Peptide compositions and methods of use thereof

Abstract

A triple agonist peptide having activity on each of a GLP-1, a glucagon, and a GIP receptor is provided. Also described are triple agonist analogs having one or more biotin moieties and / or fatty acid moieties conjugated thereto to improve bioavailability and pharmacokinetics. Compositions and formulations of these triple agonist peptides are particularly suitable for treating, ameliorating and / or preventing one or more metabolic diseases, such as obesity, diabetes or non-alcoholic fatty liver disease. Compositions and methods of use thereof for the treatment, amelioration and / or prevention of one or more neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to USSN 63 / 346,605, filed May 27, 2022, which is incorporated herein by reference in its entirety.

[0003] References to sequence listings

[0004] The sequence listing submitted as a text file named “DDP_107_PCT_ST26.xml” created on March 25, 2023 and 287,384 bytes in size is hereby incorporated by reference pursuant to 37 CFR §1.834(c)(1). Technical Field

[0005] The present invention is generally in the field of peptides and analogs thereof having activity at all of the glucagon, GLP-1 and GIP receptors, and uses thereof. Background Art

[0006] In recent years, with economic development and medical progress, the elderly population has grown rapidly. Aging increases the risk of chronic diseases such as dementia, heart disease, type 2 diabetes, arthritis, and cancer. As complications associated with obesity, heart disease and cerebrovascular disease rank first and second in mortality. Obesity is considered to be the cause of various adult diseases such as diabetes and non-alcoholic fatty liver disease.

[0007] Obesity refers to a state in which the amount of fat accumulated is higher than the normal amount and the most accurate method for assessing obesity is to measure the body fat mass. However, accurately measuring the fat mass is expensive and therefore indirect methods are used to assess it. The most commonly used indirect methods are to measure the body mass index (BMI) and waist circumference. The World Health Organization (WHO) announced a classification based on normal weight based on data that correlates BMI with the risk of death: 18.5 to 24.9 kg / m 2 , Overweight: 25 to 29.9 kg / m 2 , and obesity: 30kg / m 2 or more.

[0008] The cause of obesity is known to be an energy imbalance due to excessive calorie intake and relatively low activity, and results in an increase in body fat. However, it is difficult to specify only one factor because obesity involves various risk factors such as eating habits, lifestyle, age, ethnicity, genetic factors, etc. Obese patients are mainly advised to control their weight through a healthier diet and physical activity, but when these methods are ineffective, patients can be treated with drugs or surgery. These usually provide limited efficacy.

[0009] Diabetes is divided into insulin-dependent diabetes (type I diabetes), insulin-independent diabetes (type II diabetes) and malnutrition-related 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 reduced insulin secretion of pancreatic beta cells or increased insulin resistance of peripheral tissues due to genetic factors, metabolic factors and environmental factors. In this regard, when body fat increases, insulin sensitivity decreases. The accumulation of abdominal fat is known to be particularly related to glucose intolerance. Moreover, it is known that insulin resistance is closely related to obesity in patients with type II diabetes, wherein the more severe the obesity, the greater the insulin resistance.

[0010] Non-alcoholic fatty liver disease (NAFLD) refers to a spectrum of diseases including simple steatosis with excessive fat accumulation in liver cells independent of alcohol consumption, non-alcoholic steatohepatitis (NASH) including liver cell damage (hepatocellular ballooning), inflammation, fibrosis, and more advanced cases, cirrhosis. The prevalence of non-alcoholic fatty liver disease is rapidly increasing with the prevalence of obesity around the world, and although the prevalence of diabetes varies from country to country, diabetes accounts for about 20% to 30% of the total population in Western countries and its incidence in Korea reaches about 16%.

[0011] GLP-1 is a hormone secreted by the small intestine under the stimulation of food intake. GLP-1 promotes the secretion of insulin in the pancreas in a blood sugar-dependent manner and inhibits the secretion of glucagon, thereby contributing to the effect of lowering blood sugar levels. In addition, GLP-1 slows down digestion in the gastrointestinal tract by acting as a satiety factor, and reduces food intake by delaying the time for emptying digested food in the gastrointestinal tract. It is reported that the administration of GLP-1 to rats has the effect of inhibiting food intake and reducing body weight, and it is confirmed that these effects occur in the normal state and the obese state, thus showing the potential of GLP-1 as a medicament for treating obesity.

[0012] GIP is one of the gastrointestinal hormones secreted in response to food intake, and in the case of GLP-1, it is a hormone composed of 42 amino acids secreted by intestinal K cells. It is reported that GIP performs the function of promoting insulin secretion in the pancreas in a blood sugar-dependent manner and helps lower blood sugar levels, thereby showing effects such as increasing the activation of GLP-1 and anti-inflammatory effects.

[0013] When blood sugar levels drop due to reasons such as drugs, diseases, hormones or enzyme deficiency, glucagon is produced in the pancreas. Glucagon signals the breakdown of glycogen in the liver to induce the release of glucose and raise blood sugar levels to normal levels. In addition to the effect of increasing blood sugar levels, glucagon suppresses appetite in animals and humans and activates hormone-sensitive lipase in fat cells to promote fat breakdown and energy expenditure, thereby showing anti-obesity effects.

[0014] Based on the effects of GLP-1 in controlling blood sugar levels and reducing body weight, GLP-1 is being developed as a therapeutic agent for treating diabetes and obesity. Exendin-4, which is prepared from lizard venom and has about 50% amino acid homology with GLP-1, is being developed as a therapeutic agent for treating similar diseases. However, it is reported that therapeutic agents containing GLP-1 and exendin-4 show side effects such as vomiting and nausea (Syed Y Y., Drugs, 2015 Jul;75(10):1141-52).

[0015] In order to maximize weight loss and as an alternative to GLP-1 based therapeutics, research has been devoted to dual agonists that bind to both the GLP-1 receptor and the glucagon receptor. Due to activation of the glucagon receptor, these have been shown to be more effective in weight loss than when GLP-1 is used alone (Jonathan W et al., Nat Chem Bio., 2009 Oct (5); 749-757).

[0016] In the research involving triple agonists that bind to GLP-1, GIP and glucagon receptors simultaneously, efforts have been made to increase resistance to dipeptidyl peptidase IV (DPP-IV) that decomposes gastrointestinal hormones to eliminate their activity by replacing the amino acid sequence, and then adding acyl groups to specific regions thereof to increase the half-life of the triple agonists (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 no triple agonist showed different activity ratios with the three different types of receptors.

[0017] It is therefore 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, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and / or obesity.

[0018] 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 sugar levels and reduce body weight without causing side effects.

[0019] It is a further object of the present invention to provide compositions and methods that can provide effective glucose control with the benefit of weight loss and a favorable side effect profile.

[0020] It is a further object of the present invention to provide therapeutic agents with a prolonged duration of action. Summary of the invention

[0021] Triple agonist peptides are provided that are active at each of the GLP-1, glucagon, and GIP receptors. Typically, these triple agonist peptides have the following polypeptide formula I

[0022] X1X2X3GTFTSDX 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

[0023] X 30 X 31 SX 33 X 34 X 35 PP X 38 X 39 X 40 (SEQ ID NO: 131),

[0024] Where X1 is H or Y;

[0025] X2 is A or 2-aminoisobutyric acid (Aib);

[0026] X3 is Q or E;

[0027] X 12 is R, W or K;

[0028] X 13 is L or Y;

[0029] X 19 It is Q, A or T;

[0030] X 21 is D or L;

[0031] X 22 is F or R;

[0032] X 23 is V, G or D;

[0033] X 25 is W, Y or A;

[0034] X 26 is L or D;

[0035] X 27 is I, L, M, G or P;

[0036] X 30 is G or P;

[0037] X 31 is P or S;

[0038] X 33 is S or G;

[0039] X 34 is G or A;

[0040] X 35 is A or P;

[0041] X 38 is P or S;

[0042] X 39 Does not exist, is S or C;

[0043] X 40 Does not exist, it is C or K;

[0044] The C-terminus has an optional amide modification, and

[0045] X 10 , X 16 , X 17 , X 18 , X 20 , X 24 and X 28 X is independently any of the 20 amino acids except cysteine. 17 , X 18 , X 20 is an unnatural amino acid. In some embodiments, wherein X 17 , X 18 , X 20 is an unnatural amino acid, X 17 , X 18 , X 20 is independently one of the following: methoxythiobutyric acid, 2-aminoisobutyric acid, and α-methylarginine.

[0046] In other embodiments:

[0047] X 10 is Y, W, K, F, H, S, L, A, E, M, Q or D;

[0048] X 16 is Y, Q, G, K, S, R, F, P or A;

[0049] X 17 is M, Y, Q, K, S, W, P, D, A, F or methoxythiothreitol;

[0050] X 18 is A, I, M, W, T, D, Y or methoxythiothreitol;

[0051] X 20 is R, Q, H, G, A, P, N, K, Aib, or α-methylarginine;

[0052] X 24 is Q, D, K, L, N, W or M; and

[0053] X 28 It is N, E, G, D, H or Q.

[0054] In preferred embodiments, the triple agonist peptides have the amino acid sequence of any one of SEQ ID NOs: 1-130.

[0055] 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.

[0056] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 134: YAibQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 135).

[0057] Also described are triple agonist analogs having one or more biotin moieties and / or fatty acid moieties conjugated thereto to improve 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 the 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 the amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to allow conjugation with the one or more biotin moieties and / or one or more fatty acids or derivatives thereof. In a preferred embodiment, one or more amino acid residues of lysine at position 10, lysine at position 12, lysine at position 17, one or more C-terminal cysteine ​​residues are introduced into the amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to allow conjugation with the one or more biotin moieties and / or one or more fatty acids or derivatives thereof. Exemplary biotin moieties suitable for conjugation are N-biotinyl-N'-(6-maleimidocaproyl)hydrazide, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-CONH2, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2, propionic acid-N-hydroxysuccinimide ester-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2, and 3-maleimidopropionate-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2.

[0058] Exemplary fatty acids suitable for conjugation, optionally via one or more hydrophilic spacers such as γGlu or 8-amino-3,6-dioxaoctanoic acid, are C16-C22 fatty acids. In some embodiments, these fatty acids or derivatives thereof 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 of the triple agonist peptides or analogs thereof and methods of use thereof are also described.

[0059] Provided is a method for treating one or more diseases of a subject in need thereof, the one or more diseases being selected from the group consisting of: obesity, diabetes, and non-alcoholic fatty liver disease. These methods include administering a pharmaceutical formulation of an effective amount of a triple agonist peptide or its analog 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 that can effectively induce weight loss, reduce body fat, reduce food intake, improve glucose homeostasis, or a combination thereof in normal or obese patients. In certain embodiments, the subject suffers from 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 that can effectively inhibit or reduce the serum level of one or more of the following: alanine aminotransferase, aspartate aminotransferase, triglycerides, gamma-glutamyl transferase, total cholesterol, low-density lipoprotein, fasting blood glucose, or a combination thereof. In preferred embodiments, the pharmaceutical formulation is administered in an amount effective to reduce one or more of steatosis, inflammation, ballooning, fibrosis, cirrhosis, or a combination thereof in a subject suffering from NAFLD.

[0060] Typically, the drug formulation is administered by enteral administration and parenteral administration (e.g., oral administration or subcutaneous administration). In certain embodiments, the drug formulation is administered in the form of pills, capsules, tablets, liquids and suspensions. In certain embodiments, the drug formulation is administered at the following intervals: 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 drug formulation is administered to a subject once a week, for up to 6 months or for a duration of one day to 10 days, one week to 10 weeks, one month to 10 months or one year to 10 years, including end values. In certain embodiments, the drug formulation is administered to a human subject with the following dosage: 0.001mg / kg of the subject's body weight to 10mg / kg of the subject's body weight, including end values. In a preferred embodiment, the drug formulation is administered to a human subject with the following dosage: 0.01mg / kg of the subject's body weight to 1mg / kg of the subject's body weight, including end values. In further embodiments, the pharmaceutical formulation is administered to a human subject at a dose of 1.0 mg to 100 mg, inclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the amino acid sequence used for peptide design of phage display library.

[0062] Figure 2is a bar graph showing luminescence in three target cell lines GLP-1R+ cells, GCGR+ cells, GIPR+ cells and two control cell lines (cells HEK293 and CHO cells) using cAMP activity assay in the presence of round 1 and round 2 enriched phage pools (1-1-P enriched pool, 1-2-P enriched pool, 1-3-P enriched pool, 2-1-P enriched pool, 2-2-P enriched pool and 2-3-P enriched pool). Unscreened library phage and helper phage M13KO7 were also used as control phage. A PBS control was also included.

[0063] Figures 3A-3C Figure 2 shows the expression of 18 unique peptides with C-terminal cysteine ​​identified from phage display in three target cell lines GLP-1R+ cells ( Figure 3A )、GCGR+ cells( Figure 3B ) and GIPR+ cells ( Figure 3C ) in the glowing bar graph.

[0064] Figures 4A-4C Schematic representation of biotin moiety, fatty acid moiety, or biotin and fatty acid moiety conjugated to triple agonist peptides. These modifications are referred to as type A (lipid at Cys40, Figure 4A ), type B (lipid at Cys40 and biotin at Lys12, Figure 4B ) and type C (lipid at Lys12 and biotin at Cys40, Figure 4C ).

[0065] Figures 5A-5C Figure 2 shows the results of the phage display assay using 18 unique peptides lipidated at the C-terminal cysteine ​​at four different concentrations of 1 nM, 10 nM, 100 nM and 300 nM in three target cell lines, GLP-1R+ cells ( Figure 5A )、GCGR+ cells( Figure 5B ) and GIPR+ cells ( Figure 5C ) in the glowing bar graph.

[0066] Figure 6A-6B Figure 2 shows the weight change in grams of mice over a 4-day period ( Fig. 6A ) or as a percentage of starting body weight ( Figure 6B ), where these mice were treated with: vehicle, peptides 5A, 12A administered subcutaneously at 20 nmol / kg once a day or 30 nmol / kg subcutaneously every other day, peptide DD01 (administered subcutaneously at 40 nmol / kg every other day), and Semaglutide (30 nmol / kg, administered subcutaneously every other day) as a positive control.

[0067] Figures 7A-7C Figure 3 shows the GLP-1R+ cells ( 0.3 nM, 1 nM, 10 nM, 100 nM and 1000 nM) after incubation with peptides 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 and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) at five different concentrations. Fig. 7A )、GCGR+ cells( Figure 7B ) and GIPR+ cells ( Figure 7C ) is a bar graph showing the percentage (%) of cAMP release in the cells. Fig. 7A The relative cAMP release of GLP-1 at 10 nM is shown as the percentage (%) of cAMP release, and the EC of GLP-1 50 The value is 0.227nM. Figure 7B The relative cAMP release of glucagon at 100 nM is shown as the percentage (%) of cAMP release and the EC of glucagon at 100 nM is shown as 50 The value is 1.271nM. Figure 7C The relative cAMP release of GIP at 33 nM is shown as the percentage (%) of cAMP release, and the EC of GIP is 50 The value is 1.784nM.

[0068] Figures 8A-8C Figure 5 shows the activity of peptides 5-1A, 5-2A, 5-3A, 5-4A, 5-5A and 5-6A and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) in three target cell lines GLP-1R+ cells ( Fig. 8A )、GCGR+ cells( Figure 8B ) and GIPR+ cells ( Figure 8C EC values ​​of GLP-1, glucagon, and GIP in GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The values ​​are 0.09 nM, 0.99 nM and 0.24 nM respectively.

[0069] Figures 9A-9C Figure 1 shows the activity of peptides 12-1A, 12-2A, 12-3A, 12-4A, 12-5A, 12-6A and 12-7A and their corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) in three target cell lines GLP-1R+ cells ( Fig. 9A )、GCGR+ cells( Fig. 9B ) and GIPR+ cells ( Fig. 9C EC values ​​of GLP-1, glucagon, and GIP in GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The values ​​are 0.09 nM, 0.99 nM and 0.24 nM respectively.

