GLP-1, GIP and GCG triple receptor agonist as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polypeptide drugs for metabolic diseases, in particular to a glucagon-like peptide (GLP-1), a glucose-dependent insulinotropic polypeptide (GIP) and a glucagon (GCG) triple receptor agonist (protein). According to the triple receptor agonist protein provided by the technical scheme, weight reduction is achieved, meanwhile, lower side effects are shown, and drug compliance is enhanced. The triple receptor stimulant researched and developed by the technical scheme can be applied to preparation of medicines with the effects of losing weight, reducing lipid or reducing blood sugar, solves the technical problem that the existing GLP-1, GIP and GCG triple receptor stimulants are difficult to reduce side effects while reducing body weight, and breaks through the weight loss mode that the traditional medicines depend on appetite suppression; due to the unique action mechanism and good safety, a safer and more effective treatment scheme is provided for patients, and the medicine has wide market prospects and social benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs for metabolic diseases, and in particular to a GLP-1, GIP, and GCG triple receptor agonist, and a preparation method and application thereof. Background Art

[0002] The glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon receptor (GCGR) have become important incretin drug targets for the treatment of diabetes, obesity, and other metabolic diseases. GLP-1R, GIPR, and GCGR are all G protein-coupled receptors that regulate glycolipid and amino acid metabolism. The GLP-1R / GIPR / GCGR network is crucial for maintaining blood glucose homeostasis. GLP-1R promotes insulin secretion, while GIPR increases insulin secretion during hyperglycemia and stimulates glucagon release during hypoglycemia. GCGR participates in blood glucose and energy regulation. Glucagon-like peptide-1 (GLP-1) is an incretin secreted by intestinal L cells. GLP-1 receptor agonists can stimulate insulin secretion in a glucose-dependent manner, regulate appetite and energy metabolism, and lower blood glucose and body weight. Similar to GLP-1, glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) are peptide hormones responsible for glucose homeostasis. GIP is a polypeptide hormone secreted by intestinal K cells that promotes insulin secretion to maintain glucose homeostasis and promotes the breakdown and deposition of white adipose tissue, complementing the effects of GLP-1 receptor agonists. GCG, secreted by pancreatic α-cells, stimulates energy expenditure, lowers blood lipids, and inhibits gastric emptying and appetite, thereby reducing weight. In hypoglycemic states, GIP can also promote GCG secretion, elevating blood sugar levels and significantly reducing the risk of hypoglycemia.

[0003] In recent years, GLP-1 receptor agonists (GLP-1RAs) and GLP-1 / GIP dual receptor agonists have made significant progress in the treatment of metabolic diseases, showing promising results in type 2 diabetes mellitus (T2DM), obesity, cardiovascular protection, and non-alcoholic fatty liver disease (NAFLD / NASH). Targeting the gut hormone effect, GLP-1 receptor agonists such as liraglutide, dulaglutide, and semaglutide have been successfully developed for the treatment of type 2 diabetes. Telpotide, developed by Eli Lilly and Company and officially marketed in China as the first GLP-1 / GIP dual receptor agent, activates two gut hormone receptors to synergistically regulate blood glucose and body weight. These peptide drugs have demonstrated promising results in both blood glucose control and weight loss. However, existing GLP-1 receptor agonists (such as semaglutide) and GLP-1 / GIP dual receptor agonists (such as telpotide) are prone to gastrointestinal adverse reactions (such as nausea, vomiting, and diarrhea) and increased heart rate (2-4 beats per minute) in clinical use, especially during the dose-escalation phase. Therefore, how to reduce the side effects of similar peptide drugs is an urgent problem to be solved in this field. Based on the above reasons, it is urgent to optimize the GLP-1R, GIPR, and GCGR triple agonists to reduce their side effects while maintaining their therapeutic effects. Summary of the Invention

[0004] The purpose of the present invention is to provide a GLP-1, GIP, GCG triple receptor agonist that exhibits the advantage of reducing weight while reducing side effects, so as to solve the technical problem that existing GLP-1, GIP, GCG triple receptor agonists are difficult to reduce weight while reducing side effects.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] GLP-1, GIP, GCG triple receptor agonist, its amino acid sequence is:

[0007] X1X2QGT FTSDY SIX 13 LD X 16 X 17 AX 19 X 20 AFIEY LLEGGPSX 33 GA PPPS;

[0008] Wherein, X1 is H or Y; X2 is Aib or G or V; X 13 is L or α-MeL; X 16 K or R or G or S; X 17 K or I or C; X 19 A or Q; X 20 A or Q; X33 K or C.

[0009] Further, X 16 or / and X 33 There are side chain groups attached.

[0010] Furthermore, the side chain group is as shown in Formula I, or as shown in Formula V, or as shown in Formula VI:

[0011]

[0012]

[0013] wherein a is 7-10, b is 1-4, and A is maleimide, succinimide, or halogenated hydrocarbon; preferably, a is 8, 9, or 10, and b is 2 or 4.

