Agonist peptides for modulating GLP-1r, GIPR, and GCGR, compositions and methods thereof

Abstract

The present disclosure provides agonist peptides against incretin hormone receptors for the management of metabolic disorders including Type 2 Diabetes, Obesity, and / or co-morbidities. Also provided are compositions and methods for treatment and management of Type 2 Diabetes, Obesity, and / or co-morbidities.

Description

AGONIST PEPTIDES FOR MODULATING GLP-1R, GIPR, AND GCGR, COMPOSITIONS AND METHODS THEREOF FIELD OF INVENTION

[0001] The present disclosure broadly relates to the field of agonist peptides. In particularly, the invention relates to peptides having agonist activity for therapeutic application. The present invention also relates to compositions and methods for treatment and management of Type 2 Diabetes, Obesity, and / or co-morbidities. BACKGROUND OF INVENTION

[0002] In cretins, the hormones released by the endocrine cells in epithelium of the small intestine, plays an essential role in the regulation of blood glucose levels, energy expenditure and fat deposition. These hormones stimulate the ?-cells in pancreas to release insulin upon sensing the presence of excess glucose in the intestinal lumen.

[0003] Therapeutic approaches involving incretin, incretin mimetics and analogues have been implemented to manage Type 2 Diabetes (T2DM) and Obesity. T2DM, characterized by insulin resistance and relative insulin deficiency, frequently coexists with obesity, marked by excessive adiposity with about two thirds of the T2DM cases diagnosed already having obesity. Historically, managing T2DM and obesity has relied on lifestyle modifications, pharmacotherapy, and, in severe cases, bariatric surgery. However, existing interventions exhibit limitations in efficacy, tolerability, and long-term adherence. Weight loss alone through medication or lifestyle changes is not enough to reduce the progression of T2DM and Obesity. Consequently, there is a burgeoning interest in innovative therapeutic approaches targeting the intricate interplay between T2DM and obesity. Exogeneous insulin therapy was one promising approach to overcome hyperglycemia, but it led to weight gain. Hence, focus shifted to devise therapies that can deal with both hyperglycemia and adiposity. Peptide-based therapies have emerged as promising avenues due to their specificity and potential to modulate key metabolic pathways.

[0004] Peptide based therapies include incretin mimetics. Incretin mimetics having mono agonist and dual agonist activity have previously been developed. However, there is a need for effective and long-acting peptides and therapeutic approaches for improving clinical outcomes and enhancing quality of life for affected individuals while lowering the global burden of T2DM and Obesity.SUMMARY OF THE INVENTION

[0005] In an initial aspect of the present disclosure, there is provided a peptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 38, SEQ ID NO. 39, and SEQ ID NO.40.

[0006] In another aspect of the present disclosure, there is provided a composition comprising the peptide as disclosed herein, and at least one pharmaceutically acceptable carrier.

[0007] In yet another aspect of the present disclosure, there is provided a method for modulating glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR, comprising providing an effective amount of the peptide as disclosed herein.

[0008] In an aspect of the present disclosure, there is provided a method for treatment or management of obesity in a subject, comprising providing an effective amount of the peptide as disclosed herein, to the subject.

[0009] In an aspect of the present disclosure, there is provided a method for treatment or management of Type 2 Diabetes in a subject, comprising providing an effective amount of the peptide as disclosed herein, to the subject.

[0010] In an aspect of the present disclosure, there is provided a method for preparation of the peptide, said method comprising: (a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-natural amino acid; (b) optionally incorporating at least one modified amino acid residue;and cleaving and recovering the peptide to obtain the peptides of the present disclosure.

[0011] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF FIGURES

[0012] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0013] Figure 1 depicts a schematic representation of the amino acid structures of the peptides, in accordance with the embodiments herein.

[0014] Figure 2 depicts the change in body weight in diet-induced obese (DIO) mice after twice weekly subcutaneous dosing of ND-MC-ZB-43 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0015] Figures 3(a) and 3(b) depicts the results of glucose metabolism in DIO mice in response to treatment with the peptide ND-MC-ZB-43. Figure 3(a) shows the results of Oral Glucose Tolerance Test (OGTT) taken at day 28 after twice weekly 0.5 mg / kg body weight ND-MC-ZB-43 treatment. Figure 3(b) depicts the results of Insulin Tolerance Test (ITT) at day 28 after twice weekly administration at 0.5 mg / kg body weight of ND-MC-ZB-43 treatment, in accordance with the embodiments herein.

[0016] Figures 4(a) and 4(b) depict the results of metabolic markers at day 28 after twice weekly administration at 0.5 mg / kg body weight of ND-MC-ZB-43 treatmentDIO mice. Figure 4(a) shows the results of the serum metabolic markers fasting glucose levels, total cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels in treated mice. Figure 4(b) depicts the HbAlc levels in treated mice, in accordance with the embodiments herein.

[0017] Figures 5(a), and 5(b) show the results of serum clinical metabolic parameters: leptin (Figure 5(a)), and adiponectin (Figure 5(b))), at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-43 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0018] Figures 6(a) and 6(b) show the results of visceral fat accumulation at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-43, at 0.5 mg / kg body weight. Figure 6(a) depicts the results of gonadal fat accumulation in the treated mice and Figure 6(b) depicts the results of retroperitoneal fat accumulation in the treated mice, in accordance with the embodiments herein.

[0019] Figure 7 shows the results of hepatic steatosis scoring by oil red O staining at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-43 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0020] Figure 8 depicts the body weight changes over time following treatment with ND-MC-ZB-43, in accordance with the embodiments herein.

[0021] Figures 9depict the dosage dependent effect of ND-MC-ZB-43 on glucose metabolism. Figure 9 shows the fasting glucose levels in DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mg / kg dose (mpk)) for 4 weeks (n = 7 per group for Glucose and n=5 for HbAlc ), in accordance with the embodiments herein.

[0022] Figures 10(a) and 10(b) depict the dosage dependent effect of ND-MC-ZB-43 on glucose metabolism. Data shows the results of Intraperitoneal glucose tolerance test (IpGTT) in DIO mice following 4 weeks of twice-weekly treatment with ND-MC-ZB-43 (0.01-0.3 mg / kg), in accordance with the embodiments herein.

[0023] Figures 11(a) to 11(c) depict the dose dependent effect of ND-MC-ZB-43 in serum lipid markers, Triglycerides (Figure 11(a)), Total cholesterol (Figure 11(b)), , and LDL (Figure 11(c)) at dosages of 0.01-0.3 mpk twice weekly for 4 weeks, in accordance with the embodiments herein.

[0024] Figures 12(a) to 12(e) depict the Luminex quantification of circulating metabolic hormones in DIO mice following ND-MC-ZB-43 treatment. Plasma adiponectin (A), secretin (B), leptin (C), insulin (D), and glucagon (E) concentrations were measured using a Luminex multiplex immunoassay after chronic administration of ND-MC-ZB-43 at 0.01, 0.1, or 0.3 mpk, in accordance with the embodiments herein.

[0025] Figures 13(a) to 13(d) show the histopathological images of liver from DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mpk) for 4 weeks. Liver sections were stained with hematoxylin and eosin (H&E) and captured at 20* magnification, in accordance with the embodiments herein.

[0026] Figures 14(a) to 14(d) show the representative Oil Red O-stained liver sections from DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mpk) for 4 weeks, demonstrating dose-dependent reduction in hepatic lipid accumulation compared with vehicle control, in accordance with the embodiments herein.

[0027] Figures 15(a) to 15(d) show the Representative histopathological images of pancreas from DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mpk) for 4 weeks. Pancreatic sections were stained with hematoxylin and eosin (H&E) and captured at 20* magnification, in accordance with the embodiments herein.

[0028] Figure 16 depicts the change in body weight in diet-induced obese (DIO) mice after twice weekly subcutaneous dosing of ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0029] Figure 17 shows the results of Oral Glucose Tolerance test (OGTT) at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0030] Figure 18 shows the results of Insulin Tolerance test (ITT) at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0031] Figure 19 shows the results of serum clinical metabolic parameters fasting glucose levels, total cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels in treated mice. Measurements were taken at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with embodiments herein.

[0032] Figures 20(a), 20(b), and 20(c) show the results of serum clinical metabolic parameters: leptin (Figure 20(a)), adiponectin (Figure 20(b)), and glucagon (Figure 20(c)), at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0033] Figures 21(a) and 21(b) show the results of visceral fat accumulation at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45, at 0.5 mg / kg body weight. Figure 21(a) depicts the results of gonadal fat accumulation in the treated mice and Figure 21(b) depicts the results of retroperitoneal fat accumulation in the treated mice, in accordance with the embodiments herein.

[0034] Figure 22 shows the results of hepatic steatosis scoring by oil red O at day 28 after twice weekly treatment of diet-induced obese (DIO) mice with ND-MC-ZB-45 at 0.5 mg / kg body weight, in accordance with the embodiments herein.

[0035] Figure 23 shows the results of a Surface Plasmon Resonance (SPR) sensorgram showing immobilisation of GIPR onto a CM5 chip (Fc=l) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and post-immobilisation stabilisation phases, in accordance with the embodiments herein.

[0036] Figure 24 shows (A) the results of a single-concentration binding SPR sensorgram of LY (positive control) at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic SPR sensorgram of LY (0, 4.1, 12, 37, 111, 333, 1000 nM) binding to immobilised GIPR, with 1:1 Langmuir fitting used to determine ka, kd, and KD (B), in accordance with the embodiments herein.

[0037] Figure 25 shows (A) the results of a single-concentration binding sensorgram of Peptide 20 at 50 pM and 5 pM injected over immobilised GIPR, and (B) shows the results of multi-concentration kinetic sensorgram of Peptide 20 (20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GIPR, in accordance with the embodiments herein.

[0038] Figure 26 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-43 at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi -concentration kinetic sensorgram of ND-MC-ZB-43 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0039] Figure 27 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-45 at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi -concentration kinetic sensorgram of ND-MC-ZB-45 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0040] Figure 28 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-43b at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-43b (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0041] Figure 29 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-44b at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-44b (0, 62.5,125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0042] Figure 30 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-45b at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45b (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0043] Figure 31 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-45c at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45c (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0044] Figure 32 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-45d at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45d (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0045] Figure 33 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-45e at 50 pM and 5 pM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45e (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0046] Figure 34 shows the results of a SPR sensorgram showing immobilisation of GLP-1R onto a CM5 chip (Fc=3) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and post-immobilisation stabilisation phases, in accordance with the embodiments herein.