[0070] Figures 10A-10B Figure 2 shows the weight change in grams of mice over a 4-day period ( Fig. 10A ) or as a percentage of starting body weight ( Fig. 10B ), wherein these mice were treated with the following: peptides 5A, 5-1A, 5-2A, 5-3A, 5-4A and 5-5A were administered subcutaneously once a day at 20 nmol / kg, and peptide DD01 (administered subcutaneously at 40 nmol / kg every other day) as a positive control.

[0071] Figures 11A-11D Figure 2 shows the weight change in grams of mice over a 3-day period ( Fig.11A ) or as a percentage of starting body weight ( Fig. 11B ), Blood glucose % change ( Fig. 11C ) and changes in food intake ( Fig.11D ), wherein these mice were treated with peptides 5A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A and 12-7A administered subcutaneously once daily at 20 nmol / kg.

[0072] Figures 12A-12C Figure 3 shows the GLP-1R+ cells ( 0.3 nM, 1 nM, 10 nM, 100 nM and 300 nM or 1000 nM) after incubation with peptides 5A, 5-1A, 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E and 5-1F and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control). Fig. 12A )、GCGR+ cells( Fig. 12B ) and GIPR+ cells ( Fig. 12C ) Bar graph of cAMP release in .

[0073] Figures 13A-13D Figure 2 shows the weight change in grams of mice over a 4-day period ( Fig.13A ) or as a percentage of starting body weight ( Fig. 13B ), Blood glucose % change ( Fig. 13C ) and changes in food intake ( Fig.13D), wherein these mice were treated with peptides 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E and 5-1F administered subcutaneously once daily at 20 nmol / kg.

[0074] Figures 14A-14G Figure 2 shows the weight change in grams of mAMLN mice over a 2-week period ( Fig.14A ) or as a percentage of starting body weight ( Fig. 14B ), Blood glucose % change ( Fig. 14C )、liver weight( Fig.14D ), triglyceride content ( Fig.14E ) and TGF-β1 mRNA levels ( Fig.14F ) and the mRNA levels of ACTA2 ( Figure 14G ), where these mice were treated with peptides 5-1A, 5D, 5-2D, 5-1D and 5-3D and positive controls DD01 and EL.EX.14. Food and vehicle controls are also included. Mean SEM, *p relative to G1, # p relative to G2. (One-way ANOVA, Tukey's multiple comparison test).

[0075] Figures 15A-15C Figure 3 shows the GLP-1R+ cells ( ) after incubation with SEQ ID NO: 77, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 and 96 and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) at five different concentrations ranging from 0.01 nM to 100 nM. Fig.15A )、GCGR+ cells( Fig. 15B ) and GIPR+ cells ( Fig. 15C ) Bar graph of cAMP release in .

[0076] Figures 16A-16C Figure 2 shows the GLP-1R+ cells (Figure 3) after incubation with SEQ ID NO: 66, 97 and 98 and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) at five different concentrations ranging from 0.01 nM to 100 nM. Fig.16A )、GCGR+ cells( Fig. 16B ) and GIPR+ cells ( Fig. 16C ) Bar graph of cAMP release in .

[0077] Figures 17A-17C Figure 2 shows the weight change in grams of HFD mice over a 2-week period ( Fig.17A ), Blood glucose % change ( Fig. 17B ), cumulative food intake from baseline in the subsequent 2 weeks ( Fig. 17C ), wherein these mice were treated with SEQ ID NO: 77, 86, 87, 89 and 90. Vehicle controls are also included. Mean SEM, # p relative to G1. One-way ANOVA, Dunnett's multiple comparison test.

[0078] Fig.18 is a graph showing the half-life determined by the remaining amount of SEQ ID NOs: 77, 86, 87, 89, 90 and 91 for FaSSIF / P.

[0079] Figures 19A-19D Figure 2 shows the weight change in grams of CDA-HFD mice over a 4-week period ( Fig.19A ), ALT level changes ( Fig.19B ), AST level changes ( Fig.19C ) and LDL level changes ( Fig.19D ), where these mice were treated with SEQ ID NO: 77, 87, 89, 90, 95 and 96. Food and vehicle controls are also included. Mean SEM, *p vs. G2. One-way ANOVA, Dunnett's multiple comparison test.

[0080] Figures 20A-20C Figure 2 shows the GLP-1R+ cells (Figure 2 ) after incubation with SEQ ID NO: 77, 89, 90, 99, 100, 101, 102, 103, 104 and 105 and the corresponding natural ligands (GLP-1, glucagon and GIP) (as positive control) at five different concentrations ranging from 0.01 nM to 100 nM. Fig. 20A )、GCGR+ cells( Fig. 20B ) and GIPR+ cells ( Fig. 20C ) Bar graph of cAMP release in .

[0081] Figures 21A-21B is a graph showing the performance of FaSSIF / P ( Fig.21A ) and trypsin ( Fig. 21B ) A graph showing the half-lives determined by the remaining amounts of SEQ ID NOs: 77, 99, 100 and 101.

[0082] Figures 22A-22F Figure 2 shows the weight change in grams of CDA-HFD mice over a 4-week period ( Fig.22A ), including ALT( Fig. 22B )、AST( Fig. 22C ) and LDL( Fig.22D ) including serum parameter levels, triglyceride content ( Fig.22E ) and NAFLD activity score (NAS) ( Fig.22F ), where these mice were treated with SEQ ID NO: 77, 99, 100, 101, 102 and 103. Food and vehicle controls are also included. Mean SEM, *p vs. G2. One-way ANOVA, Dunnett's multiple comparison test; ％ p, G7 relative to G8, $ p, G3 relative to G7, & p, G3 versus G8, unpaired two-tailed Student's test.

[0083] Figures 23A-23H Figure 2 shows the weight change in grams of mAMLN mice over a 4-week period ( Fig.23A ), liver weight and liver-to-body weight ratio ( Fig. 23B ), visceral fat weight and visceral fat to body weight ratio ( Fig.23C ), including ALT( Fig.23D )、AST( Fig.23E ) and LDL( Fig.23F ) including serum parameter levels, triglyceride content ( Figure 23G ) and histological scores for steatosis, lobular inflammation, and ballooning ( Fig.23H ), these mice were treated with SEQ ID NO: 77, 89, 97, 104 and 101 and tirzepatide as a positive control. Food and vehicle controls were also included. Mean SEM, *p relative to G2, #p relative to G8, one-way ANOVA, Dunnett's multiple comparison test.

[0084] Figures 24A-24C Figure 2 shows the GLP-1R+ cells ( ) after incubation with SEQ ID NO: 66, 97, 101, 106, 107, 108, 109, 110, 111 and 112 and the corresponding natural ligands (GLP-1, glucagon and GIP) and tesiparatide (as a positive control) at six different concentrations from 0.001 nM to 100 nM. Fig.24A )、GCGR+ cells( Fig. 24B ) and GIPR+ cells ( Fig.24C ) in Figure 2.

[0085] Figures 25A-25G Figure 2 shows the weight change in grams of CDA-HFD mice over a 4-week period ( Fig.25A ), liver weight and liver-to-body weight ratio ( Fig.25B ), including ALT( Fig.25C )、AST( Fig.25D ) and LDL)( Fig.25E) including serum parameter levels, triglyceride content ( Fig.25F ) and NAFLD activity score ( Figure 25G ), where these mice were treated with SEQ ID NO: 97, 110, 108, 111 and 112 and tesiparatide as a positive control. Food and vehicle controls were also included. Mean SEM, *p relative to G2, $ p relative to G8, one-way ANOVA, Dunnett's multiple comparison test. DETAILED DESCRIPTION

[0086] I. Definitions

[0087] 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 "prophylactic agent" generally refers to an agent that can be administered to prevent a disease or to prevent certain conditions.

[0088] As used herein, the term "pharmaceutically acceptable salt" refers to derivatives of the compounds defined herein, wherein the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or 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 conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example, formed from non-toxic inorganic or organic acids. Such conventional nontoxic salts include those derived from such inorganic acids as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, naphthalenesulfonic, methanesulfonic, ethanedisulfonic, oxalic and isethionate.

[0089] The phrases "pharmaceutically acceptable" or "biocompatible" refer to those compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions or vehicles such as liquid or solid fillers, diluents, solvents or encapsulating materials involved in carrying or transporting 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.

[0090] The term "therapeutically effective amount" refers to the amount of a therapeutic agent that produces some desired effects at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount can vary according to such factors 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 determine the effective amount of a particular compound empirically without having to conduct excessive experiments. In certain embodiments, the term "effective amount" refers to the amount of a preventive or therapeutic agent used to reduce or mitigate the risk of suffering from liver disease / disorders or to reduce or mitigate one or more symptoms of liver disease / disorders (such as reducing inflammation in the liver). Other desired results also include reducing and / or inhibiting serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG) and total cholesterol (TC), fat accumulation or fatty degeneration, inflammation, ballooning, fibrosis, long-term morbidity and mortality. The effective amount can be administered once or multiple times.

[0091] The term "inhibit" or "reduce" in the context of inhibition refers to reducing or lowering activity and quantity. This can be a complete inhibition or reduction of activity or quantity, or a partial inhibition or reduction. The 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 long-acting GLP-1r agonist can inhibit or reduce the activity and / or quantity of activated microglia by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95% or 99% compared to the activity and / or quantity of the same cells in an equivalent tissue of a subject who has not received a long-acting GLP-1r agonist or has not been treated with it. In some embodiments, inhibition and reduction are compared at the mRNA, protein, cell, tissue and organ levels.

[0092] The terms "treating" or "treatment" refer to improving, alleviating, or reducing one or more symptoms of a disease, disorder, and / or condition in a person who may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed as having the disease, disorder, and / or condition; reducing disease symptoms, inhibiting the disease, disorder, and / or condition, such as hindering its progression; and alleviating the disease, disorder, or condition, such as causing the disease, disorder, and / or condition to regress. Treating a disease or condition includes ameliorating at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected, such as treating pain in a subject by administering an analgesic, even if such an agent does not treat the cause of the pain. Desirable therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a liver disease / disorder are alleviated or eliminated, including, but not limited to, reduction and / or inhibition of elevations in transaminases including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), reduction of cancer cell proliferation in the case of liver cancer, improvement of the quality of life of patients suffering from the disease, reduction of the dose of other drugs required to treat the disease, slowing of the progression of the disease, and / or prolonging the survival of the individual. The term "improvement" refers to a reduction, inhibition, attenuation, alleviation, cessation, or stabilization of the development or progression of a disease.

[0093] The terms "prevent", "prevention", and "preventing" mean administering a composition or method to a subject or system at risk of or susceptible to one or more symptoms caused by a disease or condition to reduce the likelihood that the subject will develop one or more symptoms of the disease or condition, or to reduce the severity, duration, or onset of one or more symptoms of the disease or condition.

[0094] The term "biodegradable" generally refers to a material that will degrade or corrode under physiological conditions into smaller units or chemicals that can be metabolized, eliminated, or excreted by a subject. Degradation time is a function of composition and morphology.

[0095] The term "protein" or "polypeptide" or "peptide" refers to any chain of more than two natural or non-natural amino acids, regardless of the post-translational modifications (e.g., glycosylation or phosphorylation) that constitute all or part of the naturally occurring or non-naturally occurring polypeptide or peptide.

[0096] The terms "biotinylation" or "biotinylated" refer to the process and product of covalent attachment of one or more biotin moieties or derivatives thereof to molecules and macrostructures, such as therapeutic proteins.

[0097] The terms "lipidation" and "lipidated" refer to the process and products of covalent attachment of one or more fatty acid moieties or their derivatives to molecules and macrostructures, such as therapeutic proteins.

[0098] The term "PEGylation" refers to the process of covalent and non-covalent attachment or fusion of polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as drugs, therapeutic proteins or vesicles.

[0099] Use of the term "about" is intended to describe values ​​that are within a range of approximately + / - 10% above or below the stated value; in other embodiments, these values ​​may range within a range of approximately + / - 5% above or below the stated value.

[0100] II. Composition

[0101] Compositions are provided that include isolated peptides active at the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insulinotropic polypeptide (GIP) receptor.

[0102] A. Triple Agonist Peptides

[0103] In some embodiments, the triple agonist peptide has the following amino acid sequence of polypeptide formula (I):

[0104] X1X2X3GTFTSDX 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

[0105] X 30 X 31 SX 33 X 34 X 35 PP X 38 X 39 X 40 (SEQ ID NO: 131),

[0106] Where X1 is H or Y;

[0107] X2 is A or 2-aminoisobutyric acid (Aib);

[0108] X3 is Q or E;

[0109] X 12 is R, W or K;

[0110] X 13 is L or Y;

[0111] X 19 It is Q, A or T;

[0112] X 21 is D or L;

[0113] X 22 is F or R;

[0114] X 23 is V, G or D;

[0115] X 25 is W, Y or A;

[0116] X 26 is L or D;

[0117] X 27 is I, L, M, G or P;

[0118] X 30 is G or P;

[0119] X 31 is P or S;

[0120] X 33 is S or G;

[0121] X 34 is G or A;

[0122] X 35 is A or P;

[0123] X 38 is P or S;

[0124] X 39 Does not exist, is S or C;

[0125] X 40 Does not exist, it is C or K;

[0126] The C-terminus has an optional amide modification, and

[0127] X 10 , X 16 , X 17 , X 18 , X 20 , X 24 and X 28, any of the 20 amino acids except cysteine. In some embodiments, X 17 , X 18 , X 20 is an unnatural amino acid. In some embodiments, wherein X 17 , X 18 , X 20 is an unnatural amino acid, X 17 , X 18 , X 20 is independently one of the following: methoxythiobutyric acid, 2-aminoisobutyric acid, and α-methylarginine.

[0128] In other embodiments,

[0129] X 10 is Y, W, K, F, H, S, L, A, E, M, Q or D;

[0130] X 16 is Y, Q, G, K, S, R, F, P or A;

[0131] X 17 is M, Y, Q, K, S, W, P, D, A, F or methoxythiothreitol;

[0132] X 18 is A, I, M, W, T, D, Y or methoxythiothreitol;

[0133] X 20 is R, Q, H, G, A, P, N, K, Aib, or α-methylarginine;

[0134] X 24 is Q, D, K, L, N, W or M; and

[0135] X 28 It is N, E, G, D, H or Q.

[0136] 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.

[0137] In one embodiment, the triple agonist peptide does not have the amino acid sequence of SEQ ID NO: 135: YAibQGTFTSDYSKLLDYMMQRDFVQWLLEGGPSSGAPPPSK (SEQ ID NO: 135).

[0138] In some embodiments, the polypeptide of Formula (I) has the amino acid sequence of any one of SEQ ID NOs: 1-130.

[0139] 1. Modification of triple agonist peptides

[0140] The direct use of natural peptides as biopharmaceuticals is often limited by their very short systemic half-life (caused by rapid metabolism, enzymatic degradation, and efficient renal clearance of smaller proteins and peptides). Modifications to exenatide, such as semaglutide, liraglutide, and NLY01, extend the half-life and pharmacokinetics of the active agent. Therefore, additional modifications are made to further improve oral bioavailability, stability, and / or pharmacokinetics.

[0141] In some embodiments, the triple agonist peptide has an amino acid sequence of any one of SEQ ID NOs: 1-130. In preferred embodiments, the triple agonist peptide is a triple agonist analog modified with one or more biotin moieties and / or one or more fatty acids, optionally with one or more spacers, to achieve desired pharmacokinetics, stability, and bioavailability. In other embodiments, the triple agonist peptide is a triple agonist analog modified 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 desired pharmacokinetics, stability, and bioavailability. In additional embodiments, the triple agonist peptides disclosed herein are modified with C-terminal amidation.