[0014] Further, Formula I is any one of Formula II, Formula III, Formula IV and Formula VII;

[0015]

[0016] Furthermore, its amino acid sequence and side chain modifications are:

[0017] YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 The modification of the position is as shown in the side chain of formula III;

[0018] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The modification of the position is as shown in the side chain of formula III;

[0019] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 Bit and X 33 The modification of the position is as shown in the side chain of formula III;

[0020] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII;

[0021] Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The modification of the position is as shown in the side chain of formula III;

[0022] Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The position is modified with a side chain as shown in formula IV;

[0023] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The modification of the position is as shown in the side chain of formula III;

[0024] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula VI;

[0025] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula V;

[0026] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula II;

[0027] Or YAibQGT FTSDY SIα-MelLD KKAAQ AFIEY LLEGGPSCGAPPPS, and X 33 The position is modified with a side chain as shown in formula VII;

[0028] Or YAibQGT FTSDY SILLD KIAQA AFIEY LLEGGPSCGAPPPS, and X 33 The position is modified with a side chain as shown in formula VII;

[0029] Or YAibQGT FTSDY SILLD GIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The modification of the position is as shown in the side chain of formula III;

[0030] Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII;

[0031] Or YVQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33The modification of the position is as shown in the side chain of formula III;

[0032] Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The modification of the position is as shown in the side chain of formula III;

[0033] Or YGQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The modified position has a side chain as shown in formula VII.

[0034] Furthermore, the GLP-1, GIP, and GCG triple receptor agonist has an amino acid sequence of:

[0035] YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The modified position has a side chain as shown in formula III (analog 2).

[0036] Furthermore, the GLP-1, GIP, and GCG triple receptor agonist has an amino acid sequence of:

[0037] YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGAPPPS, and X 33 The modified position has a side chain as shown in formula VII (analog 4).

[0038] Furthermore, the GLP-1, GIP, and GCG triple receptor agonist has an amino acid sequence of:

[0039] YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The modified position has a side chain as shown in formula III (analog 5).

[0040] The present technical solution also provides a preparation method of a GLP-1, GIP, GCG triple receptor agonist, which comprises the following steps in sequence: preparing a precursor of a GLP-1, GIP, GCG triple receptor agonist, modifying side chain groups, and chemically coupling special amino acids; modifying the side chain groups in X 33 Bit or X 16 amino acids.

[0041] Furthermore, the nucleotide sequence for expressing the GLP-1, GIP, and GCG triple receptor agonist precursor is integrated into a vector to obtain an expression vector; an engineered cell containing the expression vector is constructed; the engineered cell is induced to express the protein; and the protein expressed by the engineered cell is recovered from inclusion bodies, subjected to ion exchange chromatography, enzyme digestion, and reverse phase chromatography to obtain the GLP-1, GIP, and GCG triple receptor agonist precursor.

[0042] This technical solution also provides a method for preparing a GLP-1, GIP, and GCG triple receptor agonist. The polypeptide sequence of the GLP-1, GIP, and GCG triple receptor agonist is synthesized by solid-phase synthesis of the polypeptide or recombinantly expressed to obtain the GLP-1, GIP, and GCG triple receptor agonist.

[0043] This technical solution also provides an application of a GLP-1, GIP, and GCG triple receptor agonist in the preparation of drugs for weight loss, lipid reduction, or blood sugar reduction.

[0044] The technical principle of this technical solution is:

[0045] The present invention relates to the technical field of polypeptide drugs for the treatment of metabolic diseases, and particularly targets the three major metabolic regulation targets: glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon receptor (GCGR). The invention provides a triple receptor agonist of glucagon-like peptide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG), aiming to solve the problem that existing drugs have side effects while ensuring therapeutic effects.

[0046] The GLP-1, GIP, and GCG triple receptor agonist of the present invention achieves weight loss while minimizing side effects through structural optimization. In terms of preparation, the present invention utilizes either recombinant expression or solid-phase synthesis. The former involves constructing an expression vector and inducing expression in engineered cells, while the latter involves direct synthesis of the polypeptide sequence and subsequent side-chain modification. Both methods yield a highly pure and active triple receptor agonist.

[0047] The beneficial effects of this technical solution are:

[0048] (1) Excellent efficacy: In vitro and in vivo activity experiments have demonstrated that the triple receptor agonists provided by the present invention exhibit comparable or superior efficacy to semaglutide and tilpotide in the treatment of metabolic diseases such as obesity. In particular, in multiple-dose studies, analogs 4 and 2 both demonstrated excellent weight loss effects, with a weight loss ratio of approximately 30%, superior to semaglutide and tilpotide.

[0049] In addition to its excellent weight loss effects, the triple receptor agonist provided by this invention also has a beneficial effect on blood sugar control. Experimental animals were subcutaneously injected with agonist analog 4, fasted after administration, and then injected with glucose at designated time points for blood sugar measurement. The results showed that the agonist in this protocol effectively enhanced glucose metabolism and exhibited significant hypoglycemic effects, demonstrating potential application value.

[0050] (2) Significantly reduce side effects: Compared with existing GLP-1 receptor agonists and GLP-1 / GIP dual receptor agonists, the triple receptor agonist provided by the present invention has significant advantages in reducing gastrointestinal adverse reactions (such as nausea, vomiting, diarrhea) and other side effects. In particular, in the multiple-dose experiment on mice, analog 4 still achieved a 29% weight loss effect while maintaining a high diet, which is much higher than semaglutide and tilportide, proving that it has broken through the traditional drug-dependent appetite suppression weight loss model. In addition, in the multiple-dose experiment on rats and beagles, analogs 2 and 4 both showed a significant effect of alleviating the side effect of reduced appetite, and the side effects were smaller than those of LY3437943.

