Glp-1 / gip / amylin receptor triple agonists and uses thereof

Abstract

This invention discloses a GLP-1 / GIP / amylin receptor triple agonist and its applications, belonging to the field of biomedical technology. The GLP-1 / GIP / amylin receptor triple agonist of this invention possesses multi-receptor agonistic activity and selectivity, efficiently activating GLP-1, GIP, AMY1, AMY3, and calcitonin receptors, while exhibiting no significant agonistic activity against glucagon receptors. Simultaneously, the GLP-1 / GIP / amylin receptor triple agonist can significantly and persistently inhibit food intake, reduce weight, and improve blood lipid levels, lowering serum triglycerides and cholesterol. Furthermore, it has a long in vivo half-life and favorable long-acting pharmacokinetic characteristics, showing potential for development into a long-acting formulation for the prevention and / or treatment of obesity, diabetes, cardiovascular diseases, and other conditions or diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a GLP-1 / GIP / amyloid receptor triple agonist and its applications. Background Technology

[0002] Overweight and obesity are manifestations of abnormal or excessive accumulation of body fat and are significant risk factors for many serious chronic diseases, including type 2 diabetes mellitus (T2DM), heart disease, and stroke, which are leading causes of death worldwide. The World Health Organization has declared obesity a global epidemic, highlighting the crucial importance of its prevention and treatment for both individuals and society. When lifestyle interventions such as diet and exercise fail to effectively control weight, drug treatments (such as semaglutide and orlistat) can provide some weight loss, but these effects are often difficult to maintain long-term and have limited effectiveness in severe obesity. In such cases, bariatric surgery becomes necessary. While it offers significant weight loss, it is an invasive procedure with high risks and high costs. Therefore, developing more safe and effective treatments for obesity is of great value in improving current treatment levels.

[0003] Glucagon-like peptide-1 (GLP-1) is a peptide hormone containing 30 or 31 amino acids, synthesized and secreted by L cells in the small intestine. As an incretin, GLP-1 lowers blood glucose levels in a glucose-dependent manner by increasing insulin secretion. However, endogenous GLP-1 is readily degraded in vivo by dipeptidyl peptidase-IV (DPP-IV), with a half-life of less than 2 minutes. Currently, several long-acting GLP-1 receptor agonists are used in the treatment of type 2 diabetes mellitus (T2DM), such as semaglutide (Ozempic®) and liraglutide (Victoza®). It is important to note that as the dosage of GLP-1 receptor agonists increases, tolerability becomes a major limiting factor for further improvement in efficacy. Common adverse reactions at high doses include gastrointestinal reactions such as nausea and vomiting.

[0004] Glucose-dependent insulinotropic peptide (GIP) is a peptide hormone synthesized and secreted by enteroendocrine K cells, containing 42 amino acids. GIP secretion is primarily stimulated by fat and carbohydrate intake; plasma GIP levels significantly increase after the ingestion of these nutrients. The half-life of GIP is approximately 7 minutes in healthy individuals, but shortens to about 5 minutes in patients with type 2 diabetes mellitus (T2DM). As an incretin hormone, GIP has a stronger insulinotropic effect than GLP-1 in healthy individuals, but this effect is significantly weakened in T2DM patients. Studies have shown that long-acting GIP analogs can improve blood sugar and have a certain weight loss effect, but their weight loss effect is not as good as that of long-acting GLP-1 analogs (PA Mroz, B. Finan, V. Gelfanov, B. Yang, MH Tschöp, RD DiMarchi, D. Perez-Tilve, Optimized GIPanalogs promote body weight lowering in mice through GIPR agonism notantagonism, Molecular Metabolism, 20 (2019) 51-62). Further research has shown that combining GIP with GLP-1 analogues can synergistically enhance weight loss effects (PK Nørregaard, MA Deryabina, P. Tofteng Shelton, JU Fog, JR Daugaard, P.-O. Eriksson, LF Larsen, L. Jessen, A novel GIP analogue, ZP4165, enhances glucagon-like peptide-1-induced body weight loss and improves glycaemic control in rodents, Diabetes, Obesity and Metabolism, 20 (2018) 60-68). Therefore, GIP receptor agonists are considered an important potential option for enhancing the weight loss effects of GLP-1 drugs.Currently, peptide drugs with dual agonist activity against both GLP-1 and GLP-1 receptors, such as tirzepatide, have been demonstrated in multiple clinical studies to be superior to GLP-1 receptor agonists in terms of weight loss and glycemic control, showcasing the clinical advantages of synergistic dual-target effects (JP Frias, MANauck, J. Van, ME Kutner, X. Cui, C. Benson, S. Urva, RE Gimeno, Z. Milicevic, D. Robins, A. Haupt, Efficacy and safety of LY3298176, a noveldual GIP and GLP-1 receptor agonist, in patients with type 2 diabetes: arandomised, placebo-controlled and active comparator-controlled phase 2 trial, The Lancet, 392 (2018) 2180-2193).