[0142] The selection of suitable functional groups for modification is based on the type of available reactive groups on the molecule that will be coupled to the biotin moiety and / or fatty acid. Typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used as site-specific conjugation. In a preferred embodiment, the reactive amino acids are lysine and cysteine.

[0143] In preferred embodiments, one or more biotin moieties and / or one or more fatty acids or derivatives thereof are conjugated to the amino acid sequence of any one of SEQ ID NOs: 1-130 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 one or more C-terminally inserted cysteine ​​or lysine residues. In other embodiments, one or more cysteine ​​and lysine residues are introduced into the amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to facilitate conjugation with the biotin moiety and / or fatty acid or derivative thereof. In specific embodiments, the triple agonist peptide has the amino acid sequence of any one of SEQ ID NOs: 1-130.

[0144] a. Biotinylation

[0145] Biotin modification of exendin derivatives has been previously described, for example in International Publication Nos. WO2009107900A1, WO 2020242268A1, and WO 2021107519A1.

[0146] Korean Patent Registration No. 10-0864584 describes an exendin-4 derivative in which biotin is modified in the lysine residue of exendin-4, which can be orally administered and has improved bioavailability in the small intestine. However, in this case, there are problems that biotin is conjugated to various lysine positions of exendin-4 to form various isomers, thereby reducing the reaction rate and yield, and biotin is conjugated to the lysine position at the N-terminus, which is the active site of exendin-4, to inhibit the activity of exendin-4.

[0147] Therefore, in a preferred embodiment, one or more biotin moieties are conjugated to an amino acid (eg, cysteine ​​or lysine) at a suitable position to provide excellent oral bioavailability without inhibiting the activity of the triple agonist peptide.

[0148] In some embodiments, the triple agonist peptides have improved in vivo oral bioavailability compared to the same triple agonist peptides not conjugated to one or more biotin moieties. In preferred embodiments, the biotin-conjugated triple agonist peptides retain a substantial portion of the activity of the same triple agonist peptides without one or more biotin moieties conjugated thereto.

[0149] In some embodiments, the biotin moiety conjugated to one or more amino acid residues (eg, cysteine ​​or lysine) of the triple agonist peptide is represented by Formula A below.

[0150] [General formula A]

[0151]

[0152] in,

[0153] X is a functional group that can be conjugated to a polypeptide,

[0154] Y is a spacer,

[0155] Z is a bonding unit,

[0156] B can be represented by the following chemical formula A-1,

[0157] [Chemical formula A-1]

[0158]

[0159] T is the terminal group,

[0160] m is an integer from 1 to 10,

[0161] n is an integer from 1 to 10, and

[0162] p is an integer 0 or 1.

[0163] In some embodiments, when n=0, Y can be directly connected to B or T.

[0164] In some embodiments, the polypeptide to which the biotin moiety is conjugated is a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 130. Alternatively, one or more cysteine ​​or lysine residues are inserted internally at any position within the amino acid sequence of any one of SEQ ID NOs: 1 to 130 to facilitate conjugation with biotin.

[0165] In some embodiments, the biotin moiety is conjugated to the triple agonist peptide by 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 by one additional cysteine ​​residue added to the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 1-130. In another embodiment, the biotin moiety is conjugated to the triple agonist polypeptide by one additional lysine residue added to the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 1-130. In some embodiments, the amino acid at the second position of SEQ ID NOs: 1-18 is substituted with 2-aminoisobutyric acid (Aib). In some embodiments, the biotin moiety is conjugated to the triple agonist polypeptide by one or more internal lysine residues (e.g., lysine 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.

[0166] In the general formula A representing the biotin moiety, X is a functional group capable of conjugating to the cysteine ​​of a polypeptide. Although not limited thereto, for example, the functional group may be maleimide, amine, succinimide, N-hydroxysuccinimide, aldehyde or carboxyl, and more specifically maleimide.

[0167] In one embodiment, when the functional group X in Formula A is conjugated to cysteine ​​or lysine of a polypeptide, the structure can be maintained, removed, or modified.

[0168] In the general formula A, Y may be a spacer and may have a structure having cleavability in vivo. Although not limited thereto, for example, Y is a directly bonded or substituted or unsubstituted alkylene group, wherein the alkylene group may include at least one of -O-, -C(=O)NR-, -C(=O)O-, -C(=O)-, -NR-, and -NOR-, and R may be hydrogen and a substituted or unsubstituted alkyl or aryl group.

[0169] In one embodiment, the spacer may include a structure represented by the following formula.

[0170]

[0171] In some embodiments, in Formula A, Z is a binding unit capable of binding to B, and may include, for example, an amino acid, a polypeptide, an alkyleneamine, or a polyamidoamine structure, but is not limited thereto.

[0172] Although not limited thereto, for example, the amino acid may be lysine, 5-hydroxylysine, 4-oxolysine, 4-thiolysine, 4-selenolysine, 4-thiohomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid, cis-4-dehydrolysine, 6-N-methyllysine, diaminopimelic acid, ornithine, 3-methylornithine, α-methylornithine, citrulline or homocitrulline, arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, ornithine, proline, serine, or threonine.

[0173] When n is 0, B can be directly bound to Y (spacer).

[0174] In some embodiments, in Formula A, T is a terminal group, and although not limited thereto, may be, for example, hydrogen or NH2.

[0175] When p is 0, B may be terminal.

[0176] In one embodiment, in the above general formula A, m may be an integer of 1 to 10, and specifically may be an integer of 1 to 8, 1 to 5, and 1 to 4.

[0177] In one embodiment, the biotin moiety can be represented by the following general formula 1A:

[0178] [Formula 1A]

[0179]

[0180] in,

[0181] Lys is lysine,

[0182] T is hydrogen or NH2,

[0183] q is an integer from 1 to 5,

[0184] r is an integer from 0, 1 to 3, and

[0185] B, n, m and p are as defined in Formula A above.

[0186] In one embodiment, the biotin moiety can be represented by the following general formula 2A or 3A:

[0187] [Formula 2A]

[0188]

[0189] in,

[0190] Lys is lysine,

[0191] T is hydrogen or NH2,

[0192] R3 is hydrogen or -SO3-,

[0193] q is 0 or an integer from 1 to 4, and

[0194] B, n, m and p are as defined in Formula A above.

[0195] [Formula 3A]

[0196]

[0197] in,

[0198] R1 is a direct bond or NH,

[0199] R3 is hydrogen or -SO3-, and

[0200] B and m are as defined in Formula A above.

[0201] In one embodiment, the biotin moiety can be represented by the following structures I-III.

[0202] Structure I

[0203]

[0204] Structure II

[0205]

[0206] Structure III.

[0207]

[0208] Exemplary biotin derivatives are shown in Tables 1 and 2 below.

[0209] Table 1. Examples of biotin derivatives.

[0210]

[0211]

[0212] Table 2. Examples of biotin derivatives.

[0213]

[0214]

[0215] B39 and B40 use desthiobiotin, an analog of biotin, as shown in structure IV.

[0216] Structure IV. Desthiobiotin

[0217]

[0218] In some embodiments, the biotin analog NHS-desthiobiotin as shown in Structure V is used for conjugation.

[0219] Structure V. NHS-dethiobiotin

[0220]

[0221] b. Lipidation

[0222] Lipidated peptides have increased lipophilicity, increased half-life in vivo (enabling once daily oral administration) and reduced pharmacokinetic variability at steady state. In some embodiments, additional amino acids are added to the C-terminus of the triple agonist peptide to enable conjugation of one or more fatty acid molecules with increased linker stability. In a preferred embodiment, the amino acid is cysteine ​​or lysine.

[0223] In some embodiments, the fatty acid moiety is conjugated to the triple agonist peptide by 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 by an additional cysteine ​​residue added to the C-terminus of the amino acid sequence of SEQ ID NO:1-130. In some embodiments, the amino acid at the second position of SEQ ID NO:1-18 is replaced by 2-aminoisobutyric acid (Aib). In some embodiments, the fatty acid moiety is conjugated to the triple agonist polypeptide by one or more internal lysine residues (e.g., lysine at position 10, lysine at position 12, lysine at position 17 and / or lysine at position 20, lysine at position 24) of any one of SEQ ID NO:1-130.

[0224] The first lipidated biologic drug to receive regulatory approval was insulin detemir in 2004. Insulin detemir, a basal insulin for the treatment of diabetes, consists of desB30 human insulin conjugated to myristic acid (C14) via the Nε-amine of LysB29.

[0225] Current lipidated biopharmaceuticals have a hydrophilic spacer between the lipid and peptide moieties, typically γGlu and / or OEG (8-amino-3,6-dioxooctanoic acid), 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 that is marketed for the treatment of diabetes and obesity. The liraglutide sequence is identical to that of native GLP-1, except that Lys34Arg is substituted, which enables selective palmitoylation of the Nε of Lys26 via the γGlu spacer (Lau J. et al., Journal of Medicinal Chemistry (J. Med. Chem.) 2015, 58 (18), 7370-7380). Due to albumin binding and slow absorption, the half-life of liraglutide (11-15 hours, subcutaneous) is significantly prolonged compared to native GLP-1 (1-1.5 hours, subcutaneous). Preferred fatty acids for lipidation have evolved from dietary fatty acids used in detemir and liraglutide to non-dietary dicarboxylic acid fatty acids for once-daily basal insulin degludec and weekly GLP-1 analog semaglutide. Degludec is lipidated with palmitic acid diacid separated by γGlu at LysB29. The peptide backbone of semaglutide is similar to that of liraglutide, except that alanine 8 is replaced by 2-aminoisobutyric acid (Aib), which reduces the degradation caused by dipeptidyl peptidase IV (DPP-4) (Lau, J. et al., Journal of Medicinal Chemistry (2015), 58 (18), 7370-7380). Semaglutide is lipidated with octadecane dioic acid at Lys26 through a spacer including γGlu and two OEG units, which results in an albumin affinity that is 5.6 times that of liraglutide. High albumin affinity and DPP-4 resistance give semaglutide a half-life of about 1 week (subcutaneous) in humans (van Witteloostuijn, SB; Pedersen, SL; Jensen, KJ Chem Med Chem 2016, 11, 1-23). ​​Impressively, this extended half-life is achieved without reducing GLP-1 receptor potency compared to the native ligand. Recently, lipidation has also been shown to be a viable strategy for extending the half-life of larger proteins, as demonstrated by the once-weekly human growth hormone somapacitan. Somapacitan's lipidation includes a significantly longer spacer region and a non-carboxylic acid fatty acid with a tetrazole head group.

[0226] Thus, in some embodiments, the triple agonist peptide is conjugated to one or more fatty acid chains, preferably with 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 may include dietary fatty acids, such as those used in detemir and liraglutide, and preferred fatty acids for lipidation are non-dietary dicarboxylic fatty acids, such as those used in degludec and semagglutide.

[0227] Exemplary fatty acid derivatives are shown in Table 3 below.

[0228] Table 3. Examples of fatty acid derivatives

[0229] Fatty acid number structure F1 C16-NHS F2 C16-MAL F3 C18-NHS F4 C18-MAL F5 C16-γGlu-NHS F6 C16-γGlu-MAL F7 C18-γGlu-NHS F8 C18-γGlu-MAL F9 C18-γGlu-OEG-NHS F10 C18-γGlu-OEG-MAL F11 C18-γGlu-2OEG-NHS F12 C18-γGlu-2OEG-MAL F13 C20-γGlu-2OEG-NHS F14 C20-γGlu-2OEG-MAL F15 C18-γGlu-2OEG-TFP F16 C18-γGlu-2OEG-NPC F17 C20-γGlu-2OEG-NPC

[0230] In preferred embodiments, the triple agonist peptide is lipidated and / or biotinylated. In specific 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 (A, B, C, D, E, and F) listed in Table 23 using one or more biotin derivatives listed in Tables 1 and 2 and / or one or more fatty acid derivatives listed in Table 3.

[0231] c. PEGylation

[0232] In some embodiments, the triple agonist peptide is modified with polyethylene glycol, polyethyleneimine, or a derivative thereof. In a preferred embodiment, the triple agonist peptide is modified by pegylation at a site similar to the biotinylation and lipidation sites. Modifications can alter pharmacokinetics, pharmacodynamics, stability, and bioavailability.

[0233] Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. The structure of PEG is (note the repeated elements in brackets): H-(O-CH2-CH2) n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. PEG, PEO or POE refer to oligomers or polymers of ethylene oxide. The three names are chemically synonymous, but historically PEG is more favored in the biomedical field, while PEO is more popular in the field of polymer chemistry. Because different applications require different polymer chain lengths, but PEG tends to refer to oligomers and polymers with molecular weights lower than 20,000g / mol, PEO refers to polymers with molecular weights higher than 20,000g / mol, and POE refers to polymers of any molecular weight. PEG and PEO are liquids or low melting point solids, depending on their molecular weight. PEG is prepared by the polymerization of ethylene oxide and is commercially available in a wide molecular weight range of 300g / mol to 10,000,000g / mol. Although it is found that PEG and PEO with different molecular weights are used for different applications, and have different physical properties (e.g., viscosity) due to chain length effects, their chemical properties are almost the same. Different forms of PEG may also be used, depending on the initiator used for the polymerization process 2, the most common initiator being the monofunctional methyl ether PEG, or methoxy poly(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, allowing the crystal structure to be determined by x-ray diffraction. Due to the difficulty in purifying and isolating pure oligomers, the price of this quality is typically 10-10,00 times that of polydisperse PEG.

[0234] PEG also has different geometric shapes. Branched PEG has three to ten PEG chains emanating from a central core group. Star PEG has 10 to 100 PEG chains emanating from a central core group. Comb PEG has multiple PEG chains that are usually grafted onto a polymer backbone. The number usually included in the PEG name indicates its average molecular weight (e.g., the average molecular weight of a PEG of n=9 is about 400 Daltons and is labeled PEG 400). Most PEGs include molecules with a molecular weight distribution (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), and the ratio of the weight average molecular weight to the number average molecular weight is called the polydispersity index (Mw / Mn). MW and Mn can be measured by mass spectrometry.

[0235] In certain embodiments, polyethylene glycol or its derivative is linear or branched type, and for branched type, preferably dimerization or trimerization can be used, and more preferably trimerization can be used. Specifically, polyethylene glycol derivative is for example methoxy polyethylene glycol succinimidyl propionate, methoxy polyethylene glycol N-hydroxysuccinimide, methoxy polyethylene glycol propionaldehyde, methoxy polyethylene glycol maleimide or the multi-branched type of these derivatives. Preferably, polyethylene glycol derivative is linear methoxy polyethylene glycol maleimide, branched methoxy polyethylene glycol maleimide or trimerization methoxy polyethylene glycol maleimide, more preferably trimerization methoxy polyethylene glycol maleimide.

[0236] PEG is a particularly attractive polymer for conjugation. Specific features of the PEG moiety that are relevant for pharmaceutical applications are: water solubility, high fluidity in solution, lack of toxicity and low immunogenicity, ease of clearance from the body, and altered distribution in vivo.

[0237] Pegylation (also commonly referred to as stylized PEGylation) is the process of 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 is then described as pegylation (PEGylation). Pegylation is conventionally achieved by incubating reactive derivatives of PEG with target molecules. The covalent attachment of PEG to drugs or therapeutic proteins can mask the medicament from the influence of the host immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the medicament, which prolongs its circulation time by reducing renal clearance. Pegylation can also provide water solubility for hydrophobic drugs and proteins.

[0238] PEGylation can improve the safety and efficacy of many therapeutic agents such as peptides, proteins, and antibody fragments. It causes changes in physiochemical properties including conformation, electrostatic binding, hydrophobicity, etc. These physical and chemical changes increase the systemic retention of the therapeutic agent. In addition, it can affect the binding affinity of the therapeutic moiety to cellular receptors and can alter the absorption and distribution patterns.