[0051] (3) Broad application prospects: The triple receptor agonist in this scheme has demonstrated significant weight loss effects and the ability to lower blood sugar and regulate glucose metabolism. It can be further explored in the treatment of metabolic diseases such as diabetes and obesity, as well as fatty liver disease, dyslipidemia, non-alcoholic steatohepatitis and other diseases. Its unique mechanism of action and good safety profile provide patients with a safer and more effective treatment option, with broad market prospects and social benefits.

[0052] In summary, the GLP-1, GIP, and GCG triple receptor agonist of the present invention has significant innovation and practicality in the field of metabolic disease treatment, and provides a new solution to the problem that existing drugs are difficult to reduce side effects while ensuring efficacy. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0054] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below.

[0055] The term "GLP-1, GIP, GCG triple receptor agonist" refers to a triple receptor agonist that can simultaneously activate the glucagon-like peptide-1 receptor (GLP-1R), the glucose-dependent insulinotropic polypeptide receptor (GIPR), and the glucagon receptor. This unique mechanism makes this agonist show potential in the treatment of type 2 diabetes and obesity.

[0056] The term "Tirzepatide" refers to a new injectable medication that acts as a dual incretin receptor agonist, activating both the glucagon-like peptide-1 receptor (GLP-1R) and the glucose-dependent insulinotropic polypeptide receptor (GIPR). This dual mechanism of action makes tirzepatide significantly effective in treating type 2 diabetes and also has a positive effect on weight loss. The use of this medication may cause gastrointestinal side effects such as nausea, vomiting, diarrhea, constipation, indigestion, and stomach pain.

[0057] The term "semaglutide" refers to a highly potent GLP-1 receptor agonist that primarily lowers blood sugar levels by stimulating insulin secretion, reducing glucagon secretion, and slowing gastric emptying. Gastrointestinal reactions such as nausea, vomiting, and diarrhea may occur with this drug.

[0058] The term "liraglutide" refers to a long-acting human GLP-1 analog that helps lower blood sugar levels by activating the GLP-1 receptor to increase insulin secretion and reduce unnecessary glucagon release. Gastrointestinal reactions such as nausea, vomiting, and diarrhea may occur with this drug.

[0059] The term "dulaglutide" refers to a GLP-1 receptor agonist that promotes insulin secretion and inhibits glucagon secretion, particularly when blood sugar levels are high. Gastrointestinal reactions such as nausea, vomiting, and diarrhea may occur with this drug.

[0060] The term "glucagon-like peptide-1 receptor (GLP-1R)": GLP-1R is primarily located on pancreatic beta cells, and its natural ligand is glucagon-like peptide-1 (GLP-1). When blood glucose levels rise, GLP-1 is secreted by intestinal L cells, binds to and activates GLP-1R, thereby promoting insulin synthesis and release and inhibiting glucagon secretion. Furthermore, GLP-1R activation can delay gastric emptying, increase satiety, and help control food intake.

[0061] The term "glucose-dependent insulinotropic polypeptide receptor (GIPR)" refers to a receptor expressed on pancreatic beta cells, whose natural ligand is glucose-dependent insulinotropic polypeptide (GIP). Similar to GLP-1, GIP is also involved in regulating postprandial insulin secretion, but its role is more limited to stimulating insulin secretion when blood glucose levels are high.

[0062] The term "glucagon receptor (GCGR)" refers to the GCGR, which is primarily found in the liver and other target tissues. Its natural ligand is glucagon. Glucagon's effects are opposite to those of insulin, promoting hepatic glycogenolysis and gluconeogenesis, thereby increasing blood glucose concentrations. Therefore, the GCGR plays a crucial role in maintaining blood glucose homeostasis.

[0063] The term "agonist" refers to a substance that can bind to and activate a receptor, thereby initiating a specific biochemical reaction or physiological effect within the cell.

[0064] The term "engineered cells" refers to cells that have been modified through genetic engineering technology. These cells can be bacteria, yeast, mammalian cells or even insect cells, with the aim of achieving specific biological functions or producing specific biological products.

[0065] The term "engineered bacteria" refers to microorganisms, usually bacteria, that have been modified through genetic engineering technology to achieve specific functions or produce specific products.

[0066] The term "competent cells" refers to cells that have been specially treated to be able to absorb and integrate foreign DNA under natural conditions.

[0067] The following is further described in detail through specific implementation methods:

[0068] Example 1: Structure of GLP-1, GIP, GCG triple receptor agonist

[0069] The amino acid sequence of the GLP-1, GIP, and GCG triple receptor agonist is as follows:

[0070] X1X2QGT FTSDY SIX 13 LD X 16 X 17 AX 19 X 20 AFIEY LLEGGPSX 33 GA PPPS.

