[0247] Changes in body weight and blood glucose in AMLN mice treated with peptides 5-1A, 5D, 5-2D, 5-1D, and 5-3D, as well as the positive controls DD01 and EL.EX.14, over a 2-week period are shown in Figures 14A - 14C. All tested peptides 5-1A, 5D, 5-2D, 5-1D, and 5-3D were able to reduce body weight and blood glucose in NASH mice.

[0248] The liver weight and triglyceride content in the liver at the end of the treatment were determined and shown in FIGS. 14D - 14E. All the tested peptides 5 - 1A, 5D, 5 - 2D, 5 - 1D, and 5 - 3D were able to reduce the liver weight and reduce the liver triglyceride content.

[0249] In addition, the expression levels of the inflammatory marker (TGF - β1) and the fibrosis marker (ACTA2, a - SMA) measured by mRNA levels indicate that the tested peptides 5 - 1A, 5D, 5 - 2D, 5 - 1D, and 5 - 3D were able to reduce both TGF - β1 and ACTA2, as shown in FIGS. 14F - 14G.

[0250] (Example 9) Peptide Screening for Improved Sites for Biotinylation Method The direct use of native polypeptides as biopharmaceuticals is often limited by their very short systemic half - life resulting from rapid metabolism, enzymatic degradation, and effective renal clearance for smaller proteins and peptides. Modifications such as biotinylation and lipidation are introduced to improve the stability, bioavailability, and in vivo absorption of these peptides. Peptides derived from SEQ ID NOs: 66 and 77 were synthesized with substitutions at several amino acids and biotinylation at five different positions to enhance the stability and oral absorption of these peptides. The sequences with lipidation at the indicated lysine residue (K * ) and biotinylation at the indicated lysine residue (B * ) or cysteine residue (B # ) and substitution of methionine with methoxynine (m * ) are in Table 30. This modification with methoxynine is to enhance the stability against oxidation. The fatty acid derivative used for conjugation here is OEG2 - γGlu - C18. The biotin derivative used for conjugation here is the biotin monomer.

[0251] An in vitro activity study was conducted to determine the biotinylation site by confirming the change in activity for each receptor according to the biotin conjugation position. The peptides were tested in vitro for activity using a cAMP assay for GLP-1R, GCGR, and GIPR as previously described. Their signals from cAMP production were compared with the native ligand peptides (GLP-1, GCG, or GIP).

[0252] Table 30. Peptide sequences with optimized biotinylation sites.

Table 30

[0253] Results SEQ ID NOs: 77, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and 96 were screened using a cAMP assay at five different concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells (Figures 15A - 15C). SEQ ID NOs: 66, 97, and 98 were screened using a cAMP assay at five different concentrations of 0.1 nM, 0.3 nM, 1 nM, 10 nM, and 100 nM in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells (Figures 16A - 16C).

[0254] SEQ ID NOs: 84, 85, 86, 87, 88, 89, 90, 91, 92, 95, and 96 were selected for full screening and screened using a cAMP assay in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells to determine their EC 50 The relative ratios of the EC 50 of each native ligand and GLP-1 to the triple agonist peptide are shown in Table 31.

[0255] As a result of comparing its activity by biotinylation of SEQ ID NO: 77 at five different positions, a biotinylation site that can minimize the loss of peptide activity was determined. The biotinylation site was confirmed again using SEQ ID NO: 66, which is an additional peptide. Based on these results in the cAMP assay, activity is maintained against three target receptors when biotinylated at K16, K17, K24, or K40.

[0256] Table 31. Ratio of native ligand and GLP-1 EC 50 to the triple agonist peptide. [Table 31]

[0257] (Example 10) In vivo efficacy study on weight loss in HFD mice Method This study was conducted to confirm the weight loss effect and select peptides to be evaluated in high-fat diet (HFD)-treated mice, which are widely used as experimental animal models in obesity and diabetes research. SEQ ID NOs: 86, 87, 89, and 90 were administered at 20 nmol / kg, s.c., Q2D for 2 weeks as shown in Table 32. The peptides were delivered with 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. Body weight change, blood glucose, and food intake were monitored Q2D. Animals were fasted for at least 4 hours before blood glucose measurement.