[0047] Figure 35 shows the results of multi-concentration kinetic sensorgram of LY (positive control; 4.1, 12, 37, 111, 333, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0048] Figure 36 shows the results of multi-concentration kinetic sensorgram of Peptide 20 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0049] Figure 37 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-43 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0050] Figure 38 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45 (0, 20, 61, 185, 555, 1666, 5000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0051] Figure 39 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-43b (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0052] Figure 40 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-44b (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0053] Figure 41 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45b (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0054] Figure 42 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45c (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0055] Figure 43 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45d (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0056] Figure 44 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45e (0, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0057] Figure 45 shows the results of a SPR sensorgram showing immobilisation of GCGR (GCGR-lig 3) onto a CM5 chip (Fc=l) via amine coupling, with sequential baseline, EDC / NHS activation, receptor injection, and postimmobilisation stabilisation phases, in accordance with the embodiments herein.

[0058] Figure 46 shows the results of multi-concentration kinetic sensorgram of LY (positive control; 0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0059] Figure 47 shows the results of multi-concentration kinetic sensorgram of Peptide 20 (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR. The low maximal binding response (~8 RU) is indicative of weak binding affinity to this receptor, in accordance with the embodiments herein.

[0060] Figure 48 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45 (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0061] Figure 49 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-43 (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0062] Figure 50 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-43b (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0063] Figure 51 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-44b (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0064] Figure 52 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45b (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0065] Figure 53 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45c (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0066] Figure 54 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45d (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0067] Figure 55 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-45e (0, 156, 312, 625, 1250, 2400, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0068] Figure 56 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-50b at 50 gM and 5 gM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50b (0, 15.5, 31.25, 62.5, 250, 500 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0069] Figure 57 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-50c at 50 gM and 5 gM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50c (0, 15.5, 31.25, 62.5, 250, 500 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0070] Figure 58 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-50d at 50 gM and 5 gM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50d (0, 15.5, 31.25, 62.5, 250, 500 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0071] Figure 59 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-50e at 50 gM and 5 gM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50e (0, 15.5, 31.25, 62.5, 250, 500 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0072] Figure 60 shows (A) the results of single-concentration binding sensorgram of ND-MC-ZB-51 at 50 gM and 5 gM injected over immobilised GIPR, and (B) the results of multi-concentration kinetic sensorgram of ND-MC-ZB-51 (0, 15.5, 31.25, 62.5, 250, 500 nM) binding to immobilised GIPR in accordance with the embodiments herein.

[0073] Figure 61 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50b (0, 31.25, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0074] Figure 62 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50c (0, 31.25, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0075] Figure 63 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50d (0, 31.25, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0076] Figure 64 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50e (0, 31.25, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0077] Figure 65 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-51 (0, 31.25, 62.5, 125, 250, 500, 1000 nM) binding to immobilised GLP-1R, in accordance with the embodiments herein.

[0078] Figure 66 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50c (0, 312, 625, 1250, 2400, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0079] Figure 67 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50b (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0080] Figure 68 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50d (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0081] Figure 69 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-50e (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0082] Figure 70 shows the results of multi-concentration kinetic sensorgram of ND-MC-ZB-51 (0, 156, 312.5, 625, 1250, 2500, 5000 nM) binding to immobilised GCGR, in accordance with the embodiments herein.

[0083] Figure 71 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-IR(A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the positive control peptide Retatrutide, in accordance with the embodiments herein.

[0084] Figure 72 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), and GIPR (B). Plots shown depict the activation of the receptors in presence of the positive control peptide Tirzepatide, in accordance with the embodiments herein.

[0085] Figure 73 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A) and GCGR (B). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-42, in accordance with the embodiments herein.

[0086] Figure 74 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GIPR (A), GCGR (B), and GLP1R (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-43, in accordance with the embodiments herein.

[0087] Figure 75 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-43(b), in accordance with the embodiments herein.

[0088] Figure 76 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB -44(b), in accordance with the embodiments herein.

[0089] Figure 77 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-45, in accordance with the embodiments herein.

[0090] Figure 78 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-45(b), in accordance with the embodiments herein.

[0091] Figure 79 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-45e, in accordance with the embodiments herein.

[0092] Figure 80 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-50(b), in accordance with the embodiments herein.

[0093] Figure 81 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-50(c), in accordance with the embodiments herein.

[0094] Figure 82 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-50(d), in accordance with the embodiments herein.

[0095] Figure 83 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-50(e), in accordance with the embodiments herein.

[0096] Figure 84 depicts results of cAMP based in-vitro assay for peptide induced receptor activation for GLP-1R (A), GIPR (B), and GCGR (C). Plots shown depict the activation of the receptors in presence of the peptide ND-MC-ZB-51, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0097] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0098] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0099] The articles “a”, “an” and “the” are used to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

[0100] The terms “comprise” or “contain” and “comprising” or “containing” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0101] The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures / combinations.

[0102] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0103] The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.

[0104] The term “peptide”, as used herein, broadly refers to a molecule having two or more amino acids residues joined together by peptide bonds. The term “peptide” and “polypeptide” are used interchangeably herein.

[0105] The term “agonist”, as used herein, refers to a substance which promotes the biological activity of another. The term agonist encompasses substances that bind to receptors and substances which promote receptor function without binding thereto. For example, a Glucagon-like peptide-1 (GLP-1) analogue binds to GLP-1 receptor (GLP-1R) to agonize GLP-1R.

[0106] Amino acid residues are generally represented by single letter and three letter abbreviations, for example “Alanine” is represented by single letter code “A” and three letter code “ala” or “Ala”. The expression “X” as used in amino acid sequences refers to any amino acid residue, including natural, non-natural, and modified amino acid residues. In the expression “Xi”, the number ‘ 1 ’ refer to the position of X amino acid in the amino acid sequence of the peptide. For example, “Xi”, denotes X amino acid at position 1, “X2” denotes X amino acid at position 2, and so on. Such representations are generally used and well understood by a person skilled in the art. The present disclosure in describing the present invention employssuch representations or phrases which is intended to mean the generally acceptable meaning in the art.

[0107] Embodiments herein disclose agonist peptides for therapeutic application. The peptides, according to embodiments herein, are incretin mimetics having agonist activity on receptors. The term “incretin”, as used herein, broadly refers to group of metabolic hormones including glucagon-like peptide- 1 (GLP-1) and glucose dependent insulinotropic peptide (GIP). Incretin hormones are naturally-occurring peptide hormones. Embodiments herein provide peptides that are capable of mimicking the activity of incretin hormones. Embodiments herein provide incretin mimetics having agonist activity on receptors, particularly incretin hormone receptors, including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor (GIPR), and glucagon receptor (GCGR). In a preferred embodiment, the peptides as disclosed herein are agonists which exhibit agonist activity on at least one, at least two, or at least three receptors including glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor (GIPR), and glucagon receptor GCGR. Further embodiments herein provide compositions comprising the peptides as disclosed herein. Also disclosed are methods for use and preparation of the peptide disclosed herein. The peptides, according to embodiments herein, are capable of regulating blood glucose and body weight in subjects. Accordingly, embodiments herein include methods for modulating receptors including GLP-1R, GIPR and GCGR, methods for treating and / or management of metabolic disorders including diabetes, obesity and / or comorbidities.

[0108] The present inventors have, by application of computational structural biology, designed unimolecular agonist peptides capable of agonist activity on at least three receptors. With the application of the machine learning (ML), molecular modelling and molecular dynamics simulations (MD), and classical physics based binding free energy calculations, the fine intricacies of peptide- receptor complex formation and protein-protein interactions were studied. This was instrumental in enhancing the binding efficiency and agonistic propertiesof the designed peptides with the three receptors, i.e. GLP-1R, GIPR and GCGR,. Accordingly, the present inventors have been able to achieve agonist peptides having improved properties.

[0109] The peptides, according to embodiments herein, are capable of binding and having selectivity to incretin hormone receptors. The peptides, as disclosed herein, are long acting with good serum half-life. Serum half-life is mainly determined by the two critical factors viz. clearance rate by the kidney and enzyme mediated degradation. It is observed that the peptides of the present invention have increased stability and solubility, showing enhanced activation of GLP-1R, reduced clearance rate, and lower chances of enzyme mediated degradation.Peptides

[0110] Embodiments herein disclose peptides having agonist properties. The peptides, according to embodiments herein, are incretin mimetics having specificity for incretin hormone receptors. The peptides, as disclosed herein, have amino acid sequences comprising natural, non-natural, and / or modified amino acid residues. The amino acid sequence of the peptides is modified to improve binding and serum half-life of the peptides. Embodiments herein further comprise agonist peptides comprising the peptides as disclosed herein. In an embodiment, there is provided an agonist peptide comprising a peptide as described herein.

[0111] In an embodiment, the peptide has an amino acid sequence of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 1. In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1:X1X2QGTFTSDX10S X12YLDE X17 X18AX20DFV X24X25LL X28GGPSSGAPPPS(SEQ ID NO. 1);wherein, Xi, X2, X10, X12, X17, Xis, X2o,X24, X25, and X28 are amino acids selected from natural, non-natural, or modified amino acid residues. Examples of natural, non-natural, and modified amino acid residues include, but are not limited to, histidine (H), tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E) or glutamine (Q), aspartic acid (D), serine (S), tryptophan (W), Alpha-aminobutyric acid (Aib), Alpha-MethylLeucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), Methyl alanine, or acylated forms thereof. Acylated forms include, but are not limited to, modified amino acids having an acyl group conjugated to the amino acid.

[0112] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein, Xi, X2, X10, X12, X17, Xis, X2o,X24, X25, and X28 are amino acids selected from a group consisting of histidine (H) or tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E) or glutamine (Q), aspartic acid (D), tryptophan (W), and serine (S).

[0113] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 1, wherein Xi is selected from Histidine (H) or Tyrosine (Y), X2 is selected from Serine (S) or a non-natural amino acid, X10 is selected from Tyrosine (Y), lysine (K), or modified lysine (Km), X12 is selected from lysine (K), Arginine (R), or modified lysine (Km), X17 is selected from Arginine (R) or Isoleucine (I), Xis is selected from Alanine (A) or Arginine (R), X20 is selected from glutamine (Q), lysine (K), or modified lysine (Km), X25 is selected from Tryptophan (W) or Tyrosine (Y), X24 is selected from glutamic acid (E) or glutamine (Q), and X28 is selected from aspartic acid (D) or serine (S).