[0071] Wherein, X1 is H or Y; X2 is Aib or G or V; X 13 is L or α-MeL (preferably L); X16 is K or R or G or S (preferably K); X 17 is K or I or C (preferably I); X 19 A or Q (preferably A); X 20 A or Q (preferably Q); X 33 is K or C (preferably K); the side chain is connected to X 33 The side chain is connected to X 33 The conventional method of the prior art is as follows: the carboxyl group at the end of the modifier is activated by succinimide (NHS) ester, and then reacts with the amino group in the R group of K to release the leaving group NHS to form an acylated product, thereby achieving the fatty acid side chain on X 33 Alternatively, the double bond of maleimide and X 33 The sulfhydryl groups react to form a stable sulfide bond, thus achieving the 33 The connection on the thiol group.

[0072] The optional side chain modifiers include: compounds represented by the general formula shown in Formula I, or compounds represented by Formula V, or compounds represented by Formula VI:

[0073]

[0074] wherein a is 7-10, b is 1-4, and A is maleimide, succinimide, or halogenated hydrocarbon;

[0075] Preferably, a is 8, 9 or 10, and b is 2 or 4.

[0076]

[0077] Formula V (Modifier e)

[0078]

[0079] Formula VI (modifier f, HOOC-PEG(20K)-NHS)

[0080] Wherein, the general formula shown in Formula I includes the following specific side chain modifiers:

[0081] Modifier b (HO-C18-Glu-AEEA-AEEA-OSU), modifier c (HO-C20-Glu-AEEA-AEEA-OSU), modifier d (HO-C22-Glu-AEEA-AEEA-OSU), modifier g (HO-C20-Glu-AEEA-AEEA-Mal).

[0082] The structural formulas are shown in Formula II, Formula III, Formula IV and Formula VII respectively.

[0083]

[0084] Formula II (Modifier b)

[0085]

[0086] Formula III (Modifier c)

[0087]

[0088] Formula IV (Modifier d)

[0089]

[0090] Formula VII (Modifier g)

[0091] Example 2: Synthesis of GLP-1, GIP, and GCG triple receptor agonists

[0092] (1) Biotechnology using microbial fermentation

[0093] The synthesis method of the GLP-1, GIP, and GCG triple receptor agonist adopts conventional means of the prior art, and the general process is as follows:

[0094] The nucleotide sequence for expressing the precursor of the triple receptor agonist of GLP-1, GIP and GCG is integrated into the vector to obtain the expression vector; the engineered cells (engineered bacteria) containing the expression vector are constructed; the engineered cells are induced to express the protein; the protein expressed by the engineered cells is recovered by inclusion body, ion exchange chromatography, enzyme digestion and reverse phase chromatography to obtain the precursor of the triple receptor agonist of GLP-1, GIP and GCG. Next, in the X of the precursor of the triple receptor agonist of GLP-1, GIP and GCG, 33 The side chains of the amino acids at the N-terminus of the GLP-1, GIP, and GCG triple receptor agonist precursors after side chain modification are coupled to a dipeptide with the sequence X1X2, where X1 is H or Y and X2 is Aib. It should be emphasized that if X2 is not Aib but is in the form of a natural amino acid (G or V), the entire polypeptide can be expressed using engineered bacteria, that is, the final N-terminal dipeptide coupling operation can be omitted.

[0095] More specifically, the synthesis process is as follows:

[0096] (1) Construction of engineered bacteria

[0097] Construction of fusion protein expression vector: Design a fusion protein expression vector based on the agonist peptide sequence, and optimize the cDNA sequence according to the codon preference of E. coli. Add specific restriction sites (Nde I and Hind III) at both ends of the target gene to facilitate insertion into the pET expression vector.

[0098] Transformation and screening: The constructed expression vector was introduced into competent E. coli BL21 (DE3) cells. Positive clones were screened using kanamycin resistance, and strains with high expression levels were selected through induction experiments as engineered strains.

[0099] Fermentation and protein expression: Use conventional fermentation technology to expand the culture of the selected engineered bacteria, adjust the temperature, pH value, dissolved oxygen content and other conditions to optimize the growth environment, and use IPTG to induce the expression of the target protein under appropriate conditions.

[0100] The bacterial cells obtained by fermentation are suspended, homogenized under high pressure, and then centrifuged to collect inclusion bodies, and the above-mentioned analogues are obtained after enzyme digestion, modification, coupling and purification.

[0101] (2) Chemical synthesis

[0102] In addition to the above methods, the polypeptides of this scheme can also be prepared using conventional solid-phase synthesis methods in the prior art and their modification sites, using standard side chain protecting groups. The solid-phase synthesis method is as follows:

[0103] (1) Resin pretreatment

[0104] Weigh Rink Amide-MBHA Resin (0.6 mmol / g loading, 1.0 g mass) into a solid-phase reactor. Add 10 mL of dichloromethane (DCM) and allow to swell at room temperature for 30 minutes. After swelling, filter and wash the resin three times with 10 mL of N,N-dimethylformamide (DMF).

[0105] (2) Fmoc deprotection

[0106] Add 10 mL of a 20% pyridine-containing DMF solution (V / V) and react at room temperature for 30 minutes to remove the Fmoc protecting group. After filtration, wash the resin twice with DMF (10 mL each time).