[0005] Amylin is a 37-amino acid peptide hormone synthesized by pancreatic β-cells and secreted synchronously with insulin. Amylin participates in energy metabolism regulation by acting on amylin receptors, which are typically composed of calcitonin receptors and RAMP proteins, and are functionally classified into AMY1, AMY2, and AMY3 subtypes. The main physiological effects of amylin include enhancing satiety and reducing appetite, delaying gastric emptying, and inhibiting glucagon secretion. It is also believed to be involved in the regulation of cardiovascular and bone metabolism systems. Clinical studies suggest that amylin receptor agonists have potential applications in the treatment of overweight / obesity and type 1 and type 2 diabetes. One of the recent research focuses has been on prolonging the in vivo duration of action of amylin through molecular modification and exploring its synergistic agonistic effect with GLP-1 receptors to enhance metabolic benefits. Patent publication number WO2022 / 129526A1 discloses the structural design and potential uses of a GLP-1 / amylin receptor dual agonist. Currently, several novel peptides designed to achieve both high oral bioavailability and simultaneous activation of GLP-1 and amylintide receptors are still under development. Meanwhile, a fixed-dose combination regimen of the GLP-1 receptor agonist semaglutide and the amylintide receptor agonist cagrillintide has been studied in overweight and obese individuals. Results suggest that this combination is superior to semaglutide alone in terms of weight loss, with good overall safety and tolerability. Nevertheless, to date, no dual GLP-1 / amylintide receptor agonist has received market approval.

[0006] Despite the availability and ongoing development of various drugs for overweight / obesity and its related complications, there is still room for improvement in the efficacy of existing treatments. Therefore, there is an urgent need to develop next-generation therapies that combine high in vitro pharmacological activity, more significant clinical weight loss benefits, and superior metabolic properties, while avoiding an increase in adverse reactions disproportionate to their efficacy. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention provides a GLP-1 / GIP / amylin receptor triple agonist and its applications. This GLP-1 / GIP / amylin receptor triple agonist exhibits excellent multi-receptor agonistic activity and selectivity, along with sustained appetite suppression, weight loss, and lipid-lowering effects, as well as long-acting pharmacokinetic characteristics, showing promising application prospects in the prevention and / or treatment of obesity and related metabolic diseases.

[0008] The technical solution of the present invention is as follows: The first aspect of this invention provides a GLP-1 / GIP / amylin receptor triple agonist polypeptide compound, said polypeptide compound having the amino acid sequence represented by SEQ ID NO:9: Tyr-Aib-Gln-Gly-Thr-Tyr-Thr-Asn-Asp-Xaa 10 -Ser-Ile-Leu-Leu-Asp-Lys-Xaa 17 -Ala-Gln-Xaa 20 -Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly-Gly-Gly-Glu-Ala-Ser-His-Leu-Ser -Thr-Ala-Gln-Thr-Ala-Arg-Leu-Ser-Ala-Glu-Leu-His-Gln-Leu-Ala-Thr-Leu-Pro-Arg-Thr-Glu-Thr-Gly-Ser-Gly-Ser-Pro-NH2 In the above sequence, Xaa 10 Selected from Val or Tyr; Xaa 17 Lys with modified side chains; Xaa 20 Selected from one of Aib, Lys, Arg, and Gln; The NH2 at the C-terminus of the sequence indicates that the polypeptide compound has an amidated C-terminus; The Lys modified by the sidechain is:

[0009] Where: n = 18.

[0010] Preferably, the Xaa 10 Val, wherein the polypeptide compound is selected from any of the amino acid sequences shown in SEQ ID NO:1-4: SEQ ID NO: 1

[0011] SEQ ID NO: 2

[0012] SEQ ID NO: 3

[0013] SEQ ID NO: 4 .

[0014] Preferably, the Xaa 10 For Tyr, the polypeptide compound is selected from any of the amino acid sequences shown in SEQ ID NO:5-8: SEQ ID NO: 5

[0015] SEQ ID NO: 6

[0016] SEQ ID NO: 7

[0017] SEQ ID NO: 8

[0018] A second aspect of the present invention protects a pharmaceutically acceptable salt of the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound described in the first aspect.

[0019] A third aspect of this invention protects a pharmaceutical preparation made from the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound described in the first aspect, the pharmaceutical preparation comprising tablets, capsules, syrups, tinctures, inhalers, sprays, injections, films, patches, powders, granules, emulsions, suppositories, and compound preparations.

[0020] A fourth aspect of the present invention protects a pharmaceutical composition comprising the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of the first aspect and a pharmaceutically acceptable excipient, or a pharmaceutically acceptable salt of the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of the second aspect and a pharmaceutically acceptable excipient.

[0021] The fifth aspect of this invention protects the use of a pharmaceutically acceptable salt of the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of the first aspect, or the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of the second aspect, or the pharmaceutical agent of the third aspect, or the pharmaceutical composition of the fourth aspect, in the preparation of a medicament for the prevention and / or treatment of obesity, diabetes, cardiovascular disease, metabolic-associated fatty liver disease, metabolic-associated steatohepatitis, and cognitive impairment.

[0022] Preferably, the cognitive impairment includes cognitive impairment caused by Alzheimer's disease.