[0114] In an embodiment, the peptide has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of:(i) X1X2QGTFTSDX10SX12YLDEX17X18AX20DFVX24X25LLX28GGPSSGAPP PS(SEQ ID NO. 1);whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is selected from Serine (S) or a non-natural amino acid,Xwis selected from Tyrosine (Y), lysine (K), or modified lysine (Km), X12 is selected from lysine (K), Arginine (R), or modified lysine (Km), X17 is selected from Arginine (R) or Isoleucine (I),Xis is selected from Alanine (A) or Arginine (R),X20 is selected from glutamine (Q), lysine (K), or modified lysine (Km), X24 is selected from glutamic acid (E) or glutamine (Q)X25 is selected from Tryptophan (W) or Tyrosine (Y), andX28 is selected from aspartic acid (D) or serine (S);(ii) X1X2QGTFTSDX10SX12X13LDKKAQX20AFIEYLLEGGPSSGAPPPS (SEQ ID NO. 38),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is a non-natural amino acid, preferably Aib,X10 is selected from lysine (K), Tyrosine (Y) or modified lysine (Km), X12 is selected from lysine (K) or Isoleucine (I),X13 is selected from Tyrosine (Y) or non-natural amino acid, preferably Alpha-MethylLeucine (crMeL), andX2ois a non-natural amino acid, preferably Aib;(iii) X1X2QGTFTSDX10 SKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO. 39),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is Serine (S) or a non-natural amino acid, preferably Aib, andXio is selected from lysine (K), Tyrosine (Y) or modified lysine (Km).(iv) X1X2QGTFTSDX10SX12X13LDKKAQX20AFIEYLLEGGPSSGAPPPSX40 (SEQ ID NO. 40),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is a non-natural amino acid, preferably Aib,Xio is selected from lysine (K), Tyrosine (Y) or modified lysine (Km), X12 is selected from lysine (K) or Isoleucine (I),X13 is selected from Tyrosine (Y) or non-natural amino acid, preferably Alpha-MethylLeucine (crMeL),X2ois a non-natural amino acid, preferably Aib,X40 is selected from lysine (K) or modified lysine (Km);or a pharmaceutically acceptable salt thereof.Modifications

[0115] The peptides, according to embodiments herein, include modified amino acid residues. These modifications may be made to amino acids in the peptides at certain positions to facilitate in improving the serum half-life of the peptide. In an embodiment, the amino acid residue is modified by conjugating with a lipid (also referred to herein as “lipid tail”) via a linker and / or a spacer. The lipid tail may bind (reversibly) to serum albumin which increases the serum half-life of the peptide.

[0116] The term “lipid”, as used herein, broadly refers to organic compounds that are soluble in non-polar solvents. The term lipid includes fatty acids and fatty diacids, and their substituted or unsubstituted forms. The term “lipid tail” and “lipid” are used interchangeably herein, and include, substituted or unsubstituted, tail of a fatty acid moiety or tail of a diacid moiety. In an embodiment, the lipid is selected from Cs- C22 fatty acids or diacids. Examples oflipid include, but are not limited to, caprylic acid (Cs), capric acid (Cio), lauric acid (C12), myristic acid (C14), palmitic acid (Cie), stearic acid (Cis), arachidic acid (C20), dodecanedioic acid (C12), tri decanedioic acid (C13), tetradecanedioic acid (C14), pentadecanoic acid (C15), hexadecanedioic acid (Cie), heptadecanedioic acid (C17), octadecanedioic acid (Cis), eicosanedioic Acid (C20), and Docosanedioate (C22). In an embodiment, the lipid is selected from a compound of Formula (a) to (i) depicted in Table 1. In an embodiment, the lipid is selected from a compound of Formula (a), (b), (c), (d), (e), (f), (g), (h), and (i) depicted in Table 1. The lipid may be attached to the amino acid via a linker and / or spacer, in accordance with the embodiment herein.

[0117] The lipid, in various embodiments herein, may comprise a compound of Formula I. In an embodiment, the lipid is a compound of Formula I.CO2H-(CH2)a-CO- Formula Iwherein ‘a’ is in the range of 8 to 22, preferably 12 to 20, more preferably 16 to 18.

[0118] The lipid, in various embodiments herein, may comprise a compound of Formula I(i). In an embodiment, the lipid is a compound of Formula I(i).CH3-(CH2)a-CO- Formula I(i)wherein ‘a’ is in the range of 8 to 22, preferably 12 to 20.

[0119] In an embodiment, the lipid is a compound of Formula I or Formula I(i).

[0120] The linker, in one embodiment herein, is attached to the lipid, preferably at the hydroxyl end of the lipid at one end and a spacer at the other end. The linker, in another embodiment herein, is attached to the lipid at one end, preferably at the hydroxyl end of the lipid and is attached at the other end to the amino acid residue. Examples of linkers include, but are not limited to, one or moregamma-glutamic acid (yGlu) moieties, and Gamma-aminobutyric acid (GABA). In an embodiment, the linker is selected from one or more yGlu moieties, Gamma-aminobutyric acid (GABA), or combination thereof. The linker may comprise of one or more moieties of yGlu. In alternate embodiments, the linker may be one or more moieties selected from benzyl-PAla, yGlu, or alpha-aminobutyric acid-yGlu (Abu-yGlu). The yGlu may be D-yGlu or L-yGlu. Accordingly, in an embodiment, the linker is at least one yGlu linker. In an embodiment, the linker is a compound of Formula (1) depicted in Table 1. In an embodiment, the linker comprises a yGlu linker, wherein the lipid is preferably attached to the yGlu linker at its a -amino group. In another embodiment, the linker comprises two yGlu moieties, wherein the lipid is preferably attached to the a -amino group of one yGlu moiety which is further attached at the carboxyl end to another yGlu moiety. Similarly, the linker may comprise two or more yGlu moieties. The linker may further be attached to a spacer. Alternatively, the linker may further be attached to the amino acid residue.

[0121] The spacer, in some embodiments herein, may be attached to the linker at one end and the amino acid residue at the other end. In other embodiment, the spacer may be attached to the lipid, preferably at the hydroxyl end of the lipid, at one end and the amino acid residue at the other end. Examples of spacers include, but are not limited to, one or more Aminoethylethanolamine (AEEA) moieties, GGGGS repeats, one or more y-Carboxylate moieties, and one or more PEG moieties. In an embodiment, the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), GGGGS repeats, one or more y-Carboxylate moieties, one or more polyethylene glycol (PEG) moieties, or combination thereof.

[0122] The term “PEG”, as used herein, refers to any water-soluble poly(ethylene oxide). The term “PEG” encompasses “Oligo(ethylene glycol)” or “OEG”, and is used interchangeably herein. Typically, PEG comprises the following structure — (OCH2CH2)b — , (also represented herein as (PEG)b ), wherein ‘b’ ranges from 1 to 4000. In an embodiment herein, PEG comprises the following structure — (PEG)b — wherein ‘b’ is in the range of 1 to 8, 2 to 8, preferably 2 to 6.

[0123] The spacer, in various embodiments herein, may comprise a compound of Formula II.

[0124] In an embodiment, the spacer is a compound of Formula II.-(amino-(PEG / AEEA)b-CH2CO2H)c- Formula II

[0125] In an embodiment, the spacer is a compound of Formula II selected from(i) -(amino-(PEG)b-CH2CO2H)c - ; or (ii) -(amino-(AEEA)b-CH2CO2H)c- , wherein ‘b’ is in the range of 1 to 10, preferably 2 to 10, more preferably 2 to 8, and ‘c’ is in the range of 1 to 10, preferably 1 to 8, more preferably 1 to 5.

[0126] In another embodiment, the spacer is y-Carboxylate.

[0127] In an embodiment, the spacer is selected from a compound of Formula (j) to (k) depicted in Table 1.

[0128] Examples of AEEA moieties include, but is not limited to, Aminoethylethanolamine, 17-Amino-10-oxo-3,6,12,15-tetraoxa- 9azaheptadecanoic acid, etc.

[0129] The spacer, according to embodiments herein, is covalently attached to the amino acid residue. In an embodiment, the amino acid residue is lysine (K). The spacer is attached to the epsilon amino group of the lysine side chain. Accordingly, in an embodiment, the modified lysine (Km) is lysine (K) attached to a lipid by a linker and spacer at the s-amino group of the lysine side chain. In another embodiment, the modified lysine (Km) is lysine (K) attached to a lipid by a spacer at the s-amino group of the lysine side chain. In a preferable embodiment, the modified lysine is lysine (K) attached to a lipid, preferably a diacid of Formula I, by at least one yGlu linker and a spacer of Formula II. In another preferable embodiment, the modified lysine is lysine (K) attached to a lipid, preferably a diacid of Formula I, by a spacer, wherein the spacer is y-Carboxylate.

[0130] The term “linker and spacer”, as used herein, refers to the combination of linker and spacer according to embodiments herein. In an embodiment, the linker and spacer is selected from yGlu-PEG, D-yGlu-(PEG)2, (PEG)2- yGlu, yGlu-(PEG)3, yGlu-(PEG)s, benzyl-PAla-(PEG)2, (yGlu)2-(PEG)2, (yGlu) 3-(PEG)2, Abu-yGlu-PEG, Abu-(yGlu)2-PEG, or Abu-(PEG)2.

[0131] In an embodiment, the linker and spacer is selected from a compound of Formula (m) to (x) depicted in Table 1. In an embodiment, the linker and spacer is selected from a compound of Formula (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w) and (x) depicted in Table 1.

[0132] In an embodiment, the modified amino acid is modified lysine of Formula III.Formula III

[0133] In another embodiment, the modified amino acid is modified lysine of Formula IV.Formula IV

[0134] In another embodiment, the modified amino acid is modified lysine of Formula V.Formula V

[0135] The modified amino acid, preferably modified lysine, may be present at one or more positions in the peptide. In an embodiment, the modified amino acid may be present at a position selected from a group consisting of 2, 10, 12, 17, 20, 21, 28, 30, 32, and 40. In an embodiment, the modified amino acid is modified lysine (Km) present at a position selected from a group consisting of 2, 10, 12, 17, 20, 21, 28, 30, 32, and 40.