[0258] Table 32. Treatment doses and regimens. [Table 32]

[0259] Results Body weight change, blood glucose, and food intake were monitored over a 2-week period in HFD mice treated with SEQ ID NOs: 77, 86, 87, 89, and 90 (Figures 17A and 17B).

[0260] The weight loss of SEQ ID NOs: 86, 87, 89, and 90 biotinylated at different sites on SEQ ID NO. 77 was compared with that of non-biotinylated SEQ ID NO. 77. SEQ ID NOs: 86 and 87 were inferior in the weight loss effect, and SEQ ID NOs: 89 and 90 were equivalent to SEQ ID NO. 77.

[0261] (Example 11) In vitro stability study against FaSSIF / P Method This study was conducted to determine the enzymatic stability of SEQ ID NOs: 77, 86, 87, 89, 90, and 91 after biotinylation in an artificial intestinal environment. The test samples were prepared by mixing a stock solution of the peptide and pancreatin dissolved in FaSSIF solution (pH 6.5) at a weight ratio of 25:1. The test samples were incubated at 37 °C for 120 minutes. Sampling from the test samples was carried out at predetermined time points (10, 30, 60, and 120 minutes) and stopped with 10% TFA solution.

[0262] Using 0.1% TFA and acetonitrile in water, analytical HPLC was performed at 35 °C on an ACQUITY Premier system (Waters, USA) equipped with an ACQUITY CSH C18 column (Waters, USA) to determine the remaining amount. The remaining amount was determined as the percentage of the peak area at the sampling time point to the peak area of the initial sample.

[0263] Results The enzymatic stability of SEQ ID NOs: 86, 87, 89, 90, and 91 was determined in an artificial intestinal environment compared with that of SEQ ID NO. 77. The half-lives determined by the remaining amount in FaSSIF / P are summarized in Table 33. As a result of this study, SEQ ID NOs: 89 and 90 showed intestinal stability not inferior to that of SEQ ID NO. 77 (Figure 18).

[0264] Table 33. In vitro stability study of SEQ ID NOs: 77, 86, 87, 89, 90, and 91 against FaSSIF / P.

Table 33

[0265] (Example 12) In vivo efficacy study in CDA-HFD mice Method

[0266] This study was conducted to evaluate the efficacy of SEQ ID NOs: 77, 87, 89, 90, 95, and 96 and to determine the effects of biotinylation and amino acid substitution on efficacy in choline-deficient L-amino acid-defined high-fat diet (CDA-HFD)-treated mice, which are widely used as experimental animal models in NAFLD / NASH research. SEQ ID NOs: 77, 87, 89, 90, 95, and 96 were administered at 20 nmol / kg, s.c., Q2D for 4 weeks as shown in Table 34. The peptides were delivered with 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group received a vehicle without peptide.

[0267] Body weight changes were monitored Q2D. After a 4-week treatment period, CDA-HFD mice were sacrificed and then serum chemical analysis was performed at Genia (Seongnam, Gyeonggi, Korea) using a HITACHI 7180 (Hitachi High-Tech Korea, Seongnam, Gyeonggi, Korea). ALT and AST for hepatocytes and several lipid markers (LDL and TG) in serum were analyzed.

[0268] Table 34. Treatment doses and regimens.

Table 34

[0269] Results

[0270] The weight changes of CDA-HFD mice treated with SEQ ID NOs: 77, 87, 89, 90, 95, and 96 over a 4-week period are shown in FIG. 19A. All tested peptides were able to reduce weight loss in the NASH mouse model.

[0271] At the end of the treatment, ALT, AST, and LDL for hepatocytes, as well as several lipid markers in serum, were analyzed and shown in FIGS. 19B-19D. All tested peptides showed an improvement in the serum lipid profile.