[0023] The beneficial technical effects of this invention are as follows: The GLP-1 / GIP / amylin receptor triple agonist of this invention can efficiently activate human GLP-1 receptor, human GIP receptor, human AMY1 receptor, human AMY3 receptor, and human calcitonin receptor, while exhibiting no significant agonistic activity against glucagon receptor, thus demonstrating good receptor agonistic activity and selectivity. Specifically, the agonistic activity of the triple agonist against GLP-1 receptor is comparable to that of the GLP-1 receptor agonist Semaglutide; its agonistic activity against AMY1 and AMY3 receptors is superior to that of natural amylin and similar to that of the long-acting amylin analog Cagrilintide; and its agonistic activity against calcitonin receptor is also similar to that of Cagrilintide and significantly superior to that of Amylin, indicating that it has excellent multi-receptor synergistic agonistic effects.

[0024] The GLP-1 / GIP / amylin receptor triple agonist of this invention also exhibits significant and sustained appetite suppression, weight loss, and lipid-regulating effects. Compared to the GLP-1 / amylin receptor dual agonist Amycretin, the triple agonist of this invention produces a longer-lasting appetite suppression effect and a more significant weight loss effect after a single dose; under repeated dosing conditions, its weight loss effect is also significantly superior to Amycretin, and it can significantly reduce serum triglyceride and cholesterol levels. Furthermore, the triple agonist of this invention has a long in vivo half-life and favorable long-acting pharmacokinetic characteristics, possessing the potential to be further developed into a long-acting formulation for the prevention and / or treatment of obesity and related metabolic diseases. Attached Figure Description

[0025] Figure 1 This represents the cumulative food intake of SD rats within 72 hours following a single subcutaneous injection of the triple agonist of this invention.

[0026] Figure 2 This represents the percentage change in body weight of SD rats within 72 hours following a single subcutaneous injection of the triple agonist of this invention.

[0027] Figure 3 The change in body weight percentage in DIO rats after 22 days of continuous subcutaneous injection of the triple agonist of this invention. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the embodiments.

[0029] Unless otherwise defined in this invention, the scientific and technical terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art.

[0030] The GLP-1 receptor, GIP receptor, and amylin receptor triple agonist described in this invention is a polypeptide compound. Its amino acid sequence is described in this specification using the amino acid residue representation methods commonly used in the art, including single-letter codes and / or three-letter codes. In addition to standard amino acid residues, the polypeptide may also contain non-natural amino acid residues, such as α-aminoisobutyric acid (Aib; also known as 2-aminoisobutyric acid).

[0031] Unless otherwise specified, the amino acid residues in the polypeptide compounds of this invention should be understood as L-configuration.

[0032] In the polypeptide compounds of the present invention, the "-NH2" portion at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0033] This invention discloses a class of compounds that exhibit agonistic activity against glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic peptide (GIP) receptor, and amylin receptor. Therefore, the compounds of this invention can be used simultaneously as GLP-1 receptor agonists, GIP receptor agonists, and amylin receptor agonists. Since these compounds can activate the above three receptors, they can also be referred to as GLP-1 / GIP / amylin receptor tri-agonists or GLP-1 / GIP / amylin receptor tri-agonist polypeptide compounds.

[0034] In this specification, "receptor agonist" or "agonist" means a ligand (e.g., a small molecule compound or peptide compound) that can bind to and activate a biological receptor, thereby triggering a corresponding biological effect. Receptor activation can be mediated by endogenous agonists (e.g., endogenous hormones) or exogenous agonists (e.g., drugs).

[0035] In this specification, "GLP-1 receptor agonist" refers to a compound that can bind to and activate the GLP-1 receptor.

[0036] In this specification, "GIP receptor agonist" refers to a compound that can bind to and activate the GIP receptor. "GLP-1 / GIP receptor dual agonist" refers to a compound that can bind to both the GIP receptor and the GLP-1 receptor simultaneously and activate both, such as Tirzepatide, which belongs to the GLP-1 / GIP receptor dual agonist category.

[0037] In this specification, "amylin receptor agonist" refers to a compound capable of binding to and activating the amylin receptor (AMY receptor) and calcitonin receptor (CTR). The amylin receptor is formed by the binding of the calcitonin receptor to any one of three receptor-modifying proteins (RAMP1, RAMP2, and RAMP3) to form a heterodimer, corresponding to three subtypes: amylin receptor 1 (AMY1 receptor), amylin receptor 2 (AMY2 receptor), and amylin receptor 3 (AMY3 receptor). Unless otherwise stated, "amylin receptor" as used herein includes at least the AMY3 receptor, and may also include AMY1 and AMY2 receptors. The agonist can activate or co-activate one or more of these subtypes of receptor.

[0038] In this specification, "pharmaceuticalally acceptable excipient" refers to an inert or substantially inert substance that is compatible with the active ingredient and suitable for administration, providing a suitable dosage form, improving processing performance, and / or enhancing ease of administration. Depending on the dosage form requirements, the pharmaceutical composition may optionally contain one or more other pharmaceutically acceptable excipients. The selection and dosage of each component can be adjusted within the conventional range in the art.