[0136] In an embodiment, the modified ammo acid is modified lysine (I m) present at a position selected from a group consisting of 10, 12, 20, and 40. In an embodiment, the peptide comprises modified lysine (Km) at a position selected from a group consisting of 10, 12, 20, and 40, wherein the modified lysine is lysine (K) attached to a lipid, preferably a diacid, by a linker and a spacer, wherein the linker is selected from one or more yGlu moieties, Gamma-aminobutyric acid (GABA), or combination thereof, and wherein the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), GGGGS repeats, y-Carboxylate, one or more PEG moieties, combination thereof.Non-natural amino acid

[0137] The peptides, according to embodiments herein, include one or more non-natural amino acids. The non-natural amino acid may help decrease / avoid enzyme mediated degradation of the peptide, thereby contributing to improvedserum half-life of the peptide. Examples of non-natural amino acids include, but are not limited to, Alpha-aminobutyric acid (Aib), Alpha-MethylLeucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), Methylalanine, or derivatives thereof. Accordingly, in an embodiment, the peptide comprises a nonnatural amino acid selected from a group consisting of Alpha-aminobutyric acid (Aib), Alpha-MethylLeucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), and Methylalanine. In a preferred embodiment, the nonnatural amino acid is Aib. In an embodiment, the peptide comprises a non-natural amino acid at position 2 of the amino acid sequence. In an embodiment, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 38, SEQ ID NO. 39, and SEQ ID NO. 40, wherein X2 is Aib. In another embodiment, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO.24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

[0138] Further, the peptide according to embodiments herein, may be amidated at the C-terminal end. The peptide having amino acid sequence as set forth in SEQ ID NO. 1 comprises a serine residue at the C-terminal end which is amidated, to preferably eliminate the negative charge and stabilize the peptide. The term “amidation”, as used herein, refers to a modification of the C-terminus of a peptide to include an amine group. Amidation may be performed by methods generally known in the field including: Rink Amide Resin-Based Approach, Pre-Functionalized Resins based approach, or On-Resin Amidation via Carbodiimide Coupling. In an embodiment, the peptide has an amino acid sequence as set forthin SEQ ID NO. 1, wherein the serine residue at position 39 is amidated at the C-terminal end.

[0139] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 36, wherein the modified lysine residue at the C-terminal end is amidated.

[0140] In an embodiment, the peptide has an amino acid sequence selected from the group consisting of:HX2QGTFTSDXioSXi2YLDEXi7Xi8AX2oDFVX24WLLX28GGPSSGAPPPS (SEQ ID NO. 2);HX2QGTFTSDYSX12YLDEX17X18AX20DFVEWLLX28GGPSSGAPPPS (SEQ ID NO. 3);HX2QGTFTSDYSX12YLDE X17X18AX20DFVEWLLDGGPSSGAPPPS (SEQ ID NO. 4); and HX2QGTFTSDYSX12YLDERAAX20DFVEWLLDGGPSSGAPPPS (SEQ ID NO.5).wherein, X2, X10, X12, X17, Xis, X2o,X24, and X28 are amino acids selected from natural, non-natural, or modified amino acid residues. Examples of natural, nonnatural, and modified amino acid residues include, but are not limited to, histidine (H), tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E) or glutamine (Q), aspartic acid (D), serine (S), tryptophan (W), Alphaaminobutyric acid (Aib), Alpha-MethylLeucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), Methylalanine, or acylated forms thereof. Acylated forms include, but are not limited to, modified amino acids having an acyl group conjugated to the amino acid.

[0141] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5, wherein, X2, X10, X12, X17, Xis, X2o,X24, and X28 are amino acids selected from a group consistingof histidine (H) or tyrosine (Y), lysine (K), arginine (R), isoleucine (I), alanine (A), glutamic acid (E) or glutamine (Q), aspartic acid (D), tryptophan (W), and serine (S).

[0142] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5, wherein, X2is selected from Serine (S) or a non-natural amino acid, Xw is selected from Tyrosine (Y), lysine (K), or modified lysine (Km), Xi2is selected from lysine (K), Arginine (R), or modified lysine (Km), X17 is selected from Arginine (R) or Isoleucine (I), Xis is selected from Alanine (A) or Arginine (R), X20 is selected from glutamine (Q), lysine (K), or modified lysine (Km), X24 is selected from glutamic acid (E) or glutamine (Q), and X2x is selected from aspartic acid (D) or serine (S).

[0143] In another embodiment, the peptide has an amino acid sequence selected from the group consisting of:HSQGTFTSDYSKYLDERAAKmDFVEWLLSGGPSSGAPPPS (SEQ ID NO. 6); HSQGTFTSDKmSKYLDEIRAKDFVQWLLDGGPSSGAPPPS (SEQ ID NO. 7); HAibQGTFTSDYSKYLDERAAKmDFVEWLLDGGPSSGAPPPS (SEQ ID NO.8);HAibQGTFTSDYSKmYLDEIRAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO. 9); HAibQGTFTSDYSKmYLDERAAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO.10);HSQGTFTSDYSKYLDERAAKmDFVEWLLSGGPSSGAPPPS-NH2(SEQ ID NO.11);HSQGTFTSDKmSKYLDEIRAKDFVQWLLDGGPSSGAPPPS-NBL (SEQ ID NO.12);HAibQGTFTSDYSKYLDERAAKmDFVEWLLDGGPSSGAPPPS-NH2(SEQ ID NO. 13);HAibQGTFTSDYSKmYLDEIRAQDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 14);HAibQGTFTSDYSKmYLDERAAQDFVEWLLDGGPSSGAPPPS-NH2(SEQ ID NO. 15);HAibQGTFTSDKmSKYLDERAAKDFVEWLLDGGPSSGAPPPS (SEQ ID NO.16);HAibQGTFTSDKmSKYLDEIRAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO.17);HAibQGTFTSDKmSKYLDERAAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO.18);HAibQGTFTSDKmSKYLDERAAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO.19);HAibQGTFTSDKmSKYLDERAAQDFVEYLLDGGPSSGAPPPS (SEQ ID NO.20);HAibQGTFTSDKmSRYLDERAAQDFVEWLLDGGPSSGAPPPS (SEQ ID NO.21);YAibQGTFTSDKmSIaMeLLDKKAQAibAFIEYLLEGGPSSGAPPPS (SEQ ID NO. 22);YAibQGTFTSDKmSIYLDKKAQAibAFIEYLLEGGPSSGAPPPS (SEQ ID NO.23);YAibQGTFTSDKmSKYLDKKAQAibAFIEYLLEGGPSSGAPPPS (SEQ ID NO. 24);YAibQGTFTSDYSIaMeLLDKKAQAibAFIEYLLEGGPSSGAPPPSKm (SEQ ID NO. 25);YAibQGTFTSDKmSKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO.26);HAibQGTFTSDKmSKYLDERAAKDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 27);HAibQGTFTSDKmSKYLDEIRAQDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 28);HAibQGTFTSDKmSKYLDERAAQDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 29);HAibQGTFTSDKmSKYLDERAAQDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 30);HAibQGTFTSDKmSKYLDERAAQDFVEYLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 31);HAibQGTFTSDKmSRYLDERAAQDFVEWLLDGGPSSGAPPPS-NH2 (SEQ ID NO. 32);YAibQGTFTSDKmSIaMeLLDKKAQAibAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO. 33);YAibQGTFTSDKmSIYLDKKAQAibAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO. 34);YAibQGTFTSDKmSKYLDKKAQAibAFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO. 35);YAibQGTFTSDYSIaMeLLDKKAQAibAFIEYLLEGGPSSGAPPPSKm-NH2 (SEQ ID NO. 36); andY Aib QGTFTSDKmSKYLDERAAQDFVQWLLEGGPSSGAPPPS-NH2 (SEQ ID NO. 37).

[0144] In another embodiment, the peptide has an amino acid sequence selected from the group consisting of:HAibQGTFTSDXioSXi2YLDERAAX2oDFVEX25LLDGGPSSGAPPPS (SEQ ID NO. 41)wherein,Xio is Tyrosine (Y), Lysine (K), or modified lysine (Km),X12 is Arginine (R), Lysine (K), or modified lysine (Km),X20 is Glutamine (Q), Lysine (K), or modified lysine (Km), andX25 is Tyrosine (Y) or Tryptophan (W); andHAibQGTFTSDYSXi2YLDERAAX2oDFVEWLLDGGPSSGAPPPS(SEQ ID NO. 42)wherein,X12 is Lysine (K) or modified lysine (Km), andX20 is Glutamine (Q), Lysine (K), or modified lysine (Km).

[0145] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.10.

[0146] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.10, wherein the serine residue at position 39 is amidated at the C-terminal end.

[0147] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.

[0148] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO.10., and SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15.

[0149] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO.34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

[0150] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25,SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO.30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

[0151] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO.29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

[0152] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, and SEQ ID NO. 36.

[0153] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, and SEQ ID NO. 36 , wherein the peptide is amidated at the C-terminal end.

[0154] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 41, and SEQ ID NO. 42.

[0155] In an embodiment, the peptide has an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 41, and SEQ ID NO. 42, wherein the serine residue at position 39 is amidated at the C-terminal end. In an embodiment, there is provided an agonist peptide comprising a peptide having an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO.14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

[0156] Table 1

[0157] Various embodiments of the peptide are disclosed herein. In an embodiment, the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 2; SEQ ID NO. 3; SEQ ID NO.4; SEQ ID NO. 5, SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO.8; SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO.11; SEQ ID NO. 12, SEQ ID NO. 13; SEQ ID NO. 14; and SEQ ID NO. 15.

[0158] Table 2 depicts the amino acid sequences of peptides in accordance with the embodiments herein.

[0159] Table 2:

[0160] Table 3 depicts the amino acid sequences of peptides in accordance with the embodiments herein.

[0161] Table 3:

[0162] Table 4 depicts the details of the peptides and acylation sites, in accordance with the embodiments herein.