[0272] (Example 13) Peptide Screening for Improved Stability by Substitution of the 20th Amino Acid and Optimization of the Lipid Structure Method It is necessary to optimize the peptide sequence for oral delivery studies by changing amino acids vulnerable to intestinal enzymes. In addition, an extended plasma half-life helps reduce variability in steady-state systemic exposure when administered orally.

[0273] Peptides derived from SEQ ID NO: 77 were synthesized with substitution of arginine, the 20th amino acid for increased intestinal stability, and optimization of the lipid structure for extended in vivo half-life. The indicated lysine residues (K * , K ** or K *** ), lipidation at the indicated lysine residues (B * ), biotinylation at the indicated lysine residues, and sequences with α-methyl-arginine (αR) are shown in Table 35. Here, the fatty acid derivatives used for conjugation were 2OEG-γGlu-C18 (K * ), 2OEG-γGlu-C20 (K ** ) or OEG-γGlu-C20 (K *** ). Here, the biotin derivative used for conjugation was the biotin monomer.

[0274] In vitro activity studies were performed to determine amino acid substitutions and lipid structures by confirming activity changes for each receptor. Peptides were tested in vitro for activity using cAMP assays against GLP-1R, GCGR, and GIPR as previously described. Their signals from cAMP production were compared to native ligand peptides (GLP-1, GCG, or GIP). Table 35. Peptide sequences [Table 35]

[0275] Results Sequences 99, 100, 101, 102, 103, 104, and 105 were screened using cAMP assays at five different concentrations in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, and compared to sequences 77, 89, and 90 (Figures 20A - 20C).

[0276] Full screening of sequences 99, 100, and 101 in the cAMP assay was performed in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells to determine their EC 50 and compared to sequence 77. The relative ratios of the EC 50 of each native ligand to that of the triple agonist peptide are shown in Table 36.

[0277] Sequences 99, 100, and 101 were synthesized with substitution of the 20th amino acid from sequence 77 to improve stability in the intestinal environment. When compared to sequence 77, the in vitro activities of sequences 99, 100, and 101 were changed by the amino acid substitution. The activity against GLP-1R slightly decreased in sequence 99, and the activities of sequence 100 against both GLP-1R and GIPR decreased compared to sequence 77. In the case of sequence 101, the activities against GLP-1R and GIPR increased.

[0278] Table 36. In vitro activities of SEQ ID NOs: 77, 99, 100, and 101. [Table 36]

[0279] (Example 14) Pharmacokinetics study in rats Method This study was conducted to evaluate the PK profiles of SEQ ID NOs: 77, 102, and 103 and compare the PK profiles of different lipidated structures of the peptides in rat plasma following IV administration. SEQ ID NOs: 77, 102, and 103 were administered at 100 nmol / kg, i.v., as shown in Table 37. The peptides were delivered in 0.02% PS80 in PBS, and the dosing volume was 2.5 mL / kg.

[0280] The animals were not fasted prior to dosing. 0.2 - 0.4 mL of whole blood was collected from the vein at each time point (0.167, 0.5, 1, 2, 4, 6, 8, 24, and 48 hours after dosing) and transferred to heparin-coated tubes. 0.1 - 0.2 mL of the resulting plasma obtained by centrifugation of the blood samples was transferred to tubes and stored at -70°C until analysis. The concentration of the peptides in plasma was determined by LC-MS / MS analysis.

[0281] Table 37. Treatment doses and regimens. [Table 37]

[0282] Results The PK parameters presented after single IV administration of SEQ ID NOs: 77, 102, and 103 are shown in Table 38.

[0283] According to the mean values of the PK parameters, SEQ ID NOs: 102 and 103 showed similar PK parameter values despite the difference in lipid structure, and SEQ ID NO: 77 had a half-life and AUC compared to SEQ ID NOs: 102 and 103.inf showed significantly lower PK parameter values in terms of dosage. Table 38. Average PK parameters of SEQ ID NOs: 77, 102, and 103 in rats after single IV administration [Table 38]

[0284] Each value represents the mean ± SD (CV%) of the provided replicates. * , PK parameters calculated in four individual subjects. Different letters (a and b) indicate statistical differences (p < 0.05) in the results of the PK parameters.