[0107] (3) Amino acid coupling

[0108] Under dry conditions, Fmoc-Ser(tBu)-OH (0.56 g, 1.8 mmol, 3.0 equiv) and 1-hydroxybenzotriazole (HoBt, 0.24 g, 1.8 mmol, 3.0 equiv) were dissolved in 5 mL of DMF and stirred for 5 minutes for activation. N,N'-diisopropylcarbodiimide (DIC, 0.28 mL, 1.8 mmol, 3.0 equiv) was added, and activation was continued for 5 minutes. The activated solution was transferred to the resin and reacted at 25°C for 2 hours. The reaction progress was monitored using the ninhydrin assay (if incomplete, the reaction time was extended). After completion of the reaction, the resin was washed once with 10 mL of DMF containing 20% ​​pyridine and then twice with DMF (10 mL each wash).

[0109] (4) Loop Iteration

[0110] Repeat steps 2-3, coupling subsequent Fmoc-protected amino acids sequentially from the C-terminus to the N-terminus according to the target sequence, until the final amino acid, Fmoc-Tyr(tBu)-OH (0.68 g, 1.8 mmol, 3.0 equivalents), is coupled. Finally, remove the Fmoc protecting group (using the same method as step 2). Wash the resin thoroughly with DMF (10 mL x 3) and set aside for subsequent cleavage and purification steps.

[0111] (5) Cracking reaction

[0112] The protected peptide resin (1.0 g, 0.6 mmol) was placed in 20 mL of cleavage solution (TFA:EDT:thioanisole = 95:2.5:2.5) and stirred at room temperature for 2.5 hours. After the reaction was complete, the resin was filtered, the cleavage solution was collected, and the resin was washed twice with 2 mL of TFA, and the filtrates were combined.

[0113] (6) Peptide precipitation and washing

[0114] Slowly add the lysate dropwise to 40 mL of methyl tert-butyl ether (MTBE) and stir on ice for 10 minutes to precipitate the peptide. Centrifuge at 4°C (5000 rpm, 5 minutes), discard the supernatant, and collect the precipitate. Wash the precipitate three times with 20 mL of MTBE, discarding the supernatant after each centrifugation. Vacuum dry for 2 hours to obtain a crude peptide white powder.

[0115] (7) Reverse-phase high performance liquid chromatography (RP-HPLC) purification column: C18 reverse-phase column (5 μm, 250 × 10 mm, ).

[0116] Mobile phase: Phase A: 0.1% TFA / water (v / v); Phase B: 0.1% TFA / acetonitrile (v / v);

[0117] Gradient elution (flow rate 4 mL / min): 0-5 min: 20% B; 5-25 min: 20%→60% B (linear gradient); 25-30 min: 60%→95% B (linear gradient); 30–35 min: 95% B (maintain).

[0118] Detection wavelength: 220nm, collect the target peak. Combine the fractions containing the target peptide, remove the acetonitrile by rotary evaporation, and freeze-dry the remaining aqueous phase. Through the above process, a high-purity target peptide (white powder) is obtained. Its structure can be confirmed by high-performance liquid chromatography and mass spectrometry analysis. It should be noted that during the solid phase synthesis of the peptide sequence, X 16 or X 33 The amino acids have been pre-attached with side chain groups by conventional methods in the prior art, so the final product is an analogue with a side chain. The process of attaching the side chain to the amino acid in the prior art is roughly as follows: the succinimide (NHS) active group at the end of the modifier reacts with the amino group to form an acylated product, thereby achieving the fatty acid side chain on X 16 or X 33 Alternatively, the double bond of maleimide and X 16 or X 33 The sulfhydryl groups react to form a stable sulfide bond, thus achieving the 16 or X 33 The connection on the thiol group.

[0119] The following examples experimentally study the technical effects of the obtained polypeptide drugs. The specific sequences and modifications of the polypeptide drugs used in the experimental studies are shown in Table 1.

[0120] Table 1: Sequence information of peptides to be tested (analogs 1-17, double underline and bold symbols represent X1 or X2 or X 13 or X 16 or X 17 or X 19 or X 20 or X 33 ; The substances corresponding to these sequences are named as analogues 1 to analogues 17 according to the numbering)

[0121]

[0122]

[0123] Example 3: In vitro activity studies

[0124] The HTRF cAMP assay was used to analyze the effects of the test compounds on different targets (expressed as EC50, half-maximal effective concentration). The experiment was performed according to the kit instructions. The experimental results are detailed in Table 2.

[0125] Table 2: In vitro activity study results (EC 50 , nM)

[0126]

[0127] From the above experimental data, we can see that analogue 2 has the most significant effect on the three receptor targets, followed by analogues 4 and 5. 33 Although modified side chains are added to the positions of the two analogs and the non-natural amino acid sites are consistent, the effects on the three targets vary significantly due to small differences in the peptide sequences. Combined with the data presented below, analog 2 has the most ideal weight loss effect after a single dose and is the most potent agonist, far exceeding other candidate analog agonists. The weight loss effects of multiple doses of analogs 2 and 4 are essentially the same, and analog 4 has the least effect on mouse food intake, indicating that it can safely and effectively reduce mouse body weight and can also serve as a potential agonist.