[0163] Table 4:Compositions

[0164] Embodiments herein provide compositions comprising the peptides or agonist peptides as disclosed herein. In an embodiment, the composition comprises at least one peptide; and at least one pharmaceutically acceptable excipient. The excipient may be a carrier, diluent, buffering agent, a tonicity modifier, an antimicrobial agent, a surfactant, and / or salt. The excipient may vary and depends on the dosage form, mode of delivery, intended therapeuticapplication, etc. In some embodiments, the buffering agent is selected from di sodium hydrogen phosphate dihydrate, di sodium hydrogen phosphate heptahydrate, sodium phosphate monobasic, potassium phosphate dibasic, sodium acetate, citric acid, sodium citrate, L-histidine, histidine hydrochloride, sodium succinate, sodium lactate, or combinations thereof. The tonicity modifier is selected from the group consisting of sodium chloride, glycerol, sucrose, mannitol, trehalose, propylene glycol, or combinations thereof. The antimicrobial preservative is selected from m-cresol, phenol, benzyl alcohol, or combinations thereof. The surfactant is selected from polysorbate 20, polysorbate 80, poloxamer 188, poloxamer 407, or combinations thereof.

[0165] In an embodiment, the composition may comprise 0.5 % w / w to 5% w / w of the peptides as disclosed herein.

[0166] In an embodiment, the composition comprises 0.05% w / w to 5% w / w of tonicity modifier with respect to the total weight of the composition, wherein the tonicity modifier comprises 0.05% w / w to 0.9% or 0.3% w / w to 0.9% w / w of ionic tonicity modifier, and / or 0.05% w / w to 0.9% or 0.3% w / w to 2.5% w / w of nonionic tonicity modifier.

[0167] In an embodiment, the buffering agent is in a concentration range of 0.01 % w / w % w / w to 2% w / w or 0.01 % w / w to 1% w / w, in respect of the total composition.

[0168] In an embodiment, the antimicrobial preservative is in a concentration range of 0.15% w / w to 0.35% w / w, in respect of the total composition.

[0169] In an embodiment, the surfactant is in a concentration range of 0.0001% w / w to 0.2 % w / w, in respect of the total composition.

[0170] Further examples of excipients include, but is not limited to, phosphate buffer saline (PBS), sodium or potassium salts, sugars, cyclodextrins,nanoparticles, and / or polymers (eg: PEG). Various other excipients are known to a person skilled in the art and may be used in various embodiments herein.

[0171] The composition may further be formulated in various dosage forms such as parenteral including subcutaneous, intravenous, intraperitoneal, or intramuscular; oral dosage forms including tablets, capsules, powders, or suspensions; transdermal; sprays, etc.Methods

[0172] The peptides, according to embodiment herein may be used for therapeutic applications. Accordingly, embodiments herein provide a method for modulating or stimulating incretin hormone receptors including glucagon-like peptide- 1 receptor (GLP-1R), or modulating gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. In an embodiment, the method for modulating or stimulating incretin hormone receptors including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR comprises providing an effective amount of the peptide as disclosed herein.

[0173] Further embodiment herein provides a method for treating a metabolic disorder. Embodiments herein further include a method for treatment and management of blood glucose levels, and reduce adiposity. Embodiments herein further include a method for treatment and management of comorbidities of diabetes including cardiovascular disease, kidney disease, retinopathy, renal diseases, non-alcoholic fatty liver, neuropathy, and mental health issues.

[0174] In an embodiment, the method for treatment and / or management of obesity in a subject, comprising administering an effective amount of the peptide as disclosed herein. In an embodiment, the method for treatment and / or management of Type 2 Diabetes in a subject, comprising administering an effective amount of the peptide as disclosed herein. The peptide, as disclosed herein, may be administered in combination with a therapeutic agent. Accordingly, in an embodiment, the method comprises administering a therapeutic agent selected fromanti-diabetic agents, sodium-Glucose Transport Protein (SGLT) inhibitors, amylin analogues, dipeptidyl peptidase IV (DPP-4) inhibitors, thiazolidinediones (TZD’s), anti-obesity drugs, bile acid sequestrants, leptin analogues, or combination thereof. Non-limiting examples of anti-diabetic agent included metformin; SGLT inhibitors includes empagliflozin, dapagliflozin, and canagliflozin; amylin analogues include cagrilintide, and pramlintide; DPP -4 inhibitors include sitagliptin, and saxagliptin; thiazolidinediones (TZD’s) include Pioglitazone. Further, Non-limiting examples of anti-obesity drugs include; phentermine-topiramate, and naltrexone-bupropion; bile acid sequestrants include Colesevelam; and Leptin analogues include metreleptin.

[0175] Embodiments herein may also be used to management of Type 2 Diabetes and / or obesity including management of symptoms and / or co-morbidities, thereof.

[0176] The term “effective amount” or “therapeutically effective amount”, used interchangeably herein, is an amount that is capable of exhibiting the desired effect in a subject. The desired effect may be stimulatory or modulatory or have agonist effect on at least one incretin hormone receptors including glucagon-like peptide- 1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR; or therapeutic effect. The effective amount may vary and depend on various factors including age of subject, severity of the diabetes / obesity, mode of delivery, etc.

[0177] The term “subject”, as used herein, refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include non-human primates, dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters, guinea pigs, and etc. The terms “patient” or “subject” are used herein interchangeably. In an embodiment, the subject is human. In an embodiment, the subject is a patient having or suspected of a metabolic disorder selected from having diabetes including Type-2 Diabetes, comorbidities of diabetes (includingcardiovascular disease, kidney disease, retinopathy, renal diseases, non-alcoholic fatty liver, neuropathy, and mental health issues); and obesity; or symptoms thereof.

[0178] The peptide, according to embodiments herein, may be prepared by synthesizing the amino acid chain by methods generally known in the field. Exemplary methods of peptide synthesis include, but is not limited to, solid phase synthesis, vapor-phase synthesis, and liquid-phase synthesis. The amino acid chain is amidated to replace the C-terminal carboxyl (-COOH) group with an amide (-CONTE). Exemplary methods that may be used for amidation of the amino acid chain include Rink Amide Resin-Based Approach; Pre-Functionalized Resins; and On-Resin Amidation via Carbodiimide Coupling. The selection of amidation method may vary and depend on Peptide sequence complexity, Desired purity and yield, and Synthetic feasibility and reagent availability.

[0179] In an example, the peptide assembly may be performed using Fmoc-SPPS on Rink Amide Resin. Standard deprotection , coupling cycles, and capping may be performed until full sequence elongation is achieved. Further, cleavage may be carried out using trifluoroacetic acid (TFA) to remove the peptide from the resin while leaving the C-terminal amidation intact. The amidated peptide may then be purified using HPLC, followed by lyophilization.

[0180] In another example, a pre-functionalized resin based on the desired peptide properties maybe selected. Fmoc-based SPPS was performed, ensuring standard deprotection coupling cycles, and capping steps. Upon peptide completion, TFA cleavage may be used to release the peptide with a C-terminal amide (-CONH2). The amidated peptide may then be purified using HPLC, followed by lyophilization.

[0181] In yet another embodiment, the peptide may be assembled using Wang Resin or another acid-labile resin. The C-terminal carboxyl (-COOH) may be activated, before cleavage, using carbodiimide coupling agents, such as: HBTU / HOBt(O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), DIC / DMAP (Diisopropylcarbodiimide / 4-Dimethylaminopyridine). The amide donor (e.g., NI in MeOH or DMF) may then be introduced to convert -COOH to -CONBL. Upon peptide completion, TFA cleavage may be used to release the peptide with a C-terminal amide (-CONH2). The amidated peptide may then be purified using HPLC, followed by lyophilization.

[0182] The amidated amino acid chain may then be acylated by lipidation of the lysine residue. The s-amino group (-NH2) in lysine provides a site for selective acylation without modifying the peptide backbone. In an example, regents such as fatty acid chlorides or NHS esters (e.g., palmitoyl chloride, stearic acid-NHS), diisopropylcarbodiimide (DIC) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-Hydroxysuccinimide (NHS) activation, or Base catalyst DIPEA (N,N-Diisopropylethylamine) and TEA (Triethylamine); and spacers and linker as described herein may be used in acylation of the amino acid chain.

[0183] In an embodiment, there is provided a method for preparation of the peptide, said method comprising: (a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-natural amino acid; (b) optionally incorporating at least one modified amino acid residue; and cleaving and recovering the peptide to obtain the peptide of the present disclosure.

[0184] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0185] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to takerestrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.Example 1 : Peptide synthesisPeptide construction:

[0186] The peptide sequences of the present disclosure were generated by solid-phase peptide synthesis using a Fmoc / tBu strategy on a PurePep™ Chorus or Prelude X peptide synthesizer (Gyros Protein Technologies). The synthesis was started from Tentagel Rink Amide resin to accommodate amidation at the C-terminus. During each standard coupling cycle, 4.8 equivalents of amino acid were activated using 2-cyano-2 (hydroxyimino)acetate (OxymaPure®) and diisopropylcarbodiimide (DIC) and the reaction mixture was agitated at 80 °C for 5 min. In the case of unnatural amino acids, an extended coupling time of 15 min was used. Capping was performed using acetic anhydride (0.5 M in DMF) to avoid the formation of impurities due to amino acid deletions. Fmoc-deprotection was conducted using piperidine (20% v / v in DMF). To allow for site-specific incorporation of the fatty acid moiety at a later stage in the synthetic process, Fmoc-Lys(ivDde)-OH (CAS 204777-78-6) was incorporated at the required K residues of the backbone sequence.

[0187] Orthogonal ivDde-deprotection of the appropriate lysine residue was conducted by agitation of the resin-bound peptide in a solution of hydrazine in DMF (5% v / v). Stepwise assembly of the side chain was then performed at this position by solid phase peptide synthesis on the PurePep™ Chorus or Prelude Xsynthesizer using the standard OxymaPure® / DIC extended coupling chemistry described above.

[0188] Cleavage: The peptides were suspended in cleave cocktail (TFA-H2O-TIS-EDT; 90.5:5.0:2.5:2.0) and stirred at room temperature for 4 h. The peptides were precipitated into ice-cold Et2O and centrifuged. The pellet was then washed with Et2O to give the crude peptide as a white solid.

[0189] Purification: The crude peptides were purified to >98% purity by reverse-phase HPLC using a Luna Cl 8 column (Phenom enex) and a water / acetonitrile gradient (containing 0.1% v / v TFA). Fractions containing the product that met purity were pooled and lyophilised. Peptide purity was examined by analytical reverse-phase HPLC (Luna C18 column; 2-90% B over 34 min; Buffer A: Water + 0.1% TFA; Buffer B: MeCN + 0.1% TFA; 214 nm) and peptide identity was confirmed by ESLMS.