[0285] (Example 15) In vitro stability study against FaSSIF / P and trypsin Methods This study was conducted to determine the enzyme stability of SEQ ID NOs: 77, 99, 100, and 101 after substitution of the 20th amino acid in an artificial intestinal environment.

[0286] For stability against FaSSIF / P, the test sample was prepared by mixing the peptide stock solution and pancreatin dissolved in FaSSIF solution (pH 6.5) at a weight ratio of 19:1. The test sample was incubated at 37 °C for 120 minutes. Sampling from the test sample was carried out at predetermined time points (10, 30, 60, and 120 minutes) and stopped with 10% TFA solution.

[0287] For stability against trypsin, the test sample was prepared by mixing the peptide stock solution and trypsin dissolved in 50 mM PBS (pH 7.8) containing 0.02% PS80 at a weight ratio of 10:1. The test sample was incubated at 37 °C for 180 minutes. Sampling from the test sample was carried out at predetermined time points (10, 30, 60, 90, 120, and 180 minutes) and stopped with 10% TFA solution.

[0288] Using 0.1% TFA and acetonitrile in water, analytical HPLC was performed at 35 °C on an ACQUITY Premier system (Waters, USA) equipped with an ACQUITY CSH C18 column (Waters, USA) to determine the residual amount. The residual amount was determined as the percentage of the peak area at the time of sample collection relative to the peak area of the initial sample.

[0289] Results The enzyme stabilities of SEQ ID NOs: 99, 100, and 101 were determined in an artificial intestinal environment in comparison to SEQ ID NO: 77. The half-lives determined by the residual amounts in FaSSIF / P and trypsin are summarized in Table 39. As a result of this study, SEQ ID NOs: 99 and 100 showed similar stability to FaSSIF / P and increased half-lives to trypsin, in comparison to SEQ ID NO: 77. SEQ ID NO: 101 showed improved stability to both FaSSIF / P and trypsin, in comparison to SEQ ID NO: 77 (Figures 21A and 21B).

[0290] Table 39. In vitro stability studies of SEQ ID NOs: 77, 99, 100, and 101 against FaSSIF / P and trypsin.

Table 39

[0291] (Example 16) In vivo efficacy study in CDA-HFD mice Methods This study was conducted to evaluate the efficacy of SEQ ID NOs: 77, 99, 100, 101, 102, and 103 and to determine the effects of amino acid substitutions and lipid structures on efficacy in CDA-HFD mice (NASH model). SEQ ID NOs: 77, 99, 100, 101, 102, and 103 were administered at 20 nmol / kg, s.c., Q2D for 4 weeks, as shown in Table 40. The peptides were delivered with 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group received a vehicle without peptide.

[0292] Weight change; serum chemistry (final); liver TG (final); and histological analysis (final) were monitored by Q2D.

[0293] Table 40. Treatment dosages and regimens.

Table 40

[0294] Results The weight changes of CDA-HFD mice treated with SEQ ID NOs: 77, 99, 100, 101, 102, and 103 over a 4-week period are shown in Figure 22A. SEQ ID NOs: 101, 102, and 103 showed reduced body weight compared to the vehicle-treated control in the NASH mouse model. In particular, a greater weight loss was observed in SEQ ID NO: 101.

[0295] At the end of treatment, ALT and AST for hepatocytes, several lipid markers (LDL and TG) in serum, and histological scores were analyzed and shown in Figures 22B - 22F. SEQ ID NOs: 101, 102, and 103 showed significant reduction of liver enzymes and hepatic steatosis compared to the vehicle-treated control. The hepatic steatosis score was significantly reduced in SEQ ID NOs: 101 and 103 compared to the CDA-HFD control. The scores for hepatocyte inflammation and NAS score were statistically reduced in SEQ ID NOs: 101, 102, and 103. SEQ ID NO: 101 had the most reduced NAS score.