[0128] Example 4: Evaluation of drug activity in mice

[0129] (1) Single-dose experiment

[0130] KM mice were used, and a quarantine period of at least 2 days and a 5-day adaptation period were performed before administration, during which sufficient food and water were given. Before the formal experiment began, the individuals were marked and weighed and randomly divided into groups. The mice were given a single dose of subcutaneous injection according to the experimental grouping, and the body weight and food intake of the mice were measured 72 hours after administration (measured at the same time every day). The average weight loss ratio and average Δ body weight of each mouse were calculated, weight loss ratio = (body weight 72 hours after administration - body weight before administration) / body weight before administration × 100%, Δ body weight = body weight 72 hours after administration - body weight before administration. There were 6 mice in each group of experiments, and the experimental data were expressed in the form of average weight loss ratio ± standard deviation and average Δ body weight ± standard deviation.

[0131] Specifically, the single administration dosage is as follows: the dosage of all analogs (peptide drugs to be tested) is 30 nmol / kg; the negative control is 0.9% NaCl. The experimental results are detailed in Table 3.

[0132] Table 3: Single-dose experimental results

[0133] Group Average Δ body weight (g) Average weight loss ratio (%) Negative control (0.9% NaCl) 0.56±0.93 1.31±2.17 Analog 1 -5.68±1.44 -13.27±3.37 Analog 2 -7.80±0.62 -18.22±1.46 Analog 3 1.11±1.13 2.58±2.65 Analog 4 -7.40±1.74 -17.29±4.07 Analog 5 0.14±0.68 0.33±1.59 Analog 6 0.48±1.16 1.12±2.72 Analog 7 1.36±0.70 2.14±1.09 Analog 8 -0.64±0.94 -1.50±2.18 Analog 9 1.46±0.47 3.40±1.11 Analog 10 -0.64±0.94 2.51±2.15 Analog 11 -5.80±0.63 -13.56±1.48 Analog 12 -0.96±0.86 -2.08±2.10 Analog 13 -1.96±0.44 -4.63±1.09 Analog 14 -0.58±0.43 -1.38±1.00 Analog 15 -0.98±0.48 -2.32±1.13 Analog 16 -1.70±0.53 -4.17±1.21 Analog 17 -1.15±0.37 -2.83±0.90

[0134] In the design of peptides with similar functions (treating diabetes, fatty liver disease, dyslipidemia, non-alcoholic steatohepatitis and obesity, etc.), in order to enhance the effect of the peptide, some non-natural amino acids, such as Aib, α-MeL, etc., are introduced into the peptide sequence. For example: X 20 If Aib is used, GIP activity, pharmacokinetics (PK), X13 The use of α-MeL13 (α-methyl-L-leucine) can enhance the activity of GCG and GIP. However, the introduction of these unnatural amino acids can complicate sequence synthesis and hinder large-scale industrial synthesis. Unlike existing technologies, the peptides in this proposal minimize the use of unnatural amino acids while maintaining efficacy.

[0135] In the process of studying polypeptide sequence and side chain modification, the inventors tried X 16 The amino acid at position 1 is used as a side chain modification site. 16 The amino acid residue at this position was modified to K for connecting the side chain, forming a sequence design as shown in SEQ ID NO.4. For details, see analogs 7 / 8 / 9 / 10. The experimental results showed that the sequence design of SEQ ID NO.4 was compatible with X 16 Side chain modifications at the 3 position of the agonist cannot effectively guarantee the weight loss effect of the product. According to the experimental results in Table 3, analogs 7-10 basically have no significant weight loss effect, and some analogs even cause weight gain in experimental mice. This shows that how to design amino acid sequences and side chain modifications to effectively ensure the agonist effect is a relatively difficult problem to solve.

[0136] Analogs 5 and 6 form a sequence as shown in SEQ ID NO.3 and are combined with X 33 The agonist obtained by side chain modification at the site is not ideal in reducing the weight of mice. It basically has no effect on reducing weight and may even cause the weight of mice to increase.

[0137] The inventors further adjusted the polypeptide sequence and compared the adjusted polypeptide sequence with X 33 The results show that the effect of analogue 2 is better than that of analogue 4 and analogue 12. The weight loss ratio of analogue 2 can reach about 18%, which is better than about 17% of analogue 4, about 2% of analogue 12 and about 4% of analogue 13. It can be seen that the specific polypeptide sequence and X 33 Only by using modified and combined sequences can the agonist effect be effectively guaranteed. In terms of the effect of single administration, SEQ ID NO.1 and SEQ ID NO.2 are the best choices.

[0138] In addition, the inventors also studied the weight loss effect of the optimal sequence SEQ ID NO.1 under different modification methods (different modification sites), see the experimental data of analogs 1-3 for details. 16 Site side chain modification and X 33 Side chain modification at both sites can reduce the weight of mice, but in X 33 The side chain modification at two sites has a better effect and is a more optimized solution. Since the side chain modification at two sites can improve the drug efficacy to varying degrees, the inventors also tried to modify the two sites of the peptide in order to improve the drug effect. The data of analog 3 showed that the double modification did not play an effective role in improving the efficacy and even increased the weight of the mice after administration. It can be seen that in X 33 The key to fully enhance the weight loss effect of agonists is to modify the fatty acids at the side chain. The inventors tried different side chain modification sites and finally determined X 33 The side chain of the amino acid at position 1 is modified, which is a new modification site that has not been reported in the prior art. 33 The side chain modification at the position, combined with a specific polypeptide sequence, can effectively enhance the weight-loss effect of the agonist.