[0039] In some embodiments, the pharmaceutical composition is a liquid formulation, such as an aqueous formulation, suitable for oral and / or parenteral administration (e.g., injection). Liquid formulations for injection can be prepared by dissolving or dispersing the active ingredient in a buffer system of suitable pH, adding isotonic adjusters, stabilizers, surfactants, and / or preservatives as needed, mixing thoroughly, then sterilizing using methods such as aseptic filtration, and filling under aseptic conditions to prepare the finished product.

[0040] Regarding the types and amounts of carriers, diluents, and other pharmaceutical excipients, as well as the formulation preparation process, those skilled in the art can refer to publicly available pharmaceutical literature for implementation, and this specification does not limit them in this regard.

[0041] The triple agonist described in this invention can be obtained using peptide preparation techniques known in the art. Unless otherwise stated, the following preparation methods are merely examples and do not constitute a limitation of this invention.

[0042] In some embodiments, the triple agonists of the present invention can be prepared by chemical methods, such as solid-phase peptide synthesis (SPPS). SPPS synthesis can employ commonly used protecting group strategies in the art, including but not limited to the t-Boc strategy or the Fmoc strategy, and can be combined with conventional coupling, deprotection, cleavage, and purification steps to obtain the target peptide and its derivatives / modifications. Besides SPPS synthesis, the peptide compounds of the present invention can also be prepared using established peptide synthesis processes such as liquid-phase peptide synthesis and fragment condensation. Specific process parameters can be selected and optimized by those skilled in the art based on the target sequence, modification type, and scale-up requirements.

[0043] In some embodiments, the triple agonist of the present invention can also be obtained via recombinant methods. For example, a nucleic acid (e.g., DNA) encoding the desired polypeptide sequence can be designed and transformed into a suitable host cell, which is then cultured under conditions suitable for polypeptide expression to obtain the expression product. Subsequently, the polypeptide can be extracted using isolation and purification methods known in the art, and further processed as needed. The host cells suitable for polypeptide expression of the present invention can be selected from prokaryotes or eukaryotic systems.

[0044] The examples or exemplary statements listed in this specification (such as "for example" or "such as") are for illustrative purposes only and do not constitute a limitation on the scope of protection of this invention. Specific embodiments are provided in this specification to further illustrate the invention, but these embodiments are not intended to be the sole limitation of the invention.

[0045] The present invention will be further described below through examples and other means.

[0046] Example 1 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 1, as shown below:

[0047] The triple agonist in this embodiment was synthesized using the standard Fmoc solid-phase peptide synthesis (Fmoc-SPPS) method, with Rink Amide MBHA resin as the solid-phase support. The specific steps are as follows: S1: Resin swelling After the weighed Rink resin was swollen and washed with dichloromethane, methanol and DMF in sequence, the peptides were assembled stepwise in a manual peptide synthesis tube.

[0048] S2: Removal of Fmoc protecting groups from resin After the resin has fully swollen, add 7 mL of 20% piperidine / DMF and react for 30 min. After the reaction, remove the solution and then wash the resin four times with DMF. After Fmoc deprotection is complete, transfer a small amount of resin to a centrifuge tube, add an appropriate amount of 5% ninhydrin / anhydrous ethanol and 80% phenol / anhydrous ethanol solution, mix well, and heat in a 110℃ metal bath for 5 min. Then observe the resin color. If the color is purple, it can be determined that the Fmoc protecting group has been successfully removed.

[0049] S3: Condensation of the first amino acid The first amino acid at the C-terminus, Fmoc-Pro-OH, was coupled to Rink resin (with the Fmoc protecting group removed) as the starting amino acid of the peptide chain. During the coupling reaction, HBTU / HOBt was used as the condensing agent, and DIPEA as the organic base catalyst. The amounts of amino acid, HBTU, HOBt, and DIPEA relative to the active groups of the resin were 4 eq, 4 eq, 4.1 eq, and 8 eq, respectively. The reaction was carried out at room temperature under nitrogen bubbling for 1–2 hours. After coupling, the Fmoc protecting group was removed with a 20% piperidine / DMF solution to obtain the free amino group, and the mixture was repeatedly washed four times with DMF until neutral.

[0050] S4: Elongation of peptide chains Subsequently, each amino acid was coupled sequentially according to the polypeptide backbone sequence. Each step used the same HBTU / HOBt / DIPEA system as described above. After coupling, the Fmoc protecting group was removed using 20% ​​piperidine / DMF. The coupling efficiency was monitored during synthesis using the ninhydrin reagent method to ensure complete reaction. Specifically, the N-terminal amino acid at position 1 was Boc-Tyr(tBu)-OH, positions 2 and 20 were Fmoc-Aib-OH, and the fatty acid-modified Lys residue at position 17 was introduced using Fmoc-Lys(Dde)-OH. Its ε-amino group on the side chain was selectively protected by Dde.

[0051] S5: Modification of fatty acid side chains After the main chain synthesis was completed, the Dde protecting group of the Lys residue at position 17 was selectively removed using a 2% hydrazine hydrate / DMF solution to expose the ε-amino group of the side chain. Subsequently, according to the requirements of the peptide structure side chain, Fmoc-OEG-OH, Fmoc-Glu-OtBu, and tert-butyl eicosanoate were sequentially coupled using the same method described above, with Fmoc removal and thorough washing performed after each reaction step.