[0296] Among the groups tested, SEQ ID NO: 101 had the best efficacy in the NASH model. The lipid structure of SEQ ID NO: 103 had better efficacy compared to SEQ ID NO: 77.

[0297] (Example 17) In vivo efficacy study in mAMLN mice Methods This study was conducted to evaluate the efficacy of SEQ ID NOs: 77, 89, 97, 104, and 101 for confirmation of the effects of biotinylation, amino acid substitution, and altered lipid structure on efficacy in mAMLN mice (obesity and NASH models). Tirzepatide, a GLP-1 and GIP dual agonist manufactured by Eli Lilly, was used as a positive control. SEQ ID NOs: 77, 89, 97, 104, 101, and tirzepatide were administered at 20 nmol / kg, s.c., Q2D for 4 weeks, as shown in Table 41. The peptides were delivered with 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. The control group was administered a solution without peptide.

[0298] Body weight change at Q2D; liver weight (final); visceral fat weight (final); serum chemistry (final); liver TG (final); inflammatory markers (final); and histological analysis (final) were monitored.

[0299] Table 41. Treatment doses and regimens.

Table 41

[0300] Results Body weight change, liver weight, visceral fat weight, ALT, AST, LDL, liver TG, and histological scores in mAMLN mice treated with SEQ ID NOs: 77, 87, 97, 104, and 101, and tirzepatide as a positive control over a 4-week period are shown in Figures 23A - 23H.

[0301] After 4 weeks of administration, SEQ ID NOs: 97 and 101, and tirzepatide showed a body weight loss similar to that of the CHOW control group. In addition, SEQ ID NOs: 97 and 101 significantly reduced liver weight, visceral fat weight, liver enzymes, and hepatic steatosis compared to the vehicle-treated control, and most of these parameters were reduced to levels similar to those of the positive control, tirzepatide.

[0302] In histological scores, hepatocyte hypertrophy significantly decreased in all treatment groups, and hepatic steatosis significantly reduced in SEQ ID NO: 89, 97, 104, and 101 treatment groups. Overall, the NAS scores for SEQ ID NO: 97 and 101 were greater than those of the tirzepatide levels and were statistically decreased.

[0303] (Example 18) Peptide screening for sequence optimization Method Based on the results of previous studies, SEQ ID NO: 66 and 101 were selected for further optimization of the peptide sequences. Peptides derived from SEQ ID NO: 66 and 101 were synthesized by substituting the 20th amino acid with αR to improve intestinal stability. Lipidation at the indicated lysine residue (K * or K ** ), biotinylation at the indicated lysine residue (B * ) and sequences with α-methyl-arginine (αR) are shown in Table 42. Here, the fatty acid derivatives used for conjugation were 2OEG-γGlu-C18 (K * ) or 2OEG-γGlu-C20 (K ** ). Here, the biotin derivative used for conjugation was the biotin monomer.

[0304] In vitro activity studies were performed to determine the optimal sequences by confirming the activity for each receptor. Peptides were tested in vitro for activity using cAMP assays for GLP-1R, GCGR, and GIPR as previously described. Their signals from cAMP production were compared with those of the native ligand peptides (GLP-1, GCG, or GIP). Table 42. Peptide sequences

Table 42

[0305] Results Sequences 106, 107, 108, 109, 110, 111, and 112 were screened at six different concentrations in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, using a cAMP assay and compared to Sequences 66, 97, and 101 and tildesepatide (Figure 24A - 24C).

[0306] Full screening of Sequences 107, 108, 110, and 112 in the cAMP assay was performed in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells to determine their EC 50 The relative ratios of the EC 50 of each native ligand to that of the triple agonist peptide are shown in Table 43.

[0307] Table 43. In vitro activities of Sequences 107, 108, 110, and 112.