[0239] PEGylation increases molecular weight, defense of metabolic sites, and inhibition of immunogenic sites, thereby increasing half-life and stability in vivo and reducing immunogenicity. In addition, since PEG increases the molecular weight of peptides and proteins, the renal excretion of peptides and proteins conjugated to PEG is reduced, so PEGylation has the advantage of increasing both pharmacokinetics and pharmacodynamics.

[0240] In some embodiments, the triple agonist peptide is modified with polyethylene glycol, polyethyleneimine or its derivatives. 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 prepared by pegylation with polyethylene glycol or its derivatives, the molecular structure of the analog can be confirmed by mass spectrometry, liquid chromatography, X-ray diffraction analysis, polarization determination and comparison between calculated and measured values ​​of representative elements constituting the pegylated triple agonist peptide.

[0241] In some embodiments, PEG or its derivatives are conjugated to triple agonist peptides by one or more additional cysteine ​​or lysine residues added to the C-terminus of the polypeptide. In one embodiment, PEG or its derivatives are conjugated to triple agonist peptides by an additional cysteine ​​residue added to the C-terminus of the amino acid sequence of SEQ ID NO: 1-53. In some embodiments, the amino acid at the second position of SEQ ID NO: 1-53 is replaced by 2-aminoisobutyric acid (Aib). In some embodiments, PEG or its derivatives are conjugated to triple agonist polypeptides by one or more internal lysine residues (e.g., 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 NO: 1-64.

[0242] B. Drug Formulations

[0243] In some embodiments, the triple agonist peptide or its analog is formulated with one or more pharmaceutical excipients, additives or fillers. For example, in some embodiments, the triple agonist peptide or its analog is formulated into a pharmaceutical formulation for administration to a subject. Compositions including the triple agonist peptide or its analog can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, which assist in processing the active compound into a pharmaceutically usable formulation.

[0244] The appropriate formulation depends on the selected route of administration. In a preferred embodiment, the composition is formulated for parenteral delivery. In a preferred embodiment, the composition is formulated for subcutaneous delivery. In certain embodiments, the composition is formulated for intravenous injection. Typically, the composition will be formulated in sterile saline or buffered solution for injection into the tissue or cell to be treated. The composition can be lyophilized and stored in a single-use vial for immediate rehydration prior to use. Other means for rehydration and administration are known to those skilled in the art.

[0245] The pharmaceutical formulation contains a combination of a triple agonist peptide or its analogues and one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH modifiers, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) and can be applied to individuals without causing undesirable biological side effects or unwanted interactions.

[0246] Typically, a pharmaceutically acceptable salt can be prepared by reacting a medicament in the form of a free acid or base with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of both water and an organic solvent; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Pharmaceutically acceptable salts include salts derived from inorganic acids, organic acids, alkali metal salts and alkaline earth metal salts of medicaments, and salts (e.g., quaternary ammonium salts) formed by reacting a drug with a suitable organic ligand. A list of suitable salts is found, for example, in Remington's Pharmaceutical Sciences, 20th edition, Lippincott Williams & Wilkins Publishing Company, Baltimore, Maryland, 2000, p. 704. Examples of ophthalmic drugs sometimes administered in the form of pharmaceutically acceptable salts include timolol maleate, brimonidine tartrate and diclofenac sodium.

[0247] 1. Dosage Unit

[0248] For ease of administration and uniformity of dosage, the composition is preferably formulated in dosage unit form. The phrase "dosage unit form" refers to physically discrete units of the conjugate suitable for the patient to be treated. However, it should be understood that the total single administration of the composition will be determined by the attending physician within the scope of reasonable medical judgment. The therapeutically effective dose can be initially estimated in a cell culture assay or in an animal (usually a mouse, rat, rabbit, dog or pig) model. Animal models are also used to achieve the desired concentration range and route of administration. Such information should then be used to determine the dosage and route that can be used for administration in humans.

[0249] 2. Formulations for administration

[0250] In some embodiments, the composition of the triple agonist peptide or its analog is formulated into a pharmaceutically acceptable formulation for administration by a specific route. In some embodiments, the composition is administered topically, for example, by direct injection into the site to be treated. In some embodiments, the composition is injected, topically applied, or otherwise directly applied to the vasculature on the vascular tissue at or near the injury, surgical operation, or implantation site. For example, in some embodiments, during a surgical procedure, the composition is topically applied to exposed vascular tissue. Typically, local administration increases the local concentration of the composition, which is greater than the concentration that can be achieved by systemic administration.

[0251] Pharmaceutical compositions formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection) and enteral routes of administration are described.

[0252] a. Enteral administration

[0253] In some embodiments, the triple agonist peptide or its analog is orally administered. For oral administration, suitable formulations include tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, lozenges, etc., and in addition to active ingredients, these formulations may include diluents (e.g., lactose, glucose, sucrose, mannitol, sorbitol, cellulose and / or glycine), slip modifiers (e.g., silicon dioxide, talc, stearates and their magnesium or calcium salts and / or polyethylene glycol). Tablets may also include binding agents such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone, and if necessary, may include disintegrants such as starch, agar, alginic acid or its sodium salt or boiling mixture and / or absorbents, coloring agents, flavoring agents and sweeteners.

[0254] In a preferred embodiment, one or more absorption or penetration enhancers are used in oral formulations. Exemplary penetration enhancers include bile acid, cholic acid, deoxycholic acid, glycocholic acid, glycocholic acid deoxycholic acid, taurochenodeoxycholic acid, taurocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, caprylocaproylpolyoxyethylene-8 glyceride, SNAC (sodium N-(8-[2-hydroxybenzoyl]amino)caprylate), propyl gallate, and salt forms thereof.

[0255] b. Parenteral Administration

[0256] In some embodiments, the triple agonist peptide or its analog is formulated into a pharmaceutically acceptable formulation for parenteral administration. The phrase "parenteral administration" is a term recognized in the art and includes modes of administration other than enteral and topical administration, such as injection, and includes but is not limited to intravenous (iv), intramuscular (im), intraperitoneal (ip), subcutaneous (sc) injection and infusion. Long-acting GLP-1r agonists can be administered parenterally, for example, by intravenous, intraperitoneal or subcutaneous routes.

[0257] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Parenteral vehicles (for subcutaneous, intravenous, intra-arterial or intramuscular injections) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters, such as ethyl oleate. Aqueous vehicles include, for example, water, alcohol / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media. Long-acting triple agonists can also be applied in the form of emulsions, such as water-in-oil. Examples of oils are oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, cod liver oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and minerals. Suitable fatty acids for parenteral formulations include, for example, oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0258] Preparations suitable for parenteral administration may include antioxidants, buffers, antibacterial agents and solutes that make the preparation isotonic with the blood of the intended recipient and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizing agents, thickening agents, stabilizers and preservatives. Intravenous vehicles may include fluid and nutrient supplements, electrolyte supplements, such as supplements based on Ringer's dextrose. Typically, water, saline, aqueous dextrose and related sugar solutions and, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly liquid carriers for injectable solutions.

[0259] Injectable pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, eds. Banker and Chalmers, pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pp. 622-630 (2009)).

[0260] III. Preparation Method

[0261] A. Methods for Preparing Triple Agonist Peptide Analogs

[0262] Triple agonists can be prepared and modified by various chemical reaction steps. Typically, methods for modifying triple agonists 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 or lipid-conjugated amino acids.

[0263] 1. Biotinylation and Lipidation

[0264] Biotin modification of exendin derivatives has been previously described, for example in International Publication Nos. WO2009107900A1, WO 2020242268A1, and WO 2021107519A1.

[0265] In some embodiments, biotinylation and lipidation are carried out simultaneously. In one embodiment, the method includes dissolving peptide C18-γGlu-2OEG-MAL (F12) and biotin-N-hydroxysuccinimide ester (B1-NHS, B38) in a dimethyl sulfoxide (DMSO) solution containing 0.3% triethylamine (TEA) (v / v). In one embodiment, the peptide and F12 are mixed at a volume ratio of 1: 1. The 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 under slight shaking. In another embodiment, B38 is added at a volume ratio of 1: 0.2, and the molar ratio is 1: 3 (peptide: biotin). In one embodiment, the mixture is reacted at 25 ° C for 60 minutes under slight shaking.

[0266] In another embodiment, the method includes dissolving the peptide, biotin-maleimide (B1-MAL, B1) and C18-γGlu-2OEG-NPC (F16) in a DMSO solution containing 0.3% TEA (v / v). In one embodiment, the peptide and B1 are mixed at a volume ratio of 1: 1. 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 under slight shaking, and then F16 is added at a volume ratio of 1: 0.2, and the molar ratio is 1: 2 (peptide: lipid). In one embodiment, the mixture is reacted at 25° C. for 90 minutes under slight shaking. Lipidated peptides and biotinylated peptides can be purified by preparative LC, and the eluent can be collected in separate fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated at 45° C. using a centrifugal evaporator for 40 minutes. The solvent can be changed into water by ultrafiltration. The purified sample can be analyzed by reverse phase HPLC to check the purity. In one embodiment, samples were freeze dried at -88°C for 18 hours and then stored at -20°C.

[0267] 2. Lipidation

[0268] In one embodiment, the method includes dissolving the peptide and C18-γGlu-2OEG-MAL (F12) in a DMSO solution containing 0.3% TEA (v / v); and each solution is mixed at a volume ratio of 1: 1. 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 under gentle shaking. In one embodiment, the lipidated peptide is purified by preparative LC, and the eluate is collected in separate fractions. In one embodiment, the ACN contained in the fractionated solution is evaporated at 45°C using a centrifugal evaporator for 40 minutes. The solvent can be changed to water by ultrafiltration. The purified sample can then be analyzed by reverse phase HPLC to check the purity. In one embodiment, the sample is freeze-dried at -88°C for 18 hours and then stored at -20°C.

[0269] IV. How to use

[0270] Pharmaceutical formulations containing one or more triple agonist peptides or analogs thereof as described herein can be administered to a subject in need thereof to treat or prevent one or more diseases.

[0271] A. Treatment methods

[0272] Methods of treating or preventing one or more metabolic diseases using triple agonist peptides are described. These methods can effectively treat or prevent one or more metabolic diseases, such as dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity and type 2 diabetes mellitus (T2DM), preferably with minimal side effects.

[0273] In a preferred embodiment, the triple agonist peptide or its analog is used with an amount and dosage regimen that effectively prevents, suppresses or alleviates one or more symptoms associated with one or more metabolic diseases such as dyslipidemia, fatty liver disease, metabolic syndrome, NAFLD, obesity and T2DM in the subject. In a preferred embodiment, the disease is fatty liver disease, obesity, NAFLD or T2DM.

[0274] At one or more time points after the initial dose, a triple agonist peptide or its analog is administered to the subject in one or more doses. Compared to a control (e.g., a subject treated with an agonist that primarily targets a single receptor for GLP-1 glucagon or a GIP receptor), the amount of the composition administered to the subject is selected to deliver an effective amount, thereby alleviating, preventing or otherwise alleviating one or more clinical or molecular symptoms of the disease or disorder to be treated. These compositions and methods are also suitable for preventive use.

[0275] These methods are also applicable to treating or preventing one or more neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease. In some embodiments, these methods include the step of administering to an individual in need thereof an effective amount of a triple agonist peptide or an analog thereof.

[0276] B. Condition to be treated

[0277] Compositions and formulations of these triple agonist peptides are effective in ameliorating or preventing one or more symptoms of metabolic or neurological diseases with minimal off-target toxicity or side effects.

[0278] In some embodiments, the subject to be treated is a human. In some embodiments, the subject to be treated is a child or infant. All methods may include the step of identifying and selecting a subject in need of treatment or a subject who will benefit from the administration of the described compositions.

[0279] 1. Obesity and type 2 diabetes

[0280] Obesity is a medical condition in which excess body fat accumulates to the point where it can have adverse effects on health, leading to reduced life expectancy and / or increased health problems. Body mass index (BMI), a measurement comparing weight to height, is a measure of height and is considered high for individuals with a BMI between 25 and 30 kg / m 2Overweight is defined as pre-obesity or overweight when the weight exceeds 30 kg / m 2 It is defined as obesity.

[0281] Obesity increases the risk of a variety of physical and psychiatric conditions. Excess weight is associated with a variety of diseases, especially cardiovascular disease, type 2 diabetes, obstructive sleep apnea, certain types of cancer, and osteoarthritis. These diseases are directly caused by obesity or indirectly through mechanisms that share common causes (such as poor diet and / or sedentary behavior). One of the strongest links is with type 2 diabetes. 64% of diabetes cases in men and 77% of diabetes cases in women are due to excess body fat. Increased body fat changes the body's response to insulin, potentially leading to insulin resistance.

[0282] The method for treating and / or preventing one or more symptoms of obesity and / or T2DM comprises administering an effective amount of a composition to a subject in need thereof, to treat and / or alleviate one or more symptoms associated with obesity and / or T2DM. In certain embodiments, the composition or its pharmaceutical formulation is administered in an amount that effectively induces weight loss, reduces body fat, reduces food intake, improves glucose homeostasis, prevents weight gain, and / or prevents an increase in body mass index, or a combination thereof, in normal or obese patients.

[0283] In some embodiments, the pharmaceutical formulation is administered to a patient suffering from obesity, an obesity-related disease or disorder, diabetes, insulin resistance syndrome, nonalcoholic steatohepatitis, cardiovascular disease, or metabolic syndrome.

[0284] In some embodiments, the pharmaceutical formulations are administered to normalize blood glucose; these formulations are preferably administered in an amount effective to reduce blood glucose levels to less than about 180 mg / dL. If necessary, the formulations may be co-administered with other anti-diabetic therapies to improve glucose homeostasis.

[0285] The pharmaceutical formulation may also be administered to a patient suffering from a disease or condition that causes obesity or predisposes the patient to obesity.

[0286] 2. Non-alcoholic fatty liver disease (NAFLD)

[0287] In preferred embodiments, these methods and compositions are used to treat or prevent nonalcoholic steatohepatitis, liver fibrosis associated with nonalcoholic steatohepatitis, primary biliary cholangitis.

[0288] In some embodiments, these compositions are used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD represents a clinical pathological disease, mainly characterized by excessive accumulation of fat in liver cells (steatosis). NAFLD covers all diseases from simple steatosis to non-alcoholic steatohepatitis (NASH), the most severe form of which may lead to life-threatening cirrhosis and hepatocellular carcinoma. It is considered to be a liver manifestation of metabolic syndrome, other pathologies of which include obesity, insulin resistance, hypertension and hyperlipidemia. Histologically, NASH is characterized by hepatic steatosis and signs of intralobular inflammation accompanied by ballooning degeneration of hepatocytes. The estimated prevalence of NASH is much lower than that of NAFLD, and is in the range of 3% to 5%. It is reported that twenty percent of NASH patients develop cirrhosis, and 30%-40% of NASH cirrhosis patients experience liver-related deaths.

[0289] NAFLD is broadly divided into 2 phenotypes, namely: non-alcoholic fatty liver (NAFL) (marked by isolated steatosis), and a more aggressive subtype, non-alcoholic steatohepatitis (NASH), characterized by cell damage, inflammatory cell infiltration, and ballooning of hepatocytes, which may further develop into fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In some embodiments, these compositions are used in an amount effective to treat or ameliorate one or more symptoms of non-alcoholic steatohepatitis (NASH).

[0290] NAFLD shows a close association with metabolic syndrome including obesity, type II diabetes, dyslipidemia, etc. based on insulin resistance. In fact, it is known that many patients with prediabetes and type II diabetes show the development of non-alcoholic fatty liver / non-alcoholic steatohepatitis, and the progression rate of cirrhosis and liver cancer (e.g., hepatocellular carcinoma) is high in these patients. At the same time, the prevalence of diabetes in patients with non-alcoholic fatty liver disease is high and the prevalence in patients with non-alcoholic steatohepatitis is significant.

[0291] The 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.

[0292] In some embodiments, these compositions are administered in an amount effective to prevent the transformation of NAFLD to NASH and to ameliorate the pathophysiology of the disease.