[0052] S6: Peptide cleavage After all amino acids and modifying groups were coupled, the resin and peptide were cleaved together with TFA / TIS / H2O (95:2.5:2.5, v / v) for 2–3 hours to achieve complete deprotection and cleavage of the peptide from the resin. The reaction solution was precipitated with ice-cold diethyl ether and then centrifuged and washed to obtain the crude peptide.

[0053] S7: Purification and Identification of Polypeptides The crude peptide was purified by semi-preparative HPLC, and then lyophilized to obtain the purified target peptide with a purity greater than 97%. The semi-preparative HPLC purification conditions were as follows: the crude peptide was dissolved in 50% methanol / water solution, purified by Shimadzu semi-preparative HPLC using a C18 column (250 mm × 20 mm), with gradient elution using an aqueous solution containing 0.1% TFA as mobile phase A and methanol as mobile phase B, a detection wavelength of 214 nm, a flow rate of 6 mL / min, and the target peak fraction was collected and lyophilized to obtain the purified peptide.

[0054] The molecular weight of the target compound was confirmed by mass spectrometry; the theoretical molecular weight is 8268.25. ESI-MS m / z: Calculated value [M+5H] 5+ 1654.7, [M+6H] 6+ 1379.0; Observation [M+5H] 5+ 1654.5, [M+6H] 6+ 1378.9.

[0055] Example 2 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 2, as follows:

[0056] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry; the theoretical relative molecular mass was 8311.32. ESI-MS m / z: Calculated value [M+5H] 5+ 1663.3, [M+6H] 6+ 1386.2; Observation [M+5H] 5+ 1663.1, [M+6H] 6+ 1386.0.

[0057] Example 3 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 3, the sequence of which is as follows:

[0058] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8339.33. ESI-MS m / z: Calculated value [M+7H] 7+ 1192.3, [M+8H] 8+ 1043.4; Observation [M+7H] 7+ 1192.2, [M+8H] 8+ 1043.3.

[0059] Example 4 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 4, as follows:

[0060] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8311.15. ESI-MS m / z: Calculated value [M+5H] 5+ 1663.2, [M+6H] 6+ 1386.2; Observation [M+5H] 5+ 1663.3, [M+6H] 6+ 1386.0.

[0061] Example 5 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 5, the sequence of which is as follows:

[0062] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8332.29. ESI-MS m / z: Calculated value [M+5H] 5+ 1667.5, [M+6H] 6+ 1389.7; Observation [M+5H] 5+ 1667.3, [M+6H] 6+ 1389.6.

[0063] Example 6 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 6, the sequence of which is as follows:

[0064] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8375.36. ESI-MS m / z: Calculated value [M+7H] 7+1197.5, [M+8H] 8+ 1047.9; Observation [M+7H] 7+ 1197.2, [M+8H] 8+ 1047.7.

[0065] Example 7 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 7, the sequence of which is as follows:

[0066] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8403.38. ESI-MS m / z: Calculated value [M+7H] 7+ 1201.5, [M+8H] 8+ 1051.4; Observation [M+7H] 7+ 1201.7, [M+8H] 8+ 1051.5.

[0067] Example 8 Synthesis of a polypeptide compound with the sequence structure SEQ ID NO: 8, as follows:

[0068] The synthesis method was the same as in Example 1. The molecular weight of the target compound was confirmed by mass spectrometry, with a theoretical relative molecular mass of 8375.19. ESI-MS m / z: Calculated value [M+7H] 7+ 1197.5, [M+8H] 8+ 1047.9; Observation [M+7H] 7+ 1197.2, [M+8H] 8+ 1047.7.

[0069] Application test case: The agonistic activity of the polypeptide compounds prepared in Examples 1-8 above was tested for GLP-1 receptor, GIP receptor, amylin receptor and calcitonin receptor.

[0070] Test Example 1: Assay of the agonistic activity of human GLP-1 receptor, human GIP receptor, and human glucagon receptor The agonistic activities of the compounds on human GLP-1 receptor (GLP-1R), human GIP receptor (GIPR), and human glucagon receptor (GCGR) were detected using the HTRF method. In the experiments, stable GLP-1 receptor expressing cell lines (CHO-GLP-1R), stable GIP receptor expressing cell lines (CHO-GIPR), and stable glucagon receptor expressing cell lines (CHO-GCGR) were cultured in F-12 medium (Hyclone) and 10% fetal bovine serum (Avantor). GLP-1R and GIPR cell lines were supplemented with 200 μg / mL or 8 μg / mL of Hygromycin B (Solarbio) and 2 μg / mL of Puromycin (Invitrogen), respectively, while the GCGR cell line was supplemented with 200 μg / mL of Hygromycin B (Solarbio). Cells were cultured at 37°C and 5% CO2. After reaching 80% confluence, they were digested with 0.25% trypsin-EDTA (Gibco) and counted. They were then seeded in 384-well plates (Greiner) at 9 μL / well (GLP-1R, GIPR) or 10 μL / well (GCGR).