Table 43

[0308] (Example 19) In Vivo Efficacy Study in CDA-HFD Mice Methods This study was conducted to evaluate the efficacy of Sequences 97, 110, 108, 111, and 112 in CDA-HFD treated mice and to determine the effect of lipid structure and biotinylation on efficacy. Sequences 97, 110, 108, 111, and 112 were administered at 20 nmol / kg, s.c., Q2D for 4 weeks, as shown in Table 44, and compared to tildesepatide. The peptides were delivered in 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group received vehicle without peptide.

[0309] Body weight change at Q2D; liver weight (final); serum chemistry (final) and liver TG (final) were monitored.

[0310] Table 44. Treatment doses and regimens.

Table 44

[0311] Results The body weight changes, liver weights, ALT, AST, LDL, and liver TG in CDA-HFD mice treated with peptides 97, 108, 110, 111, and 112 over a 4-week period and with tildepazotide as a positive control are shown in Figures 25A - 25G.

[0312] After 4 weeks of administration, all treated peptides and tildepazotide showed similar weight loss, liver weight loss, and improvement of liver-related markers.

[0313] (Example 20) Intestinal Absorption after Oral Administration in Rats Method This study was conducted to evaluate the intestinal absorption of peptides after enteral administration of oral pharmaceutical formulations. The pharmaceutical formulations were administered to rats in the form of a suspension by intraduodenal (ID) injection. Sequences 87, 89, and 90 were accurately weighed and dissolved in 10 mM PBS containing polysorbate 80 (pH 7.4, vehicle I). The mixture was vortexed for 5 minutes to obtain a clear solution. Then, 2.5 mL of the vehicle was added to the mixture and vortexed for 30 minutes to obtain a homogeneous opaque suspension. The composition of vehicle II was sodium chenodeoxycholate and propyl gallate in polysorbate 80 in PBS. The animals were not fasted prior to dosing. 0.2 - 0.4 mL of whole blood was collected from the vein at each time point (0, 0.167, 0.5, 1, 2, 4, 8, 24, and 48 hours after IV and ID administration). The collected whole blood was incubated at room temperature for 20 minutes, and the tubes were centrifuged at 13,000 rpm for 10 minutes at 4°C. 0.1 - 0.2 mL of the obtained serum was transferred to tubes and stored at -70°C until analysis. The concentration of the peptide in the serum was determined by LC-MS / MS analysis. The PK parameters were analyzed by non-compartmental analysis using Phoenix WinNonlin 5.0.1 software (Pharsight Corporation, Mountain View, CA, USA).

[0314] Table 45. Treatment doses and regimens. [Table 45]

[0315] Results The PK parameters presented after single intraduodenal administration of sequences 87, 89, and 90 are shown in Table 46. Intestinal absorption was confirmed after enteral administration of the oral pharmaceutical formulations.

[0316] According to the mean values of the PK parameters, after ID administration of sequences 87, 89, and 90 in rats, mean C maxThe values were 289.53 ng / mL, 331.73 ng / mL, and 281.58 ng / mL, but C max reached them at 0.25 hours, 0.43 hours, and 0.30 hours, respectively. After administration of the ID of SEQ ID NO: 87, 89, and 90, respectively, the t1 / 2 were 6.17, 7.24, and 8.37, and the AUCinf values were 1,861.06 ng·h / mL, 2,793.64 ng·h / mL, and 2,488.01 ng·h / mL, respectively.

[0317] Table 46. Mean PK parameters of SEQ ID NO: 87, 89, and 90 after single duodenal administration in rats.

Table 46

[0318] Each value represents the mean ± SD of the replicates provided.

[0319] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications and the materials cited therein are specifically incorporated by reference.