[0293] Methods for treating and / or preventing one or more symptoms of NAFLD or NASH generally comprise administering to a subject in need thereof an effective amount of a composition to treat and / or alleviate one or more symptoms associated with NAFLD or NASH.

[0294] In some embodiments, these compositions are administered in an amount effective to inhibit or reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC), fat accumulation or steatosis, inflammation, ballooning, fibrosis, long-term morbidity, and mortality.

[0295] 3. Neurological and neurodegenerative diseases

[0296] These compositions and formulations thereof can be used to treat one or more neurological and neurodegenerative diseases. These compositions and methods are particularly suitable for treating one or more neurological or neurodegenerative diseases associated with microglia and / or astrocyte activation. In certain embodiments, the disease or condition is selected from but not limited to neurological diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD)). In one embodiment, these compositions are used to treat Alzheimer's disease (AD) or Parkinson's disease.

[0297] Neurodegenerative diseases are chronic progressive disorders of the nervous system that affect neurological and behavioral functions and involve biochemical changes that lead to different histopathological and clinical syndromes (Hardy H et al., Science. 1998; 282: 1075-9). Abnormal proteins that resist the mechanisms of cellular degeneration accumulate within the cells. The pattern of neuronal loss is selective in the sense that one group is affected while others remain intact. Usually, there is no clear cause for this disease. Classically described diseases as neurodegenerative are Alzheimer's disease, Huntington's disease and Parkinson's disease.

[0298] Neuroinflammation mediated by activated microglia and astrocytes is a major hallmark of various neurological disorders, making it a potential therapeutic target. A number of scientific reports have shown that reducing neuroinflammation by targeting these cells in the early stages can delay the onset of the disease, and thus 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., Nature Review: Neuroscience 2007, 8, 57). Delivery of therapeutic agents across the blood-brain barrier is a challenging task. Neuroinflammation leads to damage to the blood-brain barrier (BBB). The damaged BBB in neuroinflammatory conditions can be exploited 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).

[0299] These compositions and methods can also be used to treat neurological or neurodegenerative diseases or disorders or central nervous system disorders. In a preferred embodiment, these compositions and methods are effective in treating and / or alleviating neuroinflammation associated with neurological or neurodegenerative diseases or disorders or central nervous system disorders. These methods generally include administering an effective amount of the composition to the subject to increase cognition or reduce cognitive decline, increase cognitive function or reduce cognitive decline, increase memory or reduce memory decline, increase ability or learning ability or reduce ability or learning ability decline or a combination thereof.

[0300] Neurodegeneration refers to the gradual loss of neuronal structure or function, including neuronal death. For example, these compositions and methods can be used to treat subjects with diseases or conditions such as Parkinson's disease (PD) and PD-related conditions, 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 dementia, Alpers' Disease, Batten Disease, cerebro-oculofacial-skeletal syndrome, corticobasal degeneration, Gerstmann-Straussler-Scheinker Disease), kuru disease, Leigh's disease, monolimbic muscular atrophy, multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis (MS), neurodegeneration with brain iron deposition, opsoclonus myoclonus, posterior cortical atrophy, primary progressive aphasia, progressive supranuclear palsy, vascular dementia, progressive multifocal leukoencephalopathy, dementia with Lewy bodies (DLB), lacunar syndrome, hydrocephalus, Wernicke-Korsakoff's syndrome, postencephalitic dementia, cancer and chemotherapy-related cognitive impairment and dementia, and depression-induced dementia and pseudodementia. In a preferred embodiment, the disease or condition is Alzheimer's disease (AD) or Parkinson's disease.

[0301] Criteria for evaluating improvements in specific neurological factors include methods for evaluating cognitive skills, motor skills, memory abilities, and the like, 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, and can be appropriately selected to diagnose the state of specific neurological damage. In order to evaluate changes in Alzheimer's disease or related neurological changes, the selected evaluation or evaluation test is performed before the start of administration of these compositions. After this initial evaluation, a treatment method for administering the composition is started and continued at different time intervals. At a selected time interval after the initial assessment of neurological deficit damage, the same evaluation or evaluation test is used again to re-evaluate changes or improvements in selected neurological criteria.

[0302] The individual is preferably an adult, and more preferably a person over the age of 30 who has lost a certain amount of neurological function due to Alzheimer's disease or dementia. Generally speaking, neural loss means any neural loss at the cellular level, including loss of neurites, neural tissue or neural networks.

[0303] In other embodiments, the methods include selecting a subject who may benefit from treatment with the compositions.

[0304] These compositions and formulations are suitable for reducing or preventing one or more pathological processes associated with the development and progression of PD. Therefore, methods for treating, reducing and preventing pathological processes associated with PD include administering these compositions in an amount and dosage regimen that is effective in reducing microglial activation, abnormal accumulation of α-synuclein, neurofibrillary tangles in the brain and / or improving tremors, stiffness, slowness of movement and difficulty walking in individuals with PD. Methods for alleviating, preventing or reversing motor dysfunction in individuals with PD are provided. These methods include administering an effective amount of a composition including one or more long-acting GLP-1r agonists to a subject in need. In a preferred embodiment, these methods include administering to a subject an effective amount of a composition including one or more triple agonist peptides or a pharmaceutically acceptable salt thereof having an amino acid sequence of any one of SEQ ID NOs: 1-64.

[0305] C. Dosage and Effective Amount

[0306] Dosage and dosing regimen depend on the severity of the condition and / or the method of administration, and can be determined by those skilled in the art. A therapeutically effective amount of a triple agonist peptide or its pharmaceutical formulation used in the treatment of fatty liver disease, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity and / or type 2 diabetes (T2DM) is generally sufficient to reduce or alleviate one or more symptoms associated with the disease or condition.

[0307] Preferably, these compositions do not target or otherwise modulate the activity or number of healthy cells that are not within or unrelated to the diseased or target tissue, or target or modulate at a reduced level compared to the level of the target cells. In this way, byproducts and other side effects associated with the composition are reduced.

[0308] The actual effective amount may vary depending on factors including the specific agent being administered, the specific composition being formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or condition of the subject being treated. In some embodiments, the dose of the triple agonist analog or its pharmaceutical formulation may be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, from about 0.01 mg / kg body weight to about 10 mg / kg body weight, and from about 0.05 mg / kg body weight to about 5 mg / kg body weight. In other embodiments, the dose is the absolute amount of the triple agonist analog or its pharmaceutical formulation administered to the subject in a single dose, such as from about 0.1 mg to about 100 mg. For example, in some embodiments, the dose of the triple agonist analog or its pharmaceutical formulation 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, such as 20 mg, 30 mg, 40 mg, 50 mg or 100 mg. In an exemplary embodiment, the dose of the triple agonist analog is 5 mg, administered once a week. Generally, for intravenous injection or infusion, the dose may be lower than for oral administration.

[0309] Typically, the time and frequency of administration will be adjusted to balance the efficacy of a given treatment schedule with the side effects of a given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple administrations, such as hourly, daily, weekly, monthly or yearly administration.

[0310] The triple agonist analog or its pharmaceutical formulation can be administered daily, biweekly, weekly, every two weeks, monthly or less frequently in an amount that provides a therapeutically effective increase in blood levels of the therapeutic agent. In the case of administration other than by the oral route, these compositions can be delivered over a period of more than one hour (e.g., 3-10 hours) to produce a therapeutically effective dose within a 24 hour period. Alternatively, the composition can be formulated for controlled release, wherein the composition is administered in a single dose repeated on a weekly or less frequent schedule.

[0311] Dosages may vary and may be administered in one or more daily doses, daily, twice weekly, weekly, biweekly, monthly or less frequently. Guidance on appropriate dosages for a given class of drug products can be found in the literature. The optimal dosing regimen can be calculated based on measurements of drug accumulation in the subject or patient. The optimal dose, dosing method, and repetition rate can be readily determined by a skilled artisan. The optimal dose may vary based on the relative potency of the individual drug compositions and may generally be based on EC values ​​found to be effective in in vitro and in vivo animal models. 50 Come and evaluate.

[0312] In some embodiments, the composition is administered to the subject for 1 to 20 years, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years. Optionally, the composition is administered for 10 years. In one embodiment, the therapeutic effect lasts for at least 1 year.

[0313] In a preferred embodiment, the triple agonist analog is orally administered in an amount of about 1 mg to about 100 mg (including end values), preferably about 5 mg to about 50 mg (including end values). 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.

[0314] In other preferred embodiments, the triple agonist analog is administered parenterally (e.g., subcutaneously) at a concentration of about 0.1 mg / mL to about 10 mg / mL (including end values), preferably about 1 mg / mL to about 5 mg / mL (including end values). 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.

[0315] In some embodiments, the regimen includes one or more cycles of therapy followed by a round of drug holiday (e.g., no drug). The drug holiday can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days; or 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0316] In some embodiments, the amount of a triple agonist analog administered to a subject after an initial dose varies over time. Thus, in some embodiments, the amount of a triple agonist analog administered to a subject after an initial dose varies over time.

[0317] D. Combination Therapies and Procedures

[0318] These compositions can be used alone or in combination with one or more conventional therapies. Preferred examples of other therapeutic agents include other conventional therapies known in the art for treating desired diseases, disorders or conditions. The (these) other therapeutic agents, preventive agents or diagnostic agents may have the same or different mechanisms of action. In certain embodiments, the combination produces an additive effect on the treatment of a disease or condition. In certain embodiments, the combination produces a more than additive effect on the treatment of a disease or condition.

[0319] E. Control

[0320] The treatment results of the triple agonist analog or its pharmaceutical formulation can be compared with a control or reference. The term "control" or "reference" refers to a standard for comparison. The term "change compared to the control" sample or subject is understood to have a statistically different level from a sample from a normal, untreated or control sample. Control samples include, for example, 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 capabilities of those skilled in the art. The analyte can be a naturally occurring substance (e.g., an antibody, a protein) characteristically expressed or produced by a cell or organism or a substance produced by a reporter gene construct (e.g., β-galactosidase or luciferase). Depending on the method used for detection, the amount and measurement of the change can be different. The determination of statistical significance (e.g., the number of standard deviations constituting the mean of a positive result) is within the capabilities of those skilled in the art. Suitable controls are known in the art and include, for example, untreated subjects or untreated cells or the same individual before treatment.

[0321] V. Kit

[0322] The composition can be packaged in a kit. The kit can include a single dose or multiple doses of a composition including one or more triple agonist peptides or their analogs or their pharmaceutical formulations, and instructions for administering the compositions. In a preferred embodiment, the triple agonist peptides or their analogs have the amino acid sequence of any one of SEQ ID NOs: 1-130. Specifically, the instructions direct that an effective amount of the composition be administered to an individual suffering from a specified specific symptom, disease, defect or injury. The composition can be formulated as described above with reference to a specific method of treatment, and can be packaged in any convenient manner.

[0323] The present invention will be further understood by reference to the following non-limiting examples.

[0324] Examples

[0325] Example 1: Screening for triple agonists using phage display

[0326] method

[0327] Screening using phage display

[0328] Custom peptide libraries were constructed using phage display. Phage display technology was used to identify peptides that bind to human GLP-1 receptor, human glucagon receptor, and human GIP receptor. 18 unique peptide sequences were identified from phage display. The library was screened against the target using trimer codons. Trimer codon technology allows the generation of random regions with each amino acid position randomly grouped by defined amino acid composition as well as region length variation. In addition, this method produces less bias. Lead peptide sequences were identified after 3-4 rounds of biopanning. Biopanning is an affinity selection technique that selects peptides that bind to a given target.

[0329] cAMP screening for GLP-1R, GCGR, and GIPR

[0330] To determine the activity of triple agonists 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 assayed using a small molecule-specific cAMP assay (DiscoverX).

[0331] Cells were plated at 7 × 10 3 Cells / well were seeded in a 96-well plate and the cells were incubated overnight at 37°C and 5% CO2. For cAMP detection assays, the cell plating reagent was replaced with cAMP assay buffer. Each well was then treated with diluted peptide and incubated at 37°C in a CO2 incubator for 30 minutes.

[0332] After incubation, cAMP antibody reagent and 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 incubated for 3 hours at room temperature in the dark. Subsequently, luminescence was measured using a microplate reader device.

[0333] result

[0334] Phage display technology was used to identify peptides that bind to the human GLP-1 receptor, the human glucagon receptor, and the human GIP receptor. Phage display technology has been previously described, for example, in U.S. Pat. No. 10,493,125.

[0335] For phage libraries, peptide libraries were designed based on GLP-1, glucagon, and GIP sequences. Figure 1and other residues shown in the polypeptide formula II to construct a custom peptide library. Amino acid positions at 10, 16, 17, 18, 20, 24 and 28 are randomly grouped by trimer incorporation of 19 codons other than cysteine. Trimer codon technology allows the generation of random regions with each AA position randomly grouped by defined AA composition and region length variation. In addition, this is the best method with minimal deviation. Lead peptide sequences are generated after 3 to 4 rounds of bio-panning. Bio-panning is an affinity selection technique that selects peptides that can bind to a given target. Round 1 cell panning: GLP-1R→GCGR→GIPR; and Round 2 cell panning: GLP-1R→GCGR→GIPR.

[0336] The backbone of the triple agonist has the following amino acid sequence of polypeptide formula II:

[0337] HX2QGTFTSDX 10 SX 12 YLDX 16 X 17 X 18 AX 20 DFVX 24 WLn 27 X 28 GGPSSGAPPPSX 40 ,

[0338] wherein X2 is A or 2-aminoisobutyric acid (Aib);

[0339] X 12 is K or W;

[0340] X 27 is L, M, I, G or P;

[0341] X 40 does not exist, is C or K, and

[0342] X (10, 16, 17, 18, 20, 24, and 28) was randomized by trimer incorporation of 19 codons excluding cysteine.

[0343] cAMP assays were performed on the 1st and 2nd round enriched phage pools to detect their effects on three target cell lines and two control cell lines. Each enriched pool was tested for activation of all target cell lines and control cell lines. Unscreened library phages and helper phage M13KO7 were also used as control phages. GLP-1R+ cells were activated from the 1-2-P enriched pool to the 2-3-P enriched pool, with the 1-3-P enriched pool indicating the highest activation. GCGR+ cells were also activated from the 1-2-P enriched pool to the 2-3-P enriched pool, with the 1-3-P enriched pool indicating the highest activation. GIPR+ cells were activated from the 1-3-P enriched pool to the 2-3-P enriched pool, with the 2-2-P enriched pool indicating the highest activation. The six enriched phage pools had no significant activation of the control cells, and the control phages had no significant activation of all target cells and control cells ( Figure 2 ).

[0344] Based on the data from the cAMP assay, the 2-2-P enrichment pool was selected to perform a monoclonal phage activation test using a cAMP assay kit. From the 2-2-P phage, a monoclonal phage cAMP activity assay was performed to verify the activity. Ninety clones from the 2-2-P phage were tested, and 29 clones were identified to be able to specifically activate all three target cells (GLP-1R+, GCGR+, and GIPR+). After sequencing, 18 unique sequences were identified to be active for all three receptors GLP-1R, GCGR, and GIPR. The amino acid sequences of 18 unique peptides are listed in Table 4. C-terminal cysteine ​​is introduced into each of these 18 peptides to allow for incorporation of modifications (such as lipidation and / or biotinylation), and these sequences are listed in Table 5.

[0345] Table 4. Peptide sequences of 18 candidate peptides

[0346]

[0347]

[0348] *When aligned with the sequence of Formula I, these sequences have amino acid deletions.

[0349] Table 5. Peptide sequences of 18 candidate peptides with C-terminal cysteine

[0350]

[0351]

[0352] *When aligned with the sequence of Formula I, these sequences have amino acid deletions.

[0353] 18 individual peptides were rescreened using cAMP assay at 4 different concentrations against three target cell lines: GLP-1R+ cells, GCGR+ cells, GIPR+ cells ( Figures 3A-3C ).