[0071] cAMP signal detection was performed using the LANCE Ultra cAMP kit (Revity). The reaction buffer was prepared according to the kit instructions, containing 5 mM HEPES, 0.1% casein, and 500 μM IBMX (Sigma) Hank's balanced salt buffer. Each test compound and positive control (GIP, GLPBIO; Semaglutide, MCE; Glucagon, MCE) was diluted 10-fold with 1× stimulation buffer. The appropriate volumes of compound or control were added to cell wells, centrifuged, and incubated at 37°C for 30 minutes. Subsequently, 4 μL / well Eu-cAMP and 4 μL / well Ultra-anti-cAMP antibody (both kit components) were added, centrifuged again, and incubated at room temperature for 1 hour. After incubation, fluorescence signals were read at 665 nm and 620 nm using a PHERAstar FSX multi-plate reader (BMG, PHERAstarFSX), and the agonistic activity and ECMO of each receptor were calculated. 50 value.

[0072] Test Example 2: Assay of agonistic activity of human AMY1 receptor, human AMY3 receptor, and human calcitonin receptor (CTR) The agonistic activity of the compounds on human AMY1 receptor (AMY1R), human AMY3 receptor (AMY3R), and human calcitonin receptor (CTR) was detected using the HTRF method. Cell lines used included COS7-AMY1R, COS7-AMY3R, and COS7-CTR, all cultured in DMEM (Corning) medium supplemented with 10% fetal bovine serum (Avantor) and 2 μg / mL of Hygromycin B (Solarbio). The AMY1R and AMY3R cell lines were also supplemented with 2 μg / mL of Puromycin (Invitrogen). Cells were cultured at 37°C and 5% CO2 until 80% confluence, then digested with 0.25% trypsin-EDTA (Gibco) for cell counting. Cells were seeded at 500 cells / well, 10 μL / well, in 384-well plates (Greiner).

[0073] cAMP signal detection was performed using the LANCE Ultra cAMP kit (Revity). Hank's balanced salt buffer containing 5 mM HEPES, 0.1% casein, and 500 μM IBMX (Sigma) was prepared according to the kit instructions. Each test compound and positive control (Amylin, Cagrilintide, MCE) was diluted 10-fold with stimulation buffer. 10 μL of each test compound or positive control was added to each well, centrifuged, and incubated at 37°C for 30 minutes. Then, 4 μL / well Eu-cAMP and 4 μL / well Ultra-anti-cAMP antibody (both kit components) were added, centrifuged again, and incubated at room temperature for 1 hour. After incubation, fluorescence signals were read at 665 nm and 620 nm using a PHERAstar FSX multi-plate reader (BMG, PHERAstar FSX) to calculate the corresponding receptor agonistic activity and EC50. 50 value.

[0074] The results of Test Example 1 and Test Example 2 are shown in the table below.

[0075] Table 1: GLP-1 receptor, GIP receptor, and glucagon receptor agonistic activities of triple agonists (ECG) 50 )

[0076] Table 2: AMY1 receptor, AMY3 receptor, and calcitonin receptor agonistic activities of triple agonists (ECG) 50 )

[0077] As can be seen from the results in Tables 1 and 2, the triple agonist of the present invention can significantly activate human GLP-1 receptor, human GIP receptor, human AMY1 receptor, human AMY3 receptor and human calcitonin receptor.

[0078] Among them, the triplicate agonist of the present invention exhibits agonistic activity (EC) on human GLP-1 receptor. 50 The value was compared with the positive control compound Semaglutide (J. Med. Chem. 2015, 58, 7370). The activity of the triple agonist against the human GIP receptor is comparable to that of the positive control compound GIP (7380). Its agonistic activity against the human GIP receptor is slightly lower than that against the positive control compound GIP. For the human AMY1 and AMY3 receptors, the agonistic activity of the triple agonist of this invention is superior to that of the positive control compound Amylin, and similar to that of the positive control compound Cagrilintide (J. Med. Chem. 2021, 64, 15, 11183–11194). For the human calcitonin receptor, the agonistic activity of the triple agonist of this invention is similar to that of Cagrilintide, and significantly superior to that of Amylin. Furthermore, the triple agonist of this invention exhibits almost no agonistic activity against the human glucagon receptor (its agonistic activity is more than 8900 times lower than that of glucagon), demonstrating good receptor selectivity.

[0079] In summary, the triple agonist of this invention can efficiently and evenly activate human GLP-1, human GIP, human AMY1, human AMY3, and human calcitonin receptors, while exhibiting no significant agonistic activity against human glucagon receptors. This demonstrates that the triple agonist of this invention possesses excellent receptor agonistic activity and selectivity.

[0080] Test Example 3: Effects of the polypeptide compound of the present invention on acute feeding and body weight in SD rats The effects of the test compound on feeding behavior and weight change were evaluated using male SD rats to investigate its feeding inhibition and acute weight loss activity.