[0320] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A triple agonist peptide or analog thereof having activity for each of the following: the glucagon-like peptide 1 (GLP-1) receptor, the glucagon receptor, and the glucose-dependent insulin secretion-stimulating polypeptide (GIP) receptor, wherein the triple agonist peptide or analog thereof is X １ X ２ X ３ GTFTSDX １０ SX １２ X １３ LDX １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ X ２３ X ２４ X ２５ X ２６ X ２７ X ２８ G X ３０ X ３１ SX ３３ X ３４ X ３５ PP X ３８ X ３９ X ４０ (SEQ ID NO: 131) The formula includes the amino acid sequence, where X １ is either H or Y; X ２ is A or 2-aminoisobutyric acid (Aib); X ３ is Q or E; X 10 is any natural amino acid other than cysteine; X １２ is R, W, or K; X １３ is either L or Y; X 16 is any natural amino acid other than cysteine; X 17 is any natural amino acid other than cysteine, or a non-natural amino acid selected from the group consisting of methoxynine, 2-aminoisobutyric acid, and alpha-methylarginine; X 18 is any natural amino acid other than cysteine, or a non-natural amino acid selected from the group consisting of methoxynine, 2-aminoisobutyric acid, and alpha-methylarginine; X １９ is Q, A, or T; X20 is any natural amino acid other than cysteine, or a non-natural amino acid selected from the group consisting of methoxynine, 2-aminoisobutyric acid, and alpha-methylarginine; X ２１ is either D or L; X ２２ is either F or R; X ２３ is V, G, or D; X 24 is any natural amino acid other than cysteine; X ２５ is W, Y, or A; X ２６ is L or D; X ２７ is I, L, M, G, or P; X 28 is any natural amino acid other than cysteine; X ３０ is G or P; X ３１ is either P or S; X ３３ is either S or G; X ３４ is G or A; X ３５ is either A or P; X ３８ is either P or S; X ３９ is non-existent, S, or C; X ４０ is non-existent, C, or K; With optional amide modification of the C-terminus, Triple agonist peptide or its analogue.

2. X １０ However, it is Y, W, K, F, H, S, L, A, E, M, Q, or D; X １６ However, it is Y, Q, G, K, S, R, F, P, or A; X １７ However, it is M, Y, Q, K, S, W, P, D, A, F, or methoxynine; X １８ However, it is A, I, M, W, T, D, Y, or methoxynine; X ２０ However, it is R, Q, H, G, A, P, N, K, Aib, or alpha-methylarginine; X ２４ However, it is Q, D, K, L, N, W, or M; X ２８ However, it is N, E, G, D, H, or Q. The triple agonist peptide or its analog according to claim 1.

3. A triple agonist peptide or analog thereof according to claim 1 or 2, having one amino acid sequence of any one of SEQ ID NOs: 1 to 130.

4. The triple agonist peptide or analog thereof according to claim 1, wherein one or more of the biotin moiety, fatty acids, or polyethylene glycol and their derivatives are conjugated via one or more spacers as needed.

5. The triple agonist peptide or analog thereof according to claim 4, wherein one or more biotin moieties, fatty acids, or polyethylene glycol, and derivatives thereof are conjugated to one amino acid sequence of any one of SEQ ID NOs: 1 to 130 via one or more amino acid residues selected from the group consisting of cysteine ​​and lysine.

6. The triple agonist peptide or analog thereof according to claim 5, comprising one or more amino acid residues selected from cysteine ​​and lysine, which are introduced into any one amino acid sequence of SEQ ID NOs. 1 to 130 by substitution or insertion, wherein one or more biotin moieties, fatty acids, or polyethylene glycol, and derivatives thereof are conjugated to the peptide or analog thereof at the one or more introduced amino acid residues.

7. comprising one or more amino acid residues introduced into any one of the amino acid sequences of SEQ ID NOs: 1 to 130 by substitution or insertion, The substitution or insertion comprises one or more C-terminal cysteine ​​residues, including lysine at position 10, lysine at position 12, lysine at position 17, lysine at position 20, lysine at position 24, One or more of the biotin moieties, fatty acids, or polyethylene glycol, and their derivatives, are conjugated to the peptide or analog at one or more introduced amino acid residues. The triple agonist peptide or its analog according to claim 3.