[0354] Example 2: Screening for candidates suitable for fatty acid modification

[0355] method

[0356] Lipid modification (Type A)

[0357] The peptide and C18-γGlu-2OEG-MAL (F12) were dissolved in a DMSO solution containing 0.3% TEA (v / v). Each solution was 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: lipid). The mixture was reacted at 25°C for 30 minutes under slight shaking. The lipidated peptide was purified by preparative LC, and the eluent was collected in separate fractions. The ACN contained in the fractionated solution was evaporated at 45°C using a centrifugal evaporator for 40 minutes. The solvent was changed to water by ultrafiltration. The purified sample was analyzed by reverse phase HPLC to check the purity. The sample was freeze-dried at -88°C for 18 hours and then stored at -20°C.

[0358] Lipid and biotin modification (Type B)

[0359] Peptide, C18-γGlu-2OEG-MAL (F12) and biotin-N-hydroxysuccinimide ester (B1-NHS, B38) are dissolved in a DMSO solution containing 0.3% TEA (v / v). First, peptide and F12 are mixed at a volume ratio of 1: 1. The concentration of the peptide is 5mg / mL, and the molar ratio is 1: 2 (peptide: lipid). The mixture is reacted at 25°C for 10 minutes under slight shaking. And then, B38 is added at a volume ratio of 1: 0.2, and the molar ratio is 1: 3 (peptide: biotin). The mixture is reacted at 25°C for 60 minutes under slight shaking. Lipidated peptide and biotinylated peptide are purified by preparative LC, and the eluent is collected in separate fractions. ACN contained in the fractionated solution is evaporated at 45°C using a centrifugal evaporator for 40 minutes. The solvent is changed into water by ultrafiltration. The purified sample is analyzed by reverse phase HPLC to check purity. The samples were freeze-dried at -88°C for 18 hours and then stored at -20°C.

[0360] Lipid and biotin modification (Type C)

[0361] Peptide, biotin-maleimide (B1-MAL, B1) and C18-γGlu-2OEG-NPC (F16) were dissolved in a DMSO solution containing 0.3% TEA (v / v). 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 under slight shaking. And 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 under slight shaking. The lipidated peptide and biotinylated peptide were purified by preparative LC, and the eluent was collected in separate fractions. ACN contained in the fractionated solution was evaporated at 45 ° C using a centrifugal evaporator for 40 minutes. The solvent was changed into water by ultrafiltration. The purified sample was analyzed by reverse phase HPLC to check the purity. The samples were freeze-dried at -88°C for 18 hours and then stored at -20°C.

[0362] result

[0363] Based on the data from the cAMP assay of 18 individual peptides, peptides 5, 12, 18, and 57 were selected for comprehensive screening. Peptides 5, 12, 18, and 57 were screened using the cAMP assay to determine their ECs in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The EC values ​​of the corresponding natural ligands of these peptides are shown in Tables 6-8. 50 EC with triple agonist peptide 50 relative ratio.

[0364] Table 6. EC of human GLP-1R relative to GLP-1 50 Percent change

[0365] Peptide number <![CDATA[Percentage change (%) of human GLP-1R relative to GLP-1 50 > GLP-1 100％ 5 611％ 12 303％ 18 40％ 57 1％

[0366] Table 7. EC of human GCGR relative to GCG 50 Percent change

[0367]

[0368]

[0369] Table 8. EC of human GIPR relative to GIP 50 Percent change

[0370] Peptide number <![CDATA[Percentage change (%) of human GIPR relative to the EC of GIP 50 > GIP 100％ 5 3％ 12 4％ 18 0％ 57 0％

[0371] The direct use of natural peptides as biopharmaceuticals is often limited by their very short systemic half-life, caused by rapid metabolism, enzymatic degradation, and efficient renal clearance of smaller proteins and peptides. Modifications such as biotinylation and lipidation have been introduced to improve the in vivo stability, bioavailability, and absorption of these peptides. Figures 4A-4C Exemplary conjugation of a biotin moiety, a fatty acid moiety, or a biotin and fatty acid moiety to a triple agonist peptide is shown in Figure 1. 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).

[0372] Subsequently, the peptides in Table 5 were modified with lipid molecules conjugated via cysteine ​​added to the C-terminus of each peptide, i.e. Figure 4A Type A modification as shown. Lipidated versions of these peptides (Type A modification) were also synthesized to produce peptides 3A, 5A, 12A, 18A, 19A, 25A, 28A, 32A, 44A, 57A, 58A, 60A, 62A, 67A, 68A, 70A, 76A, and 77A. These lipidated peptides ( Figures 5A-5C ).

[0373] The lipidated peptides 5A, 12A, 18A, and 32A were screened using the cAMP assay to determine their EC activity in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The EC values ​​of the corresponding natural ligands of these peptides are shown in Tables 9-11. 50 EC with triple agonist peptide 50 relative ratio.

[0374] Table 9. EC of human GLP-1R relative to GLP-1 50 Percent change

[0375]

[0376]

[0377] Table 10. EC of human GCGR relative to GCG 50 Percent change

[0378] Peptide number <![CDATA[Percentage change (%) of human GCGR relative to GCG in terms of EC 50 > Glucagon 100％ 5A 14％ 12A 15％ 18A 0％ 32A 0％

[0379] Table 11. EC of human GIPR relative to GIP 50 Percent change

[0380] Peptide number <![CDATA[Percentage change (%) of human GIPR relative to the EC of GIP 50 > GIP 100％ 5A 5％ 12A 8％ 18A 0％ 32A 0％

[0381] Based on the cAMP assay results for peptides (5, 12, 18, 57) and lipidated peptides (5A, 12A, 18A, 32A), peptides 5 and 12 were selected for further study. Lipidation reduced glucagon and GIP activity.

[0382] Peptide 5 was further investigated using all three types of modifications (A, B, and C). Figures 4A-4C The modified peptides 5A, 5B, and 5C were screened using a cAMP assay to determine their EC in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The EC values ​​of peptide 5 and its modified forms and their corresponding natural ligands (STD) are summarized in Table 12. 50 The EC values ​​of the corresponding natural ligands (STD peptides) of these peptides are summarized in Table 13. 50 EC of each triple agonist peptide 50 Table 14 summarizes the EC values ​​of GLP-1 peptides. 50 EC of each triple agonist peptide 50 The data show that lipidation at Cys40 reduces glucagon and GIP activity. Type C (lipidation at Lys 12) shows better activity than type B.

[0383] Table 12. EC of peptide No. 5 and its modified forms 50

[0384]

[0385] Table 13. EC of STD peptides and triple agonist peptides 50 ratio

[0386]

[0387] Table 14. EC of GLP-1 peptides and triple agonist peptides 50 ratio

[0388]

[0389] Example 3: In vivo efficacy study method for weight loss in normal mice

[0390] In order to confirm the therapeutic effect of peptide in vivo, polypeptide is applied to mice to measure weight change.First, 20nmol / kg or 30nmol / kg dosage of polypeptide is applied to normal C57BL / 6 mice by subcutaneous injection respectively on day 0, day 1 and day 2 (once a day, QD) or day 0 and day 3 (every other day, Q2D).Peptide is delivered with 0.02% polysorbate 80 (PS80) in PBS, and the administration volume is 10mL / kg.Body weight is monitored once a day for 6 hours.Table 15 summarizes detailed treatment doses and schemes.

[0391] The DD01 peptide was used as a positive control and has the following amino acid sequence with a PEGylated C-terminal cysteine ​​(PEG MW 50 kDa): HAibQGT FTSDY SKYLD EQAAK EFVQW LMNTC (SEQ ID NO: 132).

[0392] Semaglutide, a GLP-1 receptor agonist from Novo Nordisk, was also used as a positive control.

[0393] Table 15. Treatment doses and schedules.

[0394]

[0395] result

[0396] Body weight changes were measured for all treatment groups ( Fig. 6A and 6B ). Superior body weight loss (BWL) was observed with daily dosing of 5A. 5A showed similar BWL at a lower dose than DD01. 12A showed similar efficacy to semaglutide.

[0397] Example 4: Design and screening of peptides that increase GIP activity

[0398] method

[0399] Peptide No. 5 and Peptide No. 12 were used for further peptide design to increase activity on GIPR. Peptide No. 5 and Peptide No. 12 and their derivatives are listed in Table 16. A C-terminal cysteine ​​was introduced into each of these peptides to allow for incorporation of modifications (such as lipidation and / or biotinylation). Also synthesized were lipidated peptides conjugated to the C-terminal cysteine, which were peptide 5-1A (SEQ ID NO: 50), peptide 5-2A (SEQ ID NO: 51), peptide 5-3A (SEQ ID NO: 52), peptide 5-4A (SEQ ID NO: 53), peptide 5-5A (SEQ ID NO: 54), peptide 5-6A (SEQ ID NO: 55), peptide 12-1A (SEQ ID NO: 56), peptide 12-2A (SEQ ID NO: 57), peptide 12-3A (SEQ ID NO: 58), peptide 12-4A (SEQ ID NO: 59), peptide 12-5A (SEQ ID NO: 60), peptide 12-6A (SEQ ID NO: 61) and peptide 12-7A (SEQ ID NO: 62).

[0400] Table 16. Peptide sequences derived from peptides 5 and 12

[0401]

[0402]

[0403] result

[0404] Peptide 5-1, peptide 5-2, peptide 5-3, peptide 5-4, peptide 5-5, peptide 5-6, peptide 12-1, peptide 12-2, peptide 12-3, peptide 12-4, peptide 12-5, peptide 12-6 and peptide 12-7 were screened in three target cell lines, GLP-1R+ cells, GCGR+ cells and GIPR+ cells using cAMP assay ( Figures 7A-7C ).

[0405] Lipidated versions of these peptides (Type A modifications) were also synthesized to produce peptides 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 in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, using a cAMP assay. Figures 8A-8C and 9A-9C).

[0406] Peptide 5A, 5-1A, 5-2A, and 5-5A were further screened using a cAMP assay to determine their ECs in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 Table 17 summarizes the EC values ​​of peptide 5A, peptide 5-1A, peptide 5-2A, peptide 5-5A and their corresponding natural ligands (STD). 50 The EC values ​​of the corresponding natural ligands (STD peptides) of these peptides are summarized in Table 18. 50 EC of each triple agonist peptide 50 Table 19 summarizes the EC values ​​of GLP-1 peptides. 50 EC of each triple agonist peptide 50 relative ratio.

[0407] Table 17. EC of peptide 5A, peptide 5-1A, peptide 5-2A, and peptide 5-5A 50

[0408]

[0409] Table 18. EC of STD peptides and triple agonist peptides 50 ratio

[0410]

[0411] Table 19. EC of GLP-1 peptides and triple agonist peptides 50 ratio

[0412]

[0413]

[0414] Example 5: In vivo efficacy study on weight loss in normal mice

[0415] method

[0416] In order to confirm the therapeutic effect of peptides in obesity, diabetes or NASH, the polypeptides were administered to mice to measure changes in food intake, blood sugar and body weight. First, 20nmol / kg or 30nmol / kg doses of polypeptides were administered to normal C57BL / 6 mice by subcutaneous injection on days 0, 1 and 2 (once a day, QD) or days 0 and 3 (every other day, Q2D). In addition, a NASH animal model was prepared by supplying a high-fat, high-fructose, high-cholesterol diet to normal C57BL / 6 mice (about 6 weeks old) for about 21 weeks and increasing the weight of the mice to about 50g on average. Thereafter, the polypeptides were administered every other day by subcutaneous injection at a dose of 20nmol / kg for 2 weeks. Body weight and food intake were measured every other day at a given time point. After a 2-week treatment period, the mice were killed and liver weight, liver triglycerides and NASH-related biomarkers were measured.

[0417] Peptides 5A, 5-1A, 5-2A, 5-3A, 5-4A and 5-5A were administered subcutaneously once daily at 20 nmol / kg as shown in Table 20. DD01, administered subcutaneously every other day at 40 nmol / kg as a positive control, was delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. Body weight was monitored once daily for 4 days.

[0418] Table 20. Treatment doses and schedules.

[0419]

[0420] Peptides 5A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A and 12-7A were administered subcutaneously once daily at 20 nmol / kg as summarized in Table 21. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. Food intake, blood glucose and body weight changes were monitored once daily.

[0421] Table 21. Treatment doses and schedules.

[0422]

[0423] result

[0424] The therapeutic effects of the peptides were evaluated in mice. The body weight of mice treated with peptides 5A, 5-1A, 5-2A, 5-3A, 5-4A and 5-5A was monitored over a 4-day period. Figures 10A-10B). A superior weight loss effect was observed in the groups treated with 5A and 5-1A. Based on body weight measurements, the therapeutic efficacy was most significant in the groups treated with peptides 5A and 5-1A, followed by 5-2A and 5-5A, then 5-4A, with the least effect observed in 5-3A.

[0425] The food intake, blood glucose and body weight changes of mice treated with peptides 5A, 12-1A, 12-2A, 12-3A, 12-4A, 12-5A and 12-7A were monitored over a period of 3 days ( Figures 11A-11D ).

[0426] Example 6: Peptide screening for improving lipidation sites

[0427] method

[0428] Peptides 5, 5-1, 5-2, 5-3, 5-4 and 5-5 were synthesized by lipidation at new positions to further improve the activity of these peptides when lipidated. Three additional sequences were also synthesized as peptides 5-6D, 5-7D and 5-3F. Table 22 shows the sequences where lipidation is present at the indicated residues (K*) and not present at Cys40. The fatty acid derivative used for conjugation here is 2OEG-γGlu-C18. Table 23 summarizes the sites and residues suitable for lipidation of these peptides.

[0429] Table 22. Peptide sequences

[0430]

[0431]

[0432] Table 23. Residues and positions suitable for modification

[0433]

[0434] result

[0435] The second peptide screen was designed to screen for peptides with increased activity on GIPR without reduced activity on glucagon and GLP-1 using peptides 5 and 12 as lead sequences. This was achieved at the peptide level, but when lipidated at the C-terminus. Therefore, the third screen was designed to maintain the potency of those peptide derivatives based on peptide 5 from the second screen, but to change the position of lipidation to improve activity.

[0436] Peptide 5A, peptide 5-1A, peptide 5D, peptide 5-1D, peptide 5-2D, peptide 5-3D, peptide 5-4D, peptide 5-5D, peptide 5-1E and peptide 5-1F were screened in three target cell lines, GLP-1R+ cells, GCGR+ cells, and GIPR+ cells, at five different concentrations of 0.3 nM, 1 nM, 10 nM, 100 nM and 300 nM or 1000 nM using a cAMP assay. Figures 12A-12C The EC values ​​of the corresponding natural ligands of these peptides are summarized in Table 24. 50 EC of each peptide used in the cAMP assay 50 The EC values ​​of GLP-1 peptides are summarized in Table 25. 50 EC of each peptide used in the cAMP assay 50 Table 26 summarizes the relative ratios of EC 50 EC of the corresponding non-lipidated peptide 50 Relative ratios. Based on the cAMP assay, the activity of these peptides towards GLP-1R was maintained when lipidated at K10, K12, K17, or C40; activity towards GCGR was maintained when lipidated at K17; and activity was minimally decreased when lipidated at K12.

[0437] Table 24. EC of natural ligands and triple agonist peptides 50 Ratio

[0438]

[0439] Table 25. EC of GLP-1 and triple agonist peptides 50 Ratio

[0440]

[0441] Table 26. Candidate triple agonist peptides and their corresponding non-lipidated forms EC 50 Ratio

[0442]

[0443] Example 7: In vivo efficacy study on weight loss in normal mice

[0444] method

[0445] Peptide 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E and 5-1F were administered subcutaneously once daily at 20 nmol / kg as shown in Table 27. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. Food intake, blood glucose and body weight changes were monitored once daily.

[0446] EL.EX.14, a triple agonist from published PCT application WO2019 / 125938, was used as a control and has the following amino acid sequence:

[0447] Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)-CO-(CH2) 18 -CO2H)AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH2 (SEQ ID NO: 133).