[0190] Figure 1 depicts a schematic representation of the amino acid structures of the peptides ND-MC-41 (SEQ ID NO. 11), ND-MC-42 (SEQ ID NO.12), ND-MC-43 (SEQ ID NO. 13), ND-MC-44 (SEQ ID NO. 14), and ND-MC-45 (SEQ ID NO. 15), in accordance with the embodiments herein.Example 2 : In vitro studies

[0191] GLP-1R, GIPR, and GCGR receptor activation was assessed in-vitro for the peptides by performing cAMP assay carried out on GLP-1R, GIPR, and GCGR positive CHO cell lines. Receptor activation efficacy of the test peptides against the receptors was assessed in vitro using the cAMP-Glo™ Max Assay (Promega, Cat. No. V1681) in Chinese Hamster Ovary (CHO) cells stably expressing human receptors GLP-1R, GIPR, and GCGR (generated by in-house stable transduction). Cells were seeded at a density of 5,000 cells per well in white, opaque 96-well plates and allowed to adhere overnight. Prior to the assay, test peptides, Retatrutide, and Tirzepatide (positive controls) were serially diluted in assay buffer to achieve a final concentration range of 2>IO1M to 3.2 / 105M. Cells were treated with the respective peptide concentrations and incubated for 1hour at 37°C. Following incubation, cAMP accumulation was measured according to the manufacturer's protocol. Luminescence was detected using a PerkinElmer VICTOR Nivo multiplate reader. Concentration-response data were analysed and ECso values were determined by nonlinear regression using a three-parameter logistic curve fit in GraphPad Prism. Data represent three independent biological replicates; each performed in triplicate technical wells. Results are expressed as relative luminescence units (RLU) plotted against the log of peptide concentration (M).

[0192] Observations: Figures 71 to 84 depict peptide induced receptor activation for GLP-1R GIPR, and GCGR. Plots shown depict the activation of the receptors in presence of peptides represented by the SEQ ID NO. 13, SEQ ID NO.15, SEQ ID NOs. 27 to 29, and SEQ ID NOs. 32 to 37. Further, the binding properties of SEQ ID NOs. 30 and 31 (ND-MC-ZB 44c and 45d) are shown in Table 5 below. As shown in Figures 71 to 84 and the table below, each of the peptides demonstrated similar or better binding affinity efficacy against all three receptors.

[0193] Table 5 depicts results of GLP-1R, GIPR, and GCGR activation as measured by cAMP Assay for ND-MC-ZB 45d.

[0194] Table 5:Example 3: Preliminary Pharmacology study on body weight and glucose levels in diet-induced obese (DIO) mice:

[0195] To evaluate the anti-obesity and metabolic effects of ND-MC-ZB-43, diet-induced obese (DIO) mice were administered 0.5 mg / kg body weight of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) alongside a vehicle control (20mM Tris HC1 (pH 8.0)) for four weeks. Efficacy was determined by monitoring percentage body weight change from baseline and assessing glucose homeostasis via Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT).

[0196] Observations: Change in body weight: Figure 2 demonstrates the change in body weight in DIO treated mice. ND-MC-ZB-43 induced a progressive and significant reduction in body weight beginning early in the treatment phase (Day 4). The peptide reached a peak efficacy between Days 19-22, achieving a substantial weight loss of approximately 30%. Despite a minor stabilization toward the end of the study, the treated group maintained a robust and sustained reduction in body mass compared to baseline, demonstrating potent weight loss activity.

[0197] Effect on Glucose Tolerance (OGTT): As shown in Figure 3 (a), ND- MC-ZB-43 treatment significantly attenuated glucose excursions following an oral glucose challenge as shown in the OGTT results taken at day 28. The treated group exhibited a markedly lower peak and a faster return to baseline. The overall glucose exposure (AUC) was reduced by approximately 51%, indicating a profound improvement in glucose tolerance.

[0198] Effect on Glucose Response During Insulin Tolerance Test (ITT):Figure 3(b) demonstrates the effect of treatment with ND-MC-ZB-43 on the Insulin Tolerance Test (ITT) taken at day 28 after treatment. As shown in Figure 3(b), treatment with ND-MC-ZB-43 significantly enhanced insulin sensitivity. Following insulin administration, the treated group showed a much more pronounced and sustained reduction in blood glucose compared to the vehicle control, which remained relatively resistant to the insulin challenge.Example 4: Preliminary pharmacological studies on metabolic markers in diet-induced obese (DIO) mice:

[0199] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-ZB-43 on major biomarkers of metabolism, DIO mice were treated with 0.5 mg / kg of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) administered subcutaneously twice weekly for four weeks. The study compared treatment effectsagainst the vehicle control 20 mM Tris HC1 (pH 8.0) through serum biochemistry, and histopathological analysis taken at day 28 after the treatment.

[0200] Observations: Effect on Clinical Metabolic Parameters: Figures 4(a) and 4(b), demonstrate the effect of treatment with ND-MC-ZB-43 on major metabolic parameters including glucose levels, total cholesterol, triglycerides, HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels taken at day 28 after treatment. As shown in Figure 4(a) and 4(b), ND-MC-ZB-43 improved both acute and chronic glycemic markers. Treated mice showed lower fasting blood glucose and a consistent reduction in HbAlc levels, suggesting enhanced long-term systemic glucose regulation. The peptide demonstrated a beneficial effect on lipid metabolism, specifically through the reduction of Triglycerides and a directional downward trend in LDL cholesterol. Total cholesterol and HDL levels remained largely comparable to controls, indicating that the lipid-lowering effect is primarily targeted toward atherogenic lipid fractions (Figure 4(a)).

[0201] Effect on Liver Enzymes and Renal Markers: Serum biochemical analysis of liver and renal functional markers in diet-induced obese (DIO) mice indicated that treatment with the weight-loss peptide did not produce major alterations in key metabolic or organ function markers compared with the vehicle control group. As shown in Table 6, renal function parameters remained comparable between groups, suggesting preserved kidney function following treatment. Similarly, systemic protein metabolism markers, including total protein and albumin, were maintained, indicating no disruption of hepatic synthetic capacity or overall metabolic homeostasis. Liver enzyme analysis showed stable Aspartate Aminotransferase (AST) levels, while Alanine Aminotransferase (ALT) displayed a modest increase in the peptide-treated group, which may reflect mild hepatic metabolic adaptation rather than overt hepatocellular injury. Overall, the biochemical profile suggests that the peptide treatment is generally well tolerated in DIO mice with no clear evidence of significant renal or hepatic toxicity.

[0202] Table 6 depicts summary of Liver and Renal function test markers at day 28 after twice weekly 0.5 mg / kg body weight ND-MC-ZB-43 treatment. Table 6:Example 5: Preliminary pharmacological studies on Serum Leptin., Adiponectin and Glucagon Levels in diet-induced obese (DIO) mice:

[0203] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-ZB-43 on serum leptin, adiponectin and glucagon levels, DIO mice were treated with 0.5 mg / kg of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) and administered subcutaneously twice weekly for four weeks. The study compared treatment effects against the vehicle control 20 mM Tris HC1 (pH 8.0) through serum biochemistry, and histopathological analysis taken at day 28 after the treatment.Observations: As shown in Figure 5(a), treatment with ND-MC-43 resulted in a significant suppression of circulating leptin. This reduction likely reflects the significant loss of adipose tissue mass and a potential reversal of leptin resistance.Whereas a moderate increase in adiponectin was observed (Figure 5(b)), suggesting improved adipose tissue function and enhanced insulin-sensitizing effects.Example 6: Preliminary pharmacological studies on organ weight of diet-induced obese (DIO) mice

[0204] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-43 on organ weight (gonadal fat and retroperitoneal fat) and histopathological analysis, DIO mice were treated with 0.5 mg / kg body weight of the peptide dissolved in 20 mM Tris HC1 (pH 8.0) and administered subcutaneously twice weekly for four weeks. The study compared treatment effects against the vehicle control 20 mM Tris HC1 (pH 8.0) and the results were taken at day 28 after the treatment.

[0205] Observations: Treatment with ND-MC-ZB-43 demonstrated a targeted effect on visceral fat accumulation. As shown in Figure 7(a), gonadal fat weights showed only a slight, non-significant reduction compared to controls, however, as shown in Figure 6(b), the impact on retroperitoneal fat was profound and statistically significant (p < 0.05). The treated group exhibited a dramatic decrease in retroperitoneal fat mass, with values consistently remaining at a fraction of those observed in the vehicle group. The high variability seen in control animals was replaced by a uniform, low fat mass in the treated group, indicating that the agonist effectively mobilizes specific deep visceral fat depots even when other peripheral depots show more moderate changes.

[0206] As shown in Figure 7, Histopathological examination of major organs including kidneys, heart, lungs, pancreas, gastrointestinal tract, and brain was done by H&E staining. The histopathological sections showed no abnormality in all animals of the ND-MC-ZB-43 group, similar to the vehicle control group. In the liver, macrovesicular hepatocellular vacuolation was observed with minimal to moderate severity (grade 1-3) and predominantly multifocal distribution, with one case of moderate diffuse involvement. Compared with the vehicle control, thehepatic vacuolation appeared comparable to slightly lower, and no additional treatment-related lesions were observed in other organs.

[0207] Further evaluation of hepatic steatosis scoring by Oil red O staining confirmed presence of severe steatosis in the vehicle control liver samples, contrary the severity of steatosis is significantly reduced in case of ND-MC-ZB-43 treated group after 28 days of treatment.Example 7: Pharmacological studies on dose range in DIO mice

[0208] In order to determine the efficacious dose range, DIO mice were treated twice weekly for four weeks with escalating doses of ND-MC-ZB-43 dissolved in 20 mM Tris HC1 (pH 8.0) (0.01, 0.1, and 0.3 mg / kg). The area under the glucose excursion curve (AUC) was calculated over 120 minutes post-glucose administration (2 g / kg, i.p.). Parameters were compared against a vehicle control (20 mM Tris HC1 (pH 8.0)) group to establish dose-response relationships for weight loss, glycemic control, and lipid metabolism.