8. The biotin moiety and its derivatives are biotin N-hydroxysuccinimide ester, N-biotinoyl-N'-(6-maleimidohexanoyl)hydrazide, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-CONH ２ , 3-maleimidepropionate-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH ２ , propionate-N-hydroxysuccinimide ester-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH ２ and 3-maleimidepropionate-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH ２ A triple agonist peptide or analog thereof according to claim 4, selected from the group consisting of the following.

9. The triple agonist peptide or analog thereof according to claim 4, wherein the fatty acids and their derivatives are C16-C22 fatty acids conjugated via one or more hydrophilic spacers.

10. The triple agonist peptide or analog thereof according to claim 9, wherein the hydrophilic spacer is γGlu or 8-amino-3,6-dioxaoctanoic acid.

11. The fatty acid or derivative thereof is C16-NHS, C16-MAL, C18-NHS, C18-MAL, C16-γGlu-NHS, C16-γGlu-MAL, C18-γGlu-NHS, C18-γGlu-MAL, C18-γGlu-NHS, C18-γGlu-OEG-MAL, C18-γGlu-2OEG-NHS, C A triple agonist peptide or analog thereof according to claim 4, selected from the group consisting of 18-γGlu-2OEG-MAL, C20-γGlu-2OEG-NHS, C20-γGlu-2OEG-MAL, C18-γGlu-2OEG-TFP, C18-γGlu-2OEG-NPC, and C20-γGlu-2OEG-NPC.

12. A pharmaceutical formulation comprising the triple agonist peptide or its analog according to claim 1.

13. A pharmaceutical preparation according to claim 12 for treating one or more diseases selected from the group consisting of obesity, diabetes mellitus, and non-alcoholic fatty liver disease, in a subject requiring treatment for one or more of the said diseases, A pharmaceutical preparation characterized in that it is administered to a target and treats or alleviates one or more symptoms of one or more of the one or more diseases.

14. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered in an amount effective in inducing weight loss, reducing body fat, reducing food intake, improving glucose homeostasis, or a combination thereof, to normal or obese subjects.

15. The pharmaceutical preparation according to claim 13, wherein the subject is suffering from non-alcoholic fatty liver disease.

16. The pharmaceutical preparation according to claim 15, wherein the non-alcoholic fatty liver disease is one or more diseases selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, cirrhosis, and liver cancer.

17. The pharmaceutical formulation according to claim 15, characterized in that the pharmaceutical formulation is administered in an amount effective in inhibiting one or more of alanine aminotransferase, aspartate aminotransferase, triglycerides, gamma-glutamyltransferase, total cholesterol, low-density lipoprotein, fasting blood glucose, or a combination thereof, or in a manner that reduces their serum levels.

18. The pharmaceutical preparation according to claim 15, characterized in that the pharmaceutical preparation is administered in an amount effective in reducing one or more of the following: steatosis, inflammation, hypertrophy, fibrosis, cirrhosis, or a combination thereof.

19. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered via a route selected from the group consisting of enteral administration and parenteral administration.

20. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered by oral administration or subcutaneous administration.

21. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered in a form selected from the group consisting of pills, capsules, tablets, liquids, and suspensions.

22. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered at intervals selected from the group consisting of once a month, once every two weeks, once a week, once every three days, once every two days, once a day, or twice a day.

23. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered to the subject once a week for a maximum of six months.

24. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered to the subject for a period of 1 to 10 days, 1 to 10 weeks, or 1 to 10 months, including the periods at both ends.

25. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered to the subject in a dose ranging from 0.001 mg per kg of body weight to 10 mg per kg of body weight of the subject, including the doses at both ends.

26. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered to the subject in a dose ranging from 0.01 mg per kg of body weight to 1 mg per kg of body weight of the subject, including the doses at both ends.

27. The pharmaceutical preparation according to claim 13, characterized in that the pharmaceutical preparation is administered to the subject in a dose of 1.0 mg to 100 mg, including the doses at both ends.

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