[0448] Table 27. Treatment doses and schedules.

[0449]

[0450]

[0451] result

[0452] The food intake, blood glucose and body weight changes of mice treated with peptides 5D, 5-1D, 5-2D, 5-3D, 5-4D, 5-5D, 5-1E and 5-1F were monitored over a period of 4 days ( Figures 13A-13D ).

[0453] The peptide showed a significant weight-loss effect.

[0454] Table 28. Weight loss of normal mice after 48 or 72 hours

[0455]

[0456]

[0457] Example 8: In vivo efficacy study in the mAMLN mouse model

[0458] method

[0459] Peptides 5-1A, 5D, 5-2D, 5-1D, 5-2D, DD01 and EL.EX.14 were administered subcutaneously at 20 nmol / kg every other day for 2 weeks as shown in Table 29. Peptides were delivered as 0.02% PS80 in PBS (DD01:F1 only). Food intake and body weight changes were monitored every other day; blood glucose (0 hours, 4 hours, 1 day, every 5 days, end); serum chemistry (end); liver TG (end); inflammatory markers (end). Animals were fasted for at least 4 hours before blood glucose measurements. The control group was administered with a vehicle without peptide.

[0460] Table 29. Treatment doses and schedules.

[0461]

[0462] result

[0463] Nonalcoholic steatohepatitis (NASH) is an obesity-associated liver disease with significant unmet medical needs. Various diet-induced NASH obesity animal models have been used for preclinical studies, target discovery, and drug development. The trans-fat-containing amylin liver NASH (AMLN) diet high in fat, fructose, and cholesterol has been widely used in ob / ob and C57BL / 6J mice to reliably induce metabolic and liver histopathological changes that recapitulate the hallmarks of NASH.

[0464] Figures 14A-14C Figure 2 shows changes in body weight and blood glucose in AMLN mice treated with peptides 5-1A, 5D, 5-2D, 5-1D and 5-3D and positive controls DD01 and EL.EX.14 over a 2-week period. All tested peptides 5-1A, 5D, 5-2D, 5-1D and 5-3D were able to reduce body weight and lower blood glucose in NASH mice.

[0465] At the end of the treatment, liver weight and liver triglyceride content were determined and are shown in Figures 14D-14E All tested peptides 5-1A, 5D, 5-2D, 5-1D and 5-3D were able to reduce liver weight and lower liver triglyceride content.

[0466] In addition, the tested peptides 5-1A, 5D, 5-2D, 5-1D and 5-3D were able to reduce both TGF-β1 and ACTA2 as indicated by the expression levels of inflammatory markers (TGF-β1) and fibrosis markers (ACTA2, a-SMA) measured at the mRNA level. Figures 14F-14G shown.

[0467] Example 9: Peptide screening for improving biotinylation sites

[0468] method

[0469] The direct use of natural polypeptides as biopharmaceuticals is often limited by their very short systemic half-life (caused by rapid metabolism, enzymatic degradation, and efficient renal clearance of smaller proteins and peptides). Modifications such as biotinylation and lipidation are introduced to improve the in vivo stability, bioavailability, and absorption of these peptides. Peptides derived from SEQ ID NOs: 66 and 77 were synthesized by substitution at several amino acids and biotinylation at five different positions to enhance the stability and oral absorption of these peptides. Table 30 shows the presence of lipidation at the indicated lysine residue (K*), at the indicated lysine residue (B*) or at the indicated cysteine ​​residue (B*). #) and a sequence in which methionine is replaced with methionine (m*). This modification using methionine is to enhance antioxidant stability. The fatty acid derivative used for conjugation here is OEG2-γGlu-C18. The biotin derivative used for conjugation here is a biotin monomer.

[0470] In vitro activity studies were performed to determine the biotinylation sites by matching the activity changes against each receptor according to the biotin conjugation position. As previously described, the activity of the in vitro test peptides against GLP-1R, GCGR and GIPR was determined using cAMP. Their signals generated from cAMP were compared with the natural ligand peptides (GLP-1, GCG or GIP).

[0471] Table 30. Peptide sequence optimized biotinylation sites.

[0472]

[0473]

[0474] result

[0475] SEQ ID NOs: 77, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 and 96 ( Figures 15A-15C ). Using cAMP assay, SEQ ID NOs: 66, 97 and 98 were screened in three target cell lines, GLP-1R+ cells, GCGR+ cells, GIPR+ cells, at five different concentrations of 0.1 nM, 0.3 nM, 1 nM, 10 nM and 100 nM ( Figures 16A-16C ).

[0476] SEQ ID NOs: 84, 85, 86, 87, 88, 89, 90, 91, 92, 95 and 96 were selected for comprehensive screening and screening using a cAMP assay to determine their ECs in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The EC values ​​of their corresponding natural ligands and GLP-1 and triple agonist peptides are shown in Table 31. 50 relative ratio.

[0477] As a result of comparing their activities by biotinylating SEQ ID NO: 77 at five different positions, the biotinylation site that minimizes the loss of peptide activity was determined. The biotinylation site was confirmed again using an additional peptide, SEQ ID NO: 66. Based on these results in the cAMP assay, the activity against these three target receptors was maintained when biotinylation was performed at K16, K17, K24, or K40.

[0478] Table 31. EC of native ligands and GLP-1 and triple agonist peptides 50 ratio.

[0479]

[0480] Example 10: In vivo efficacy study on weight loss in HFD mice

[0481] method

[0482] This study was conducted to confirm the weight loss effect of mice treated with a high fat diet (HFD) and to select peptides for evaluation. The mice are widely used as experimental animal models in obesity and diabetes research. SEQ ID NO: 86, 87, 89 and 90 were administered subcutaneously at 20nmol / kg every other day for 2 weeks, as shown in Table 32. The peptides were delivered as 0.02% PS80 in PBS, and the dosing volume was 10mL / kg. Body weight changes, blood glucose and food intake were monitored every other day. Animals were fasted for at least 4 hours before blood glucose measurements.

[0483] Table 32. Treatment doses and schedules.

[0484]

[0485] result

[0486] The body weight changes, blood glucose and food intake of HFD mice treated with SEQ ID NOs: 77, 86, 87, 89 and 90 were monitored over a 2-week period ( Fig.17A and 17B ).

[0487] The weight loss of SEQ ID NOs:86, 87, 89 and 90 (biotinylated at different sites in SEQ ID NO:77) was compared to the non-biotinylated SEQ ID NO:77. SEQ ID NOs:86 and 87 were inferior in weight loss effect, and SEQ ID NOs:89 and 90 were comparable to SEQ ID NO:77.

[0488] Example 11: In vitro stability study of FaSSIF / P

[0489] method

[0490] This study was conducted to determine the enzyme stability of SEQ ID NO: 77, 86, 87, 89, 90 and 91 in an artificial intestinal environment after biotinylation. Test samples were prepared by mixing a stock solution of peptides dissolved in FaSSIF solution (pH 6.5) and pancreatin at a weight ratio of 25:1. The test samples were incubated at 37°C for 120 minutes. Samples were taken from the test samples at predetermined time points (10 minutes, 30 minutes, 60 minutes and 120 minutes) and the incubation was stopped with a 10% TFA solution.

[0491] The residual amount was determined by analytical HPLC on an ACQUITY Premier system (Waters, USA) using an ACQUITY CSHC18 column (Waters, USA) at 35° C. using 0.1% TFA in water and acetonitrile. The residual amount was determined as the percentage of the peak area at the sampling time point relative to the peak area of ​​the initial sample.

[0492] result

[0493] The enzyme stability of SEQ ID NO: 86, 87, 89, 90 and 91 was determined in an artificial intestinal environment compared to SEQ ID NO: 77. The half-life determined by the remaining amount in FaSSIF / P is summarized in Table 33. As a result of this study, SEQ ID NO: 89 and 90 showed intestinal stability that was not inferior to SEQ ID NO: 77 ( Fig.18 ).

[0494] Table 33. In vitro stability study of SEQ ID NOs: 77, 86, 87, 89, 90 and 91 against FaSSIF / P.

[0495] SEQ ID NO. Half-life (minutes) 77 39.4 86 25.4 87 16.4 89 43.0 90 42.1 91 33.3

[0496] Example 12: In vivo efficacy study in CDA-HFD mice

[0497] method

[0498] This study was conducted to evaluate the efficacy of SEQ ID NO: 77, 87, 89, 90, 95 and 96 to determine the effects of biotinylation and amino acid substitution on the efficacy of mice treated with a choline-deficient, L-amino acid defined high-fat diet (CDA-HFD), which is widely used as an experimental animal model in NAFLD / NASH research. SEQ ID NO: 77, 87, 89, 90, 95 and 96 were administered subcutaneously at 20 nmol / kg every other day for 4 weeks, as shown in Table 34. The peptides were delivered as 0.02% PS80 in PBS, and the dosing volume was 10 mL / kg. The control group was administered by a vehicle without the peptide.

[0499] Body weight changes were monitored every other day. After the 4-week treatment period, CDA-HFD mice were sacrificed and serum chemistry analysis was performed at Genia (Seongnam, Gyeonggi, Korea) using HITACHI 7180 (Hitachi High-Tech Korea, Seongnam, Gyeonggi, Korea). ALT and AST in hepatocytes and several lipid markers (LDL and TG) in serum were analyzed.

[0500] Table 34. Treatment doses and schedules.

[0501]

[0502] result

[0503] Fig.19A Shown are changes in body weight over a 4-week period in CDA-HFD mice treated with SEQ ID NOs: 77, 87, 89, 90, 95, and 96. All peptides tested were able to reduce body weight loss in the NASH mouse model.

[0504] At the end of treatment, ALT, AST and LDL in hepatocytes and several lipid markers in serum were analyzed and Figures 19B-19D All tested peptides were shown to improve serum lipid profiles.

[0505] Example 13: Screening for peptides with improved stability by replacing the 20th amino acid and optimizing the lipid structure

[0506] method

[0507] It is necessary to optimize the peptide sequence for oral delivery studies by changing amino acids that are susceptible to intestinal enzymes. In addition, a prolonged plasma half-life helps to reduce the variability of systemic exposure at steady state when administered orally.

[0508] By replacing the 20th amino acid arginine, synthesize the peptide derived from SEQ ID NO:77, to increase intestinal stability and optimize lipid structure, thereby prolonging the half-life in vivo. Table 35 shows that there is lipidation at the indicated lysine residue (K*, K** or K***), there is biotinylation at the indicated lysine residue (B*) and the sequence with α-methylarginine (αR). The fatty acid derivative used for conjugation here is 2OEG-γGlu-C18 (K*), 2OEG-γGlu-C20 (K**) or OEG-γGlu-C20 (K***). The biotin derivative used for conjugation here is a biotin monomer.

[0509] In vitro activity studies were performed to determine the amino acid substitutions and lipid structures by demonstrating changes in activity for each receptor. As previously described, the activity of the in vitro test peptides against GLP-1R, GCGR and GIPR was determined using cAMP. Their signals generated from cAMP were compared with the natural ligand peptides (GLP-1, GCG or GIP).

[0510] Table 35. Peptide sequences

[0511]

[0512] result

[0513] Compared with SEQ ID NOs: 77, 89 and 90, SEQ ID NOs: 99, 100, 101, 102, 103, 104 and 105 ( Figures 20A-20C ).

[0514] SEQ ID NOs: 99, 100 and 101 were comprehensively screened in the cAMP assay to determine their EC activity in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells compared to SEQ ID NO: 77. 50 The EC of their corresponding natural ligands are shown in Table 36. 50 EC with triple agonist peptide 50 relative ratio.

[0515] SEQ ID NO: 99, 100 and 101 were synthesized by substituting the 20th amino acid of SEQ ID NO: 77 to improve stability in the intestinal environment. When compared with SEQ ID NO: 77, the in vitro activities of SEQ ID NO: 99, 100 and 101 were changed according to the amino acid substitution. Compared with SEQ ID NO: 77, the activity on GLP-1R was slightly reduced in SEQ ID NO: 99, and the activity on both GLP-1R and GIPR was reduced in SEQ ID NO: 100. In the case of SEQ ID NO: 101, the activities on GLP-1R and GIPR were increased.

[0516] Table 36. In vitro activities of SEQ ID NOs: 77, 99, 100 and 101.

[0517]

[0518] Example 14: Pharmacokinetic study in rats

[0519] method

[0520] This study was conducted to evaluate the PK properties of SEQ ID NOs: 77, 102, and 103 and to compare the PK properties of different lipidated structures of the peptides in rat plasma by intravenous administration. SEQ ID NOs: 77, 102, and 103 were administered intravenously at 100 nmol / kg as shown in Table 37. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 2.5 mL / kg.

[0521] Animals were not fasted before administration. At each time point (0.167 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours and 48 hours after administration), 0.2-0.4 mL of whole blood was drawn from the vein and transferred to a heparin-coated tube. 0.1-0.2 mL of plasma obtained by centrifugation of the blood sample was transferred to a tube and stored at -70°C until analysis. The concentration of peptides in plasma was determined by LC-MS / MS analysis.

[0522] Table 37. Treatment doses and schedules.

[0523]

[0524] result

[0525] Table 38 shows the PK parameters presented after a single intravenous administration of SEQ ID NOs: 77, 102 and 103.

[0526] According to the average values ​​of PK parameters, SEQ ID NO: 102 and 103 showed similar PK parameter values ​​despite the difference in lipid structure, and SEQ ID NO. 77 showed significant differences in half-life and AUC compared with SEQ ID NO: 102 and 103. inf / dose with significantly lower PK parameter values.

[0527] Table 38. Mean PK parameters of SEQ ID NO: 77, 102 and 103 in rats after a single intravenous administration

[0528]

[0529] Each value represents the mean ± SD (CV %) of the replicates provided. *, PK parameters calculated using four individual subjects. Different letters (a and b) indicate statistical differences (p<0.05) in the PK parameter results.

[0530] Example 15: In vitro stability study of FaSSIF / P and trypsin

[0531] method

[0532] This study was conducted to determine the enzyme stability of SEQ ID NOs: 77, 99, 100 and 101 in an artificial intestinal environment after substitution of the 20th amino acid.

[0533] For stability against FaSSIF / P, test samples were prepared by mixing the stock solution of peptide and pancreatin dissolved in FaSSIF solution (pH 6.5) at a weight ratio of 19:1. The test samples were incubated at 37°C for 120 minutes. Samples were taken from the test samples at predetermined time points (10 minutes, 30 minutes, 60 minutes, and 120 minutes) and the incubation was stopped with 10% TFA solution.

[0534] For stability against trypsin, test samples were prepared by mixing a stock solution of peptide and trypsin dissolved in 50 mM PBS (pH 7.8) containing 0.02% PS80 at a weight ratio of 10: 1. The test samples were incubated at 37°C for 180 minutes. Samples were taken from the test samples at predetermined time points (10 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, and 180 minutes) and the incubation was stopped with a 10% TFA solution.

[0535] The residual amount was determined by analytical HPLC on an ACQUITY Premier system (Waters, USA) using an ACQUITY CSHC18 column (Waters, USA) at 35° C. using 0.1% TFA in water and acetonitrile. The residual amount was determined as the percentage of the peak area at the sampling time point relative to the peak area of ​​the initial sample.

[0536] result

[0537] The enzyme stability of SEQ ID NO: 99, 100 and 101 compared to SEQ ID NO: 77 was determined in an artificial intestinal environment. Table 39 summarizes the half-life determined by the remaining amount in FaSSIF / P and trypsin. As a result of this study, SEQ ID NO: 99 and 100 showed similar stability against FaSSIF / P and increased half-life against trypsin compared to SEQ ID NO: 77. SEQ ID NO: 101 showed improved stability against both FaSSIF / P and trypsin compared to SEQ ID NO: 77. ( Fig.21A and 21B ).

[0538] Table 39. In vitro stability study of SEQ ID NOs: 77, 99, 100 and 101 against FaSSIF / P and trypsin.