[0209] Observations: Dose-dependent changes in body weight and food intake: As shown in Figure 8, ND-MC-ZB-43 demonstrated a robust and clear dosedependent intervention on body weight dynamics. While the vehicle control group exhibited the typical progression of the diet-induced obesity (DIO) model — gaining approximately 15% body weight over 25 days — the treated groups showed escalating levels of efficacy. The lowest dose of 0.01 mg / kg acted as a weightmaintenance therapy, effectively halting the progression of obesity and keeping mice at their baseline weight. As the dose increased to 0.1 mg / kg, the peptide induced a moderate and consistent weight loss of approximately 8%. The most significant impact was observed at the 0.3 mg / kg dose, which produced a robust and sustained reduction of approximately 18% relative to baseline. As shown in Table 7, this weight loss was closely correlated with a potent anorexic effect, as cumulative food intake decreased in a dose-responsive manner, culminating in a 30-33% reduction in consumption at the highest dose compared to controls.Table 7: Cumulative feed consumption (Day 0-25) in DIO mice in Vehicle Control and ND-MC-ZB-43-treated groups (0.01, 0.1, 0.3 mg / kg dose (mpk)).

[0210] Dose dependent effect of ND-MC-ZB-43 in glycemic control and glucose tolerance: The effect of the dosages on glucose metabolism was also assessed. As shown in Figure 9, fasting blood glucose and HbAlc levels showed significant reductions primarily at the 0.1 mg / kg and 0.3 mg / kg levels. The 0.1 mg / kg dose appeared to reach a plateau for these specific parameters, suggesting it may be the threshold dose for glycemic optimization.

[0211] Furthermore, as shown in Figures 10(a) and 10(b), ND-MC-ZB-43 significantly improved glucose handling as evident from intraperitoneal glucose tolerance test (IpGTT). The total glucose excursion (AUC) decreased progressively with increasing doses. The 0.3 mg / kg dose achieved the most significant improvement, reducing the AUC by approximately 42% compared to the vehicle.

[0212] Dose dependent effect of ND-MC-ZB-43 in serum lipid markers: As shown in Figures 11(a) to 11(c), treatment with serum lipid markers were evaluated in dose escalation groups of ND-MC-ZB-43 treated DIO mice. At 28 day post dose HDL showed significant increases were observed only at the 0.3 mg / kg dose, indicating an improvement in good cholesterol. While both LDL & total cholesterol markers showed significant, dose-related reductions starting at 0.1 mg / kg, with more pronounced effects at 0.3 mg / kg. In case of triglycerides a significant reduction was achieved at the highest dose (0.3 mg / kg), indicating improved lipid clearance.

[0213] Dose dependent effect of ND-MC-ZB-43 in serum metabolic hormone markers: The dose dependent effect of ND-MC-ZB-43 was also assessed on serum metabolic hormone markers using the Luminex multiplex immunoassay. As shown in Figures 12(a) to 12(e), treatment with ND-MC-ZB-43 induced a distinct, dose-dependent shift in the endocrine landscape of DIO mice, most notably within the adipokine axis. Leptin levels showed the most robust response, with higher doses (0.1 and 0.3 mg / kg) achieving significant to highly significant reductions, mirroring the substantial loss of adipose tissue mass. In contrast, Resistin levels exhibited an unexpected significant elevation at the 0.1 mg / kg dose, a trend that persisted but lost statistical significance at 0.3 mg / kg due to individual variability. While Insulin and Glucagon both trended downward at higher doses — suggesting a systemic reduction in metabolic strain and improved insulin sensitivity — these changes did not reach statistical significance within the 28-day window. Similarly, markers such as Adiponectin and Secretin remained relatively stable across the tested range; although Secretin showed a gradual numerical increase with escalating doses, neither marker deviated significantly from the vehicle control, indicating that these specific pathways may be less sensitive to the agonist within this dose ceiling.

[0214] Dose dependent effect of ND-MC-ZB-43 in serum metabolic hormone markers: As shown in Figures 13(a) to 13(d), histopathological examination of liver sections identified hepatocellular vacuolation as the predominant finding across all groups. In the vehicle control group, both microvesicular and macrovesicular vacuolation were observed, mainly as mild to moderate multifocal lesions. In the ND-ZB-43 treated groups (0.01, 0.1, and 0.03 mpk), similar changes occurred with microvesicular vacuolation ranging from minimal to moderate severity in focal or multifocal distribution, with occasional macro vesicular vacuolation. Hepatocellular necrosis and inflammatory foci were observed sporadically in a few animals. Overall, hepatic lesions were minimal to moderate with variable distribution across groups.

[0215] Further, as shown in Figures 14(a) to 14(d), histopathological analysis of Oil Red O-stained liver sections of liver sections from DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mpk) for 4 weeks (n=7 / group) using a semi-quantitative grading scale (1-3), showed predominantly severe hepatic lipid accumulation (grade 3) in the control group. Treatment with ND-MC-ZB-43 (0.01, 0.1, and 0.3 mpk) reduced lipid accumulation, with lesions shifting mainly to moderate (grade 2) and mild (grade 1) severity, indicating a dose-related improvement compared with control.

[0216] Dose dependent effect of ND-MC-ZB-43 in histopathology of pancreas: Further histological evaluation of pancreatic sections was conducted in DIO mice treated with ND-MC-ZB-43 (0.01-0.3 mpk) for 4 weeks. The pancreatic sections were stained with hematoxylin and eosin (H&E) and analysed. As shown in Figures 15(a) to 15(d), evaluation of pancreatic sections revealed that ND-MC-ZB-43 treatment promoted a positive trend in endocrine morphology compared to vehicle controls. While control animals exhibited a relatively restricted beta-cell area (1.09-2.55%) and lower islet density, treatment across all dose levels (0.01, 0.1, and 0.3 mg / kg) generally increased these parameters. Notably, the 0.3 mg / kg group achieved the highest observed beta-cell area of 5.58%, suggesting a dosedependent expansion or preservation of insulin-producing tissue. Predominantly moderate-to-strong staining intensity across the treated groups further indicates robust cellular functional integrity. These findings suggest that ND-MC-ZB-43 may exert a protective or regenerative effect on the pancreatic islets, potentially offsetting the islet exhaustion typically observed in chronic diet-induced obesity.Example 8: Preliminary Pharmacology study on body weight and glucose levels in diet-induced obese (DIO) mice:

[0217] To evaluate the anti -obesity and metabolic effects of ND-MC-ZB-45, DIO mice were administered 0.5 mg / kg of the peptide (dissolved in 20 mM Tris HC1 (pH 8.0)) alongside a vehicle control (20 mM Tris HC1 (pH 8.0)). Efficacy was determined by monitoring percentage body weight change from baseline andassessing glucose homeostasis via OGTT and ITT, with total glycemic excursion quantified by area under the curve (AUC).

[0218] Observations: Change in body weight: As shown in Figure 16, treatment with ND-MC-ZB-45 at 0.5 mg / kg induced a rapid and sustained divergence in body weight compared to the vehicle control. Both groups began at a comparable baseline (Day 0); however, pharmacological activity was evident as early as Day 4, with the treated group exhibiting an initial 11% reduction in body weight. The weight-reducing effect progressed sharply through the first two weeks, reaching 20% by Day 8 and 25% by Day 12. Peak efficacy was recorded at Day 15, with a maximum reduction of approximately 26% relative to baseline. Following this peak, the treated group entered a maintenance phase, sustaining a substantial weight reduction of 21-24% through the remainder of the 26-day study. In contrast, the vehicle control group exhibited a steady, progressive weight gain, ending the study at approximately 4-5% above baseline. These findings confirm that ND-MC-ZB-45 produces a robust, rapid-onset, and durable anti-obesity effect.

[0219] Effect on Glucose Tolerance (OGTT): As shown in Figure 17, ND-MC-ZB-45 treatment significantly attenuated glucose excursions following an oral glucose challenge. The treated group exhibited a markedly lower peak and a faster return to baseline. The overall glucose exposure (AUC) was reduced significantly indicating a profound improvement in glucose tolerance.

[0220] Effect on Insulin Tolerance Test (ITT): As shown in Figure 18, treatment with ND-MC-ZB-45 significantly enhanced insulin sensitivity. Following insulin administration, the treated group showed a much more pronounced and sustained reduction in blood glucose compared to the vehicle control, which remained relatively resistant to the insulin challenge.Example 9: Preliminary pharmacological studies on metabolic markers in diet-induced obese (DIO) mice:

[0221] To evaluate the efficacy of the GLP-l / GCGR / GIPR agonist ND-MC-45 on major biomarkers of metabolism, DIO mice were treated with 0.5 mg / kgof the peptide dissolved in 20 mM Tris HC1 (pH 8.0) and administered subcutaneously twice weekly for four weeks. The study compared treatment effects against the vehicle control 20 mM Tris HC1 (pH 8.0) through serum biochemistry, and histopathological analysis taken at day 28 after the treatment.

[0222] Observations: Effect on Clinical Metabolic Parameters: As shown in Figure 19 and Figure 20, treatment with ND-MC-ZB-45 at 0.5 mg / kg produced a significant improvement in the lipid and glycemic profile of DIO mice, though with some internal variability in chronic markers. As shown in Figure 19, blood glucose concentrations were consistently reduced compared to vehicle controls, indicating improved acute glucose regulation. The peptide demonstrated potent lipid-lowering effects. As shown in Figure 19, triglycerides, Total Cholesterol, and LDL concentrations were all reduced relative to controls. The reduction in LDL, in particular, points to a decrease in circulating atherogenic lipoproteins, which is a key therapeutic goal for metabolic syndrome. In contrast to the other lipid fractions, HDL levels remained largely stable. While the mean value appeared slightly lower in the treated group, significant individual overlap with the vehicle control suggests that the treatment did not induce a consistent or clinically pronounced shift in "good" cholesterol levels.