[0139] Analogs 14-17, which do not use any unnatural amino acids in their polypeptide sequences, still exhibited a moderate weight-loss effect. Analog 11, which incorporates the unnatural amino acid α-Mel, was not as effective as Analog 2 or Analog 4, further demonstrating that SEQ ID NOs. 1 and 2 are optimal choices for single-dose efficacy.

[0140] (2) Multiple-dose experiment

[0141] In addition to the single-dose experiment, multiple-dose experiments were also conducted. The experimental groups involved were: negative control (0.9% NaCl) and peptide drug treatment group, which were dosed once every 72 hours for a total of three doses; the single dose of the peptide drug treatment group was 30 nmol / kg; the peptide drugs included: semaglutide (batch number 202202AMV1), tilportide (batch number D792710D), analog 2, and analog 4. During the experiment, the average weight loss ratio and average Δ body weight in each group were counted. There were 5 mice in each group, and the experimental data were expressed in the form of average weight loss ratio ± standard deviation, average Δ body weight ± standard deviation. At the same time, the average food intake of each group of experimental animals per day was counted.

[0142] The experimental results are shown in Tables 4 and 5.

[0143] Table 4: Results of multiple-dose experimental study (average weight loss ratio and average Δ body weight)

[0144]

[0145] Table 5: Statistics of average food intake of experimental animals (unit: g)

[0146] D1 D2 D3 D4 D5 D6 D7 D8 D9 Negative control 5.50 7.16 7.04 7.76 8.32 8.54 8.74 8.78 9.16 semaglutide 3.20 5.94 7.54 1.88 5.64 7.42 4.24 7.42 9.14 Tipoleptide 0.74 1.12 2.60 1.16 2.88 5.12 4.24 6.00 6.50 Analog 2 3.45 5.10 8.03 3.53 4.98 6.60 3.50 4.45 5.62 Analog 4 5.35 8.85 10.55 5.98 8.27 10.73 5.88 8.78 10.62 Analog 11 0.70 3.82 5.40 3.68 5.12 6.23 7.99 5.44 8.67 Analog 12 2.39 4.67 8.51 2.95 7.16 8.03 4.69 8.20 9.91

[0147] Regarding the weight-loss effects of the analogs tested above, both analogs 4 and 2 demonstrated significant weight-loss effects after multiple administration, with weight-loss ratios reaching approximately 30%, surpassing those of semaglutide and tilportide, as well as analogs 11 and 12 (see Table 4 for details). It is important to note that analog 11 demonstrated a relatively good effect in single-dose experiments. However, after multiple administrations, the weight-loss effect of analog 11 deteriorated, far inferior to that of analogs 2 and 4. The inventors speculate that this is due to the experimental animals developing resistance to analog 11, as evidenced by a significant decrease in food intake and weight after the initial injection, with subsequent injections showing worsening effects. For analog 11, food intake on days 1, 4, and 7, all measured 24 hours after injection, showed an increasing trend. This phenomenon may be due to the animals developing resistance to analog 11. In contrast, food intake for analogs 2 and 4 on days 1, 4, and 7, immediately after injection, was similar, suggesting that analogs 2 and 4 are less susceptible to developing resistance. The weight loss effect of analogue 12 in a single experiment was not ideal, and its effect was not significantly improved in multiple experiments.

[0148] With respect to the food intake of experimental animals (the side effect of the drug suppressing appetite), in the multiple dosing experiment, the average daily food intake of mice was recorded synchronously to study the side effects of the drug. The experimental data in Table 5 show that the food intake of experimental animals decreased significantly on the day after each administration of analog 2 (D1, D4, D7), and the food intake of experimental animals gradually increased over time after administration. In general, the side effect of suppressing appetite of analog 2 is smaller than that of tirpotide in the prior art, and is similar to that of semaglutide (but the weight loss effect is significantly excellent). Analog 2 can effectively reduce the weight of experimental animals relative to these existing drugs under a smaller side effect (appetite suppression). Therefore, analog 2 is a relatively ideal polypeptide drug that reduces side effects and effectively controls weight. The effect of analog 4 on food intake is smaller than that of analog 2, especially in the early stage (24h after injection of D1, D4, and D7), and the inhibition of food intake is significantly lower than that of semaglutide and tirpotide. Combined with the data in Table 4, the weight loss effect of analogue 4 is significantly higher than that of semaglutide and tilpotide, demonstrating that it can still achieve unexpected weight loss effects while maintaining a suitable diet. More specifically, compared to tilpotide, where the food intake drops to a very low level after each injection, analogue 4 still achieves a weight loss effect of approximately 30% while maintaining a higher food intake, which is much higher than semaglutide (-5.27%) and tilpotide (-14.04%). This demonstrates that it breaks through the traditional drug-dependent appetite suppression weight loss model and can still achieve unexpected weight loss effects while maintaining a suitable diet. Among them, the structural differences between analogues 2 and 4 mainly lie in the amino acid residue type of X33 (K and C, respectively) and the side chain modification type on X33 (Formula III and Formula VII, respectively). These structural differences lead to differences in the effects and modes of action. However, overall, analogues 2 and 4 are superior to similar peptide products in the prior art in terms of improving weight loss effects and reducing side effects that affect appetite.