[0081] Male SD rats, weighing 230–260 g, were purchased from the Experimental Animal Center of Xuzhou Medical University (SPF grade). After arrival, the animals were housed under standard rat feed and water conditions for 3–7 days to acclimatize. The ambient temperature was 22±2℃, and the relative humidity was 45–65%, using a 12-hour inverted light cycle (9:00 AM to 9:00 PM as the dark period). After the acclimatization period, the rats were randomly divided into groups of 6 rats each, housed individually.

[0082] The groups were set up as follows: blank control group (Saline), positive control group (Amycretin, 10 nmol / kg), SEQ ID NO: 1 (10 nmol / kg), SEQ ID NO: 2 (10 nmol / kg), SEQ ID NO: 3 (10 nmol / kg), SEQ ID NO: 4 (10 nmol / kg), SEQ ID NO: 5 (10 nmol / kg), SEQ ID NO: 6 (10 nmol / kg), SEQ ID NO: 7 (10 nmol / kg), and SEQ ID NO: 8 (10 nmol / kg). Before the experiment, rats in each group were weighed to ensure a consistent baseline weight. All rats had free access to food and water throughout the experimental period.

[0083] The experiment used a single subcutaneous injection (SC) to administer the drug to the rats in the neck and back. The required test compound solutions were prepared according to body weight, using the following formula: 8 mM phosphate, 250 mM glycerol, 0.007% Tween 20, pH 7.4. After administration, the rats were returned to their original cages and allowed free access to food and water. The cumulative food intake of the SD rats was calculated by weighing the remaining feed at 24, 48, and 72 hours after administration. Rats were also weighed at 24, 48, and 72 hours to calculate weight change. By comparing the food intake and weight change rate of each group, the feeding inhibition effect and acute weight loss activity of each test compound were evaluated.

[0084] The entire experimental process adhered to ethical requirements for laboratory animals, and the animals were disposed of according to regulations after the experiment. Experimental data are expressed as mean ± standard deviation (Means ± SD). Statistical significance was assessed first using one-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons to analyze differences between groups. The results are shown in the table below.

[0085] Table 3: Cumulative food intake of rats in different treatment groups over 72 hours

[0086] In the table: *** P<0.001 vs saline, ## P<0.01 vs Amycretin, ### P<0.001 vs Amycretin Table 4: Body weight change rate of rats in different treatment groups at 72 hours

[0087] In the table: ** P<0.01 vs saline, *** P<0.001 vs saline, # P<0.05 vs Amycretin, ## P<0.01 vs Amycretin, ### P<0.001 vs Amycretin From Table 3, Table 4 and Figure 1 , Figure 2 It was found that after a single subcutaneous injection of the GLP-1 / amylin receptor dual agonist Amycretin (eBioMedicine, 2025, 118,105862) or the triple agonist of this invention into SD rats, the triple agonist of this invention significantly inhibited food intake at all time points (24, 48, and 72 hours) compared with the control group (saline group) during the 72-hour observation period (P<0.001). In contrast, the Amycretin group only showed a statistically significant inhibitory effect on food intake compared with the control group at 24 hours after administration, and there was no significant difference at the 48 and 72 hour time points.

[0088] Meanwhile, data on the rate of change in body weight in SD rats showed that, within the 72-hour observation period, the Amycretin group did not exhibit a significant weight loss effect compared to the control group. The triple agonist group of this invention significantly reduced rat body weight at all time points (24, 48, and 72 hours) compared to the control group (P<0.01 or P<0.001), and the weight loss at 24 and 48 hours was significantly greater than that of the Amycretin group (P<0.05, P<0.01, or P<0.001). These results indicate that the triple agonist of this invention, under single-dose conditions, exhibits superior sustained appetite suppression and weight reduction effects compared to Amycretin.

[0089] Test Example 4: Pharmacokinetic Study To evaluate the pharmacokinetic characteristics of the triple agonist of this invention and Amycretin, male SD rats weighing approximately 350 g (purchased from the Experimental Animal Center of Xuzhou Medical University) were randomly divided into groups of three. Amycretin or the triple agonist of this invention (dose 50 nmol / kg) was administered subcutaneously. Blood samples were collected from the ophthalmic venous plexus at different time points after administration (up to 96 hours), and collected in cryovials containing EDTA. Plasma was centrifuged at 3000 rpm. Store at 80°C. Before analysis, thaw plasma samples, add two volumes of acetonitrile containing 0.5% formic acid for protein precipitation, centrifuge at 15,000 rpm for 10 min, and collect the supernatant for LC-MS / MS analysis using multiple reaction monitoring (MRM) in positive ion electrospray ionization mode. Pharmacokinetic parameters (t... 1 / 2 The calculations were performed using BAPP software (v2.0).

[0090] Table 5: Pharmacokinetic parameters of the triple agonist in rats

[0091] As shown in Table 5, the triple agonist of the present invention exhibits a relatively long in vivo half-life in rats, which is similar to that of the publicly reported control compound Amycretin (eBioMedicine, 2025, 118, 105862). These results indicate that the triple agonist of the present invention possesses long-acting pharmacokinetic characteristics and has the potential to support a once-weekly dosing regimen in humans.