[0223] Effect on Liver Enzymes and Renal Markers: Administration of ND-MC-ZB-45 demonstrated a stable safety profile with biomarkers remaining within physiological norms. As shown in Table 8, renal health was preserved, as evidenced by stable urea and creatinine levels, suggesting no adverse impact on filtration or waste clearance. Similarly, hepatic synthetic function remained robust, with total protein and albumin concentrations showing no clinical deviations. While liver enzymes showed some variability — specifically a reduction in AST suggesting decreased hepatocellular stress and a numerical shift in ALT — the lack of systemic elevation across the group suggests these fluctuations were not indicative of toxicity.Table 8: Liver Enzymes and Renal Markers of ND-MC-ZB-45 Treated Mice Compared To Vehicle ControlExample 10: Preliminary pharmacological studies on Serum Leptin., Adiponectin and Glucagon Levels in diet-induced obese (DIO) mice:

[0224] As shown in Figures 20(a) to 20(c), administration of ND-MC-ZB-45 at 0.5 mg / kg induced significant shifts in key metabolic hormones, reflecting a profound improvement in adipose tissue health and systemic energy balance. As shown in Figure 20(a), Leptin levels exhibited a dramatic and statistically significant reduction (p < 0.001), dropping from baseline levels of -105-115 ng / mL to -20-30 ng / mL; this nearly 80% decrease serves as a powerful indicator of reduced adiposity and the potential reversal of leptin resistance. In contrast, as shown in Figure 20(b), Adiponectin levels showed a favorable increase, rising from -140-150 ng / mL to -165-175 ng / mL, suggesting improved adipose tissue function and enhanced insulin sensitization. Meanwhile, as shown in Figure 20(c), circulating Glucagon levels remained relatively stable and comparable to the vehicle control (-1200-1500 pg / mL), indicating that while the peptide engages theglucagon receptor (GCGR) to drive metabolic benefits like increased energy expenditure, it does so without inducing deleterious hyperglucagonemia.Example 11: Preliminary pharmacological studies on organ weight of diet-induced obese (DIO) mice

[0225] As shown in Figure 21 (a) and 21 (b), treatment with ND-MC-ZB-45 demonstrated a targeted effect on visceral fat accumulation. While gonadal fat weights (Figure 21(a)), showed only a slight, non-significant reduction compared to controls, the impact on retroperitoneal fat (Figure 21(b)) was profound and statistically significant (p < 0.05). The treated group exhibited a dramatic decrease in retroperitoneal fat mass, with values consistently remaining at a fraction of those observed in the vehicle group. The high variability seen in control animals was replaced by a uniform, low fat mass in the treated group, indicating that the agonist effectively mobilizes specific deep visceral fat depots even when other peripheral depots show more moderate changes.

[0226] As shown in Figure 22, histopathological assessment via Oil Red O staining confirmed that ND-MC-ZB-45 treatment significantly reduces hepatic lipid accumulation. While the vehicle control group exhibited severe hepatic steatosis, with scores nearing the maximum on the grading scale, the treated group showed a robust and statistically significant reduction (p < 0.001). Individual data points consistently trended lower than controls, indicating that the agonist effectively mobilizes ectopic fat from the liver. This reduction in steatosis scores suggests that the compound not only drives systemic weight loss but specifically targets hepatic fat metabolism, a key factor in reversing metabolic liver disease.Example 12: SPR Studies

[0227] Binding kinetics of test peptides to human GLP-1R, GIPR, and GCGR were determined by Surface Plasmon Resonance (SPR) using a Biacore T200 instrument (Cytiva). Each receptor was covalently immobilised onto a CM5 sensor chip via standard amine coupling chemistry (EDC / NHS activation) in HBS-EP+ running buffer. A reference flow cell treated with EDC / NHS but withoutreceptor was used for background subtraction. Test peptides and the reference compound LY were serially diluted in HBS-EP+ and injected over the receptor-immobilised flow cells at multiple concentrations at a flow rate of 30 pl / min during an association phase (~60 s) followed by a dissociation phase (~60 s). Referring to Figures 10 to 21, all the sensorgrams are plotted as baseline-subtracted binding response (Resonance Units, RU) versus time (seconds). Multi-concentration sensorgrams were globally fitted to a 1:1 Langmuir binding model to derive the association rate constant (ka, 1 / Ms), dissociation rate constant (kd, 1 / s), and equilibrium dissociation constant (KD, M).

[0228] Observations: The binding kinetics studies depicted in Figures 23-72 characterize the similar or better binding affinity for the peptides under evaluation with respect to the positive controls against all three receptor classes included GLP1R, GIPR and GCGR. These observations could be inferenced based on the association and dissociation profiles of these peptides relative to the positive control.Advantages of the present disclosure

[0229] The present disclosure provides agonist peptides against incretin hormone receptors, including glucagon-like peptide-1 receptor (GLP-1R), gastric inhibitory polypeptide receptor GIPR, and glucagon receptor GCGR. These peptides demonstrate good serum half-life, stability, and binding kinetics. The peptides are also effective in vivo in stabilizing glucose levels, enhancing insulin sensitivity and ameliorating the effects of metabolic disorders such as by lowering cholesterol levels and adipose tissue accumulation, among others. Also disclosed are methods for use and preparation of the peptide disclosed herein.

Claims

1. / We Claim1. A peptide having an amino acid sequence of at least 90% identity to an amino acid sequence selected from the group consisting of:(i) X1X2QGTFTSDX10SX12YLDEX17X18AX20DFVX24X25LLX28GGPSSGAPPP S(SEQ ID NO. 1);whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is selected from Serine (S) or a non-natural amino acid,Xwis selected from Tyrosine (Y), lysine (K), or modified lysine (Km), X12 is selected from lysine (K), Arginine (R), or modified lysine (Km), X17 is selected from Arginine (R) or Isoleucine (I),Xis is selected from Alanine (A) or Arginine (R),X20 is selected from glutamine (Q), lysine (K), or modified lysine (Km), X24 is selected from glutamic acid (E) or glutamine (Q),X25 is selected from Tryptophan (W) or Tyrosine (Y), andX28 is selected from aspartic acid (D) or serine (S);(ii) X1X2QGTFTSDX10SX12X13LDKKAQX20AFIEYLLEGGPSSGAPPPS (SEQ ID NO. 38),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is a non-natural amino acid, preferably Alpha-aminobutyric acid (Aib), X10 is selected from lysine (K), Tyrosine (Y) or modified lysine (Km), X12 is selected from lysine (K) or Isoleucine (I),X13 is selected from Tyrosine (Y) or non-natural amino acid, preferably Alpha-MethylLeucine (crMeL), andX2ois a non-natural amino acid, preferably Aib;(iii) X1X2QGTFTSDX10 SKYLDERAAQDFVQWLLEGGPSSGAPPPS (SEQ ID NO. 39),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is Serine (S) or a non-natural amino acid, preferably Aib, and X10 is selected from lysine (K), Tyrosine (Y) or modified lysine (Km); and(iv) X1X2QGTFTSDX10SX12X13LDKKAQX20AFIEYLLEGGPSSGAPPPSX40 (SEQ ID NO. 40),whereinXi is selected from Histidine (H) or Tyrosine (Y),X2 is a non-natural amino acid, preferably Aib,X10 is selected from lysine (K), Tyrosine (Y) or modified lysine (Km), X12 is selected from lysine (K) or Isoleucine (I),X13 is selected from Tyrosine (Y) or non-natural amino acid, preferably Alpha-MethylLeucine (crMeL),X2ois a non-natural amino acid, preferably Aib,X40 is selected from lysine (K) or modified lysine (Km);or a pharmaceutically acceptable salt thereof.

2. The peptide as claimed in claim 1, wherein the modified lysine (Km) is lysine (K) attached to a lipid, preferably a diacid, by a linker and / or a spacer,wherein the linker is selected from one or more yGlu moieties, Gamma-aminobutyric acid (GABA), or combination thereof, andwherein the spacer comprises a moiety selected from Aminoethylethanolamine (AEEA), GGGGS repeats, one or more y- Carboxylate moieties, one or more PEG moieties, or combination thereof.

3. The peptide as claimed in claim 1, wherein the non-natural amino acid is selected from a group consisting of Alpha-aminobutyric acid (Aib), Alpha- MethylLeucine (crMeL), HomoSerine (hSer), Norvaline (nVal), NorLeucine (nLeu), Diaminopimelic acid (DAP), Methionine sulfoxide (MetO), Selenocysteine (Sec), and Methylalanine.

4. The peptide as claimed in claim 1 or claim 2, wherein the modified lysine is lysine (K) attached to a diacid of Formula I by at least one yGlu linker and a spacer of Formula IICO2H-(CH2)a-CO- Formula I-(amino-(PEG / AEEA)b-CH2CO2H)c- Formula II wherein ‘a’ is in the range of 8 to 22, preferably 12 to 20, ‘b’ is in the range of 1 to 10, and ‘c’ is in the range of 1 to 10.

5. The peptide as claimed in claim 4, wherein the spacer is a compound of Formula II and is selected from:(i) -(amino-(PEG)b-CH2CO2H)c- ; or (ii) -(amino-(AEEA)b-CH2CO2H)c- , wherein ‘b’ is in the range of 1 to 10, and ‘c’ is in the range of 1 to 10.

6. The peptide as claimed in claim 1 or claim 2, wherein the modified lysine is selected from Formula III, Formula IV, or Formula V:Formula III;Formula V7. The peptide as claimed in claim 1, having an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO.4, and SEQ ID NO. 5.

8. The peptide as claimed in claim 1, wherein the peptide is amidated at the C-terminal end.

9. The peptide as claimed in claim 1, wherein the peptide is having an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ IDNO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO.28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, and SEQ ID NO. 37.

10. The peptide as claimed in claim 1, wherein the peptide exhibits agonist activity on glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and / or glucagon receptor GCGR.

11. A composition comprising the at least one peptide as claimed in claim 1, and at least one pharmaceutically acceptable excipient.

12. A method for modulating glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor GIPR, and / or glucagon receptor GCGR, comprising providing an effective amount of the peptide as claimed in claim 1 or the composition as claimed in claim 11.

13. A method for treatment or management of obesity in a subject, comprising providing an effective amount of the peptide as claimed in claim 1 to said subject.

14. The method as claimed in claim 13, further comprising administering a therapeutic agent selected from anti-obesity drugs, bile acid sequestrants, leptin analogues, or combination thereof, to said subject.

15. A method for treatment or management of Type 2 Diabetes in a subject, comprising providing an effective amount of the peptide as claimed in claim 1 to said subject.

16. The method as claimed in claim 15, further comprising administering a therapeutic agent selected from anti-diabetic agents, sodium-GlucoseTransport Protein (SGLT) inhibitors, amylin analogues, dipeptidyl peptidase IV (DPP-4) inhibitors, thiazolidinediones (TZD’s), or combination thereof, to said subject.

17. A method for preparation of the peptide as claimed in claim 1, said method comprising:(a) synthesizing the peptide by solid-phase peptide synthesis and optionally including at least one non-natural amino acid;(b) optionally incorporating at least one modified amino acid residue; and (c) cleaving and recovering the peptide to obtain the peptide as claimed in claim 1.

18. The peptide as claimed in claim 6, wherein the modified lysine is lysine (K) attached to a lipid by a y-Carboxylate spacer.

19. An agonist peptide comprising the peptide as claimed in anyone of claims 1-10.

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