[0539]

[0540] Example 16: In vivo efficacy study in CDA-HFD mice

[0541] method

[0542] This study was conducted to evaluate the efficacy of SEQ ID NO: 77, 99, 100, 101, 102 and 103 to determine the effect of amino acid substitutions and lipid structure on efficacy in CDA-HFD mice (NASH model). SEQ ID NO: 77, 99, 100, 101, 102 and 103 were administered subcutaneously every other day at 20 nmol / kg for 4 weeks as shown in Table 40. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group was administered by vehicle without peptide.

[0543] Body weight changes; serum chemistries (end); liver TGs (end); and histological analysis (end) were monitored every other day.

[0544] Table 40. Treatment doses and schedules.

[0545]

[0546] result

[0547] Fig.22A Shown are changes in body weight of CDA-HFD mice treated with SEQ ID NOs: 77, 99, 100, 101, 102, and 103 over a 4-week period. In the NASH mouse model, SEQ ID NOs: 101, 102, and 103 were shown to reduce body weight compared to vehicle-treated controls. Specifically, the greatest weight loss was observed in SEQ ID NO: 101.

[0548] At the end of treatment, ALT and AST in hepatocytes, several lipid markers (LDL and TG) in serum, histological scores were analyzed and Figures 22B-22F SEQ ID NOs: 101, 102 and 103 showed significant reductions in liver enzymes and hepatic steatosis compared to vehicle-treated controls. Hepatic steatosis scores were significantly reduced in SEQ ID NOs: 101 and 103 compared to CDA-HFD controls. Hepatic cell inflammation scores and NAS scores were statistically reduced in SEQ ID NOs: 101, 102 and 103. NAS scores for SEQ ID NO: 101 decreased the most.

[0549] Among the test groups, SEQ ID NO: 101 had the most excellent efficacy in the NASH model. Compared with SEQ ID NO: 77, the lipid structure of SEQ ID NO: 103 had superior efficacy.

[0550] Example 17: In vivo efficacy study in mAMLN mice

[0551] method

[0552] This study was conducted to evaluate the efficacy of SEQ ID NO: 77, 89, 97, 104 and 101 to demonstrate the effects of biotinylation, amino acid substitutions and altered lipid structures on efficacy in mAMLN mice (a model of obesity and NASH). Tesiparatide, a dual agonist of GLP-1 and GIP from Eli Lilly, was used as a positive control. SEQ ID NO: 77, 89, 97, 104, 101 and Tesiparatide were administered subcutaneously at 20 nmol / kg every other day for 4 weeks as shown in Table 41. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group was administered by solution without peptide.

[0553] Body weight changes; liver weight (end); visceral fat weight (end); serum chemistry (end); liver TG (end); inflammatory markers (end); and histological analysis (end) were monitored every other day.

[0554] Table 41. Treatment doses and schedules.

[0555]

[0556] result

[0557] Figures 23A-23H Shown are changes in body weight, liver weight, visceral fat weight, ALT, AST, LDL, liver TG, and histological scores of mAMLN mice treated with SEQ ID NOs: 77, 87, 97, 104, and 101 and tesiparatide as a positive control over a 4-week period.

[0558] After 4 weeks of administration, SEQ ID NOs: 97 and 101 and tesiparatide showed similar weight loss to the food 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 the positive control tesiparatide.

[0559] In the histological scores, hepatocyte ballooning was significantly reduced in all treated groups, and hepatic steatosis was significantly reduced in the groups treated with SEQ ID NOs: 89, 97, 104, and 101. Overall, the NAS scores in SEQ ID NOs: 97 and 101 were statistically reduced beyond the level of tesiparatide.

[0560] Example 18: Peptide Screening for Sequence Optimization

[0561] method

[0562] Based on the results of previous studies, SEQ ID NO:66 and 101 were selected for further optimization of peptide sequences. Peptides derived from SEQ ID NO:66 and 101 were synthesized by replacing the 20th amino acid with αR to improve intestinal stability. Table 42 shows the presence of lipidation at the indicated lysine residues (K* or K**), biotinylation at the indicated lysine residues (B*), and sequences with α-methylarginine (αR). The fatty acid derivatives used for conjugation here are 2OEG-γGlu-C18 (K*) or 2OEG-γGlu-C20 (K**). The biotin derivative used for conjugation here is a biotin monomer.

[0563] In vitro activity studies were performed to determine the best sequence by demonstrating activity against each receptor. As previously described, the activity of the peptides tested in vitro against GLP-1R, GCGR and GIPR was determined using cAMP. Their signals generated from cAMP were compared with the native ligand peptides (GLP-1, GCG or GIP).

[0564] Table 42. Peptide sequences

[0565]

[0566]

[0567] result

[0568] Compared with SEQ ID NOs: 66, 97 and 101 and tesiparatide, SEQ ID NOs: 106, 107, 108, 109, 110, 111 and 112 ( Figures 24A-24C ).

[0569] SEQ ID NOs: 107, 108, 110 and 112 were comprehensively screened in the cAMP assay to determine their EC activity in three target cell lines: GLP-1R+ cells, GCGR+ cells, and GIPR+ cells. 50 The EC of their corresponding natural ligands are shown in Table 43. 50 Relative ratio to the EC50 of the triple agonist peptide.

[0570] Table 43. In vitro activities of SEQ ID NOs: 107, 108, 110 and 112.

[0571]

[0572] Example 19: In vivo efficacy study in CDA-HFD mice

[0573] method

[0574] This study was conducted to evaluate the efficacy of SEQ ID NOs: 97, 110, 108, 111 and 112 to determine the effect of lipid structure and biotinylation on efficacy in CDA-HFD treated mice. SEQ ID NOs: 97, 110, 108, 111 and 112 were administered subcutaneously every other day at 20 nmol / kg for 4 weeks compared to tesiparatide as shown in Table 44. The peptides were delivered as 0.02% PS80 in PBS and the dosing volume was 10 mL / kg. The control group was administered by vehicle without peptide.

[0575] Body weight changes; liver weights (end); serum chemistries (end) and liver TGs (end) were monitored every other day.

[0576] Table 44. Treatment doses and schedules.

[0577]

[0578]

[0579] result

[0580] Figures 25A-25G Shown are changes in body weight, liver weight, ALT, AST, LDL and liver TG of CDA-HFD mice treated with NOs: 97, 108, 110, 111 and 112 and tesiparatide as a positive control over a 4-week period.

[0581] After 4 weeks of administration, all treated peptides and tesiparatide showed similar body weight loss, liver weight loss, and improvements in liver-related markers.

[0582] Example 20: Intestinal absorption after enteral administration in rats

[0583] method

[0584] This study was conducted to evaluate the intestinal absorption of peptides after enteral administration of oral drug formulations. The drug formulation was administered to rats by intraduodenal (ID) injection in the form of a suspension. SEQ ID_87, 89 and 90 were accurately weighed and dissolved in 10mM PBS containing polysorbate 80 (pH 7.4, vehicle I). The mixture was vortexed for 5 minutes to obtain a clear solution. Then, 2.5mL of vehicle was added to the mixture and vortexed for 30 minutes to obtain a uniform opaque suspension. The composition of vehicle II is sodium chenodeoxycholate and propyl gallate in polysorbate 80 in PBS. Animals were not fasted before administration. 0.2 to 0.4mL of whole blood was drawn from the vein at each time point (0 hours, 0.167 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours and 48 hours after intravenous or intraduodenal administration). The collected whole blood was incubated at room temperature for 20 minutes, and the tube was centrifuged at 13,000 rpm for 10 minutes at 4°C. The resulting serum 0.1 mL to about 0.2 mL was transferred to a tube and stored at -70°C until analysis. The concentration of peptides in serum was determined by LC-MS / MS analysis. PK parameters were analyzed by non-compartmental analysis using Phoenix Win Nonlin 5.0.1. software (Pharsight Corporation, Mountain View, CA, USA).

[0585] Table 45. Treatment doses and schedules.

[0586]

[0587] result

[0588] Table 46 shows the PK parameters presented after a single intraduodenal administration of SEQ ID NOs: 87, 89, and 90. Intestinal absorption was demonstrated after enteral administration of oral drug formulations.

[0589] According to the mean values ​​of PK parameters, after intraduodenal administration of SEQ ID_87, 89 and 90 in rats, the mean C max The values ​​were 289.53 ng / mL, 331.73 ng / mL and 281.58 ng / mL, and the C max After intraduodenal administration of SEQ ID_87, 89, and 90, t1 / 2 were 6.17, 7.24, and 8.37, respectively, and AUCinf values ​​were 1,861.06 ng / mL, 2,793.64 ng / mL, and 2,488.01 ng / mL, respectively.

[0590] Table 46. Mean PK parameters of SEQ ID NOs: 87, 89 and 90 in rats after a single intraduodenal administration.

[0591] PK parameters SEQ ID_87 SEQ ID_89 SEQ ID_90 Group 1 2 3 Replicas (quantity) 4 5 5 Route of administration In the duodenum In the duodenum In the duodenum <![CDATA[C max (ng / mL)]]> 289.53±109.9 331.73±124.32 281.58±88.52 <![CDATA[T max (Hours)]]> 0.25±0.17 0.43±0.15 0.30±0.18 <![CDATA[t 1 / 2 (Hours)]]> 6.17±1.36 7.24±2.05 8.37±0.92 <![CDATA[MRT inf (Hours)]]> 8.19±1.92 9.89±3.91 11.84±1.48 <![CDATA[AUC inf (ng hour / mL)]]> 1,861.06±1,056.95 2,793.64±1,464.66 2,488.01±824.48

[0592] Each value represents the mean ± SD of replicates presented.

[0593] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The publications cited herein and the materials cited therein are specifically incorporated by reference.

[0594] Those skilled 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 appended claims.

Claims

1. A triple agonist peptide or an analog thereof, wherein the triple agonist peptide or an analog thereof has activity on each of a glucagon-like peptide-1 (GLP-1) receptor, a glucagon receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the triple agonist peptide or an analog thereof comprises X1X2X3GTFTSDX 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 GX 30 X 31 SX 33 X 34 X 35 PP X 38 X 39 X 40 (SEQ ID NO:131), Where X1 is H or Y; X2 is A or 2-aminoisobutyric acid (Aib); X3 is Q or E; X 12 is R, W or K; X 13 is L or Y; X 19 It is Q, A or T; X 21 is D or L; X 22 is F or R; X 23 is V, G or D; X 25 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 Does not exist, is S or C; X 40 does not exist, or C; The C-terminus has an optional amide modification, and X 10 , X 16 , X 17 , X 18 , X 20 , X 24 and X 28 , any of the 20 amino acids except cysteine, Alternatively, X 17 , X 18 , X 20 are unnatural amino acids independently selected from the group consisting of methoxinine, 2-aminoisobutyric acid, and α-methylarginine.

2. The triple agonist peptide or analog according to claim 1, wherein 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 methoxythiothreitol; X 18 is A, I, M, W, T, D, Y or methoxythiothreitol; X 20 is R, Q, H, G, A, P, N, K, Aib, or α-methylarginine; X 24 is Q, D, K, L, N, W or M; and X 28 It is N, E, G, D, H or Q.

3. The triple agonist peptide or analog according to claim 1 or 2, which has an amino acid sequence of any one of SEQ ID NOs: 1-130.

4. A triple agonist peptide or analogue according to any one of claims 1 to 3, wherein the peptide or analogue is conjugated to one or more of a biotin moiety, a fatty acid or polyethylene glycol and derivatives thereof, optionally via one or more spacers.

5. The triple agonist peptide or analog according to claim 4, wherein the one or more biotin moieties, the fatty acid or the polyethylene glycol and derivatives thereof are conjugated to the 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.

6. A triple agonist peptide or analog according to claim 5, wherein one or more amino acid residues of cysteine ​​and lysine are introduced into the amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to allow conjugation with said one or more biotin moieties, said fatty acids or said polyethylene glycol and derivatives thereof.

7. A triple agonist peptide or analogue according to any one of claims 3 to 6, wherein one or more amino acid residues of lysine at position 10, lysine at position 12, lysine at position 17, lysine at position 20, lysine at position 24, one or more C-terminal cysteine ​​residues are introduced into the amino acid sequence of any one of SEQ ID NOs: 1-130 by substitution or insertion to allow conjugation with said one or more biotin moieties, said fatty acids or said polyethylene glycol and derivatives thereof.

8. A triple agonist peptide or analogue according to any one of claims 4 to 7, wherein the biotin moiety and its derivatives suitable for conjugation are selected from the group consisting of: biotin N-hydroxysuccinimide ester, N-biotinyl-N'-(6-maleimidocaproyl)hydrazide, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-CONH2, 3-maleimidopropionate-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2, propionic acid-N-hydroxysuccinimide ester-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2 and 3-maleimidopropionate-PEG-Lys(biotin)-Lys(biotin)-Lys(biotin)-CONH2.

9. A triple agonist peptide or analogue according to any one of claims 4 to 7, wherein the fatty acids and derivatives thereof suitable for conjugation via one or more hydrophilic spacers are C16-C22 fatty acids.

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

11. A triple agonist peptide or analogue according to any one of claims 4 to 10, wherein the fatty acid or derivative thereof suitable for conjugation is selected from the group consisting of 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-MAL, C18-γGlu-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.

12. A pharmaceutical formulation comprising the triple agonist peptide or analogue according to any one of claims 1 to 11.

13. A method of treating one or more diseases in a subject in need thereof, wherein the one or more diseases are selected from the group consisting of obesity, diabetes, and non-alcoholic fatty liver disease, the method comprising: An effective amount of the pharmaceutical formulation according to claim 12 is administered to treat or alleviate one or more symptoms of the one or more diseases.

14. The method of claim 13, wherein 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 normal or obese patients.

15. The method of claim 13 or 14, wherein the subject suffers from non-alcoholic fatty liver disease. 16 . The method 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 disease, non-alcoholic steatohepatitis, cirrhosis and liver cancer.

17. The method of claim 15 or 16, wherein the drug formulation is administered in an amount effective to inhibit or reduce serum levels of one or more of: alanine aminotransferase, aspartate aminotransferase, triglycerides, gamma-glutamyl transferase, total cholesterol, low-density lipoprotein, fasting blood glucose, or a combination thereof.

18. The method of any one of claims 15 to 17, wherein the pharmaceutical formulation is administered in an amount effective to reduce one or more of: steatosis, inflammation, ballooning, fibrosis, cirrhosis, or a combination thereof.

19. The method according to any one of claims 13 to 18, wherein the pharmaceutical formulation is administered by a route selected from the group consisting of enteral administration and parenteral administration.

20. The method of any one of claims 13 to 19, wherein the pharmaceutical formulation is administered by oral administration or subcutaneous administration.

21. The method of any one of claims 13 to 20, wherein the pharmaceutical formulation is administered in a form selected from the group consisting of a pill, a capsule, a tablet, a liquid, and a suspension.

22. The method of any one of claims 13 to 21, wherein the pharmaceutical formulation is administered at an interval 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 method of any one of claims 13 to 22, wherein the pharmaceutical formulation is administered to the subject once a week for up to 6 months.

24. The method of any one of claims 13 to 23, wherein the drug formulation is administered to the subject for a duration of one day to 10 days, one week to 10 weeks, or one month to 10 months, inclusive.

25. The method of any one of claims 13 to 24, wherein the pharmaceutical formulation is administered to a human subject at a dose of 0.001 mg / kg of the subject's body weight to 10 mg / kg of the subject's body weight, inclusive.

26. The method of any one of claims 13 to 25, wherein the pharmaceutical formulation is administered to a human subject at a dose of 0.01 mg / kg of the subject's body weight to 1 mg / kg of the subject's body weight, inclusive.

27. The method of any one of claims 13 to 26, wherein the pharmaceutical formulation is administered to a human subject at a dose of 1.0 mg to 100 mg, inclusive.

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