[0092] Test Example 5: Long-term drug administration experiment in diet-induced obese (DIO) rats Male SD rats (approximately 250 g in weight, purchased from the Experimental Animal Center of Xuzhou Medical University) were selected and housed individually in a standard animal room (temperature 22±2℃, humidity 55±10%, light cycle 12 h / 12 ​​h). They were fed a 45% high-fat diet (D12451, Research Diets, Inc.) until their weight exceeded 750 g (approximately 4-5 months) to establish the DIO rat model. Rats were matched and grouped according to body weight and body fat percentage (QMR06-090H-PRO, Suzhou Numai Analytical Instruments Co., Ltd.), with 6 rats per group. Each group of rats received a subcutaneous injection (once daily in the neck and back) of either Amycretin or the triple agonist of this invention at a dose of 10 nmol / kg. The solution formulation was: 8 mM phosphate, 250 mM glycerol, 0.007% Tween 20, pH 7.4. The control group received the same volume of saline. The administration period was 22 days, with rat weight measured every two days during this period. After the experiment, the body fat content of DIO rats in each group was measured, and they were sacrificed to collect blood. Serum was separated, and triglyceride and cholesterol levels were measured. Experimental data are expressed as mean ± standard deviation (Means ± SD). To assess statistical significance, one-way ANOVA was first used, followed by Tukey's post-hoc test for multiple comparisons to analyze differences between groups.

[0093] Table 6: Percentage change in body weight (%) and serum triglyceride and cholesterol levels in DIO rats under different treatment groups

[0094] In the table: *** P<0.001 vs saline, ### P<0.001 vs Amycretin In the table, the percentage change in body weight (%) is the percentage change in body weight of rats after 22 days of experimentation compared to the initial body weight.

[0095] As shown in Table 6 and Figure 3 As shown, during the 22-day experimental period, DIO rats receiving subcutaneous injections of Amycretin, 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, and SEQ ID NO: 8 once daily significantly reduced rat body weight compared to the control group (P<0.001), and all the triple agonists of this invention showed significantly better weight-loss effects on day 22 than the Amycretin group (P<0.001). Furthermore, the treatment groups of 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, and SEQ ID NO: 8 exhibited excellent lipid-lowering effects after the experiment, with significantly lower serum triglyceride and cholesterol levels compared to the control and Amycretin groups (P<0.001), while the Amycretin group did not show a significant lipid-lowering effect compared to the control group.

[0096] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A GLP-1 / GIP / amylin receptor triple agonist polypeptide compound, characterized in that, The polypeptide compound has the amino acid sequence represented by SEQ ID NO:9: Tyr-Aib-Gln-Gly-Thr-Phe-Asn-Asp-Xaa 10 -Ser-Ile-Leu-Leu-Asp-Lys-Xaa 17 -Ala-Gln-Xaa 20 -Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly-Gly-Gly-Glu-Ala-Ser-His-Leu-Ser-Thr-Ala-Gln-Thr-Ala-Arg-Leu-Ser-Ala-Glu-Leu-His-Gln-Leu-Ala-Thr-Leu-Pro-Arg-Thr-Glu-Thr-Gly-Ser-Gly-Ser-Pro-NH2 In the above sequence, Xaa 10 Selected from Val or Tyr; Xaa 17 Lys with modified side chains; Xaa 20 Selected from one of Aib, Lys, Arg, and Gln; The NH2 at the C-terminus of the sequence indicates that the polypeptide compound has an amidated C-terminus; The Lys modified by the sidechain is: Where: n = 18.

2. The GLP-1 / GIP / amylin receptor triple agonist polypeptide compound according to claim 1, characterized in that, The Xaa 10 Val, wherein the polypeptide compound is selected from any of the amino acid sequences shown in SEQ ID NO:1-4: SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 。 3. The GLP-1 / GIP / amylin receptor triple agonist polypeptide compound according to claim 1, characterized in that, The Xaa 10 For Tyr, the polypeptide compound is selected from any of the amino acid sequences shown in SEQ ID NO:5-8: SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 8 4. A pharmaceutically acceptable salt of the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound according to any one of claims 1 to 3.

5. A pharmaceutical preparation made from the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound according to any one of claims 1 to 3, characterized in that, The pharmaceutical preparations include tablets, capsules, syrups, tinctures, inhalers, sprays, injections, films, patches, powders, granules, emulsions, suppositories, and compound preparations.

6. A pharmaceutical composition, characterized in that, It includes the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of any one of claims 1 to 3 and a pharmaceutically acceptable excipient, or the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of claim 4 and a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient.

7. Use of a pharmaceutically acceptable salt of the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of any one of claims 1 to 3, or the GLP-1 / GIP / amylin receptor triple agonist polypeptide compound of claim 4, or the pharmaceutical agent of claim 5, or the pharmaceutical composition of claim 6, in the preparation of a medicament for the prevention and / or treatment of obesity, diabetes, cardiovascular disease, metabolic-associated fatty liver disease, metabolic-associated steatohepatitis, and cognitive impairment.

8. The use according to claim 7, characterized in that, The cognitive impairments include those caused by Alzheimer's disease.

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