[0149] In addition to analogues 2 and 4, the inventors have also conducted a large number of studies on analogues of other sequence structures. For example, analogue 11 is different from analogue 4 mainly in that X 13 Position (L in analogue 4 and α-Mel in analogue 11) and X 17 The amino acid residues at position (I for analogue 4 and K for analogue 11) are used. Although the difference in amino acid residues is relatively small, the weight loss effect (after multiple administration) and the effect of reducing the side effect of appetite suppression of analogue 11 are far inferior to those of analogue 4. In fact, the use of analogue 11 may lead to a certain degree of drug resistance in experimental animals. This shows that the design of appropriate amino acid sequences is crucial for ensuring the efficacy of peptide drugs. The fact that analogues 2 and 4 can achieve the above-mentioned excellent effects is an unexpected technical effect.

[0150] The inventors also conducted multiple dosing experiments on analogue 12. The main difference between analogue 12 and analogue 4 is that X 19 Position (A for analogue 4, Q for analogue 12) and X 20 The amino acid residues at positions (Q for analogue 4 and A for analogue 12) were replaced. Analogue 12 was inferior to analogue 11 in multiple aspects, including single-dose and multiple-dose studies, and appetite suppression in mice. These experimental results further demonstrate that the superior effects achieved by analogues 2 and 4 are unexpected technical achievements.

[0151] Example 5: Effect of drugs on rat food intake

[0152] Multiple dosing experiments were conducted on rats (n=3). The peptide drug was administered at a dose of 200 nmol / kg (0.9% NaCl in the negative control group). Starting from D1, the drugs were administered subcutaneously every 48 hours for a total of 5 doses. The average food intake of each group of experimental animals was calculated daily. The experimental results are detailed in Table 6. The experimental data showed that analogs 2 and 4 reduced the food intake of experimental animals to a certain extent compared with the negative control. However, compared with the prior art LY3437943, analogs 2 and 4 had a significantly reduced inhibitory effect on the appetite of mice. This indicates that analogs 2 and 4 developed in this protocol have fewer side effects and have achieved unexpected technical results in reducing the side effect of appetite suppression.

[0153] Table 6: Average food intake of experimental animals (g)

[0154] D2 D3 D4 D5 D6 D7 D8 D9 D10 Analog 2 19.6 24.8 24.1 24.5 17.5 26.3 14.9 20.4 23.4 Analog 4 16.7 23.3 16.9 21.3 14.5 27.7 16.4 25.3 16.2 LY3437943 3.4 0.77 2.6 2.4 1.5 2.0 2.3 0.9 1.0 Negative control 30.0 32.9 28.5 29.4 32.2 30.8 28.8 30.8 29.3

[0155] Example 6: Effects of Drugs on Dietary Amount in Beagle Dogs

[0156] A multiple-dose study was conducted in beagle dogs (n=1). The peptide drug was administered at a subcutaneous dose of 100 nmol / kg, starting on Day 1 and administered every 72 hours for a total of five doses. The experimental results are detailed in Table 7. These results demonstrate that analogs 2 and 4 exhibited less appetite-reducing side effects than LY3437943, consistent with the effect trends observed in Example 5. Compared to the prior art LY3437943, analogs 2 and 4 exhibited 1.6-1.7 times greater total food intake.

[0157] Table 7: Average food intake of experimental animals (g)

[0158]

[0159]

[0160] At the same time, the metabolic parameters of the above-mentioned experimental animals were tested. The levels of total cholesterol (CHO), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were tested after the experimental animals received 5 treatments. The data of metabolic parameters at a dose of 100 nmol / kg are provided in Table 8.

[0161] Table 8: Total cholesterol (CHO), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels in experimental animals before and after 5 treatments

[0162]

[0163] As shown in Table 8, analogs 2 and 4 have significant effects on lowering cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein.

[0164] Example 7: Pharmacokinetic (PK) study of drugs in rats

[0165] Male rats were administered a 400 nmol / kg subcutaneous injection. 0.2 ml of whole blood (EDTA anticoagulant tube) was collected from each rat at 0 h, 2 h, 6 h, 10 h, 24 h, 34 h, 48 h, 72 h, 96 h, 120 h, and 144 h after administration. The blood was processed to obtain plasma samples, which were then analyzed by LC-MS / MS. The plasma concentration-time curves were analyzed using Graphpad Pism 9.5 software, and PK parameters and half-lives were calculated. The PK parameters calculated using the above method are shown in Table 9, which shows the maximum plasma concentrations (C max ), peak time (T max ), half-life (T 1 / 2 ), area under the drug-time curve (AUC 0-last ), the above analogs all showed prolonged pharmacokinetic profile.

[0166] Table 9: Pharmacokinetic (PK) parameters of drugs in rats

[0167]

[0168] Example 8: Glucose tolerance assessment in normal KM mice

[0169] The blood glucose and body weight of KM mice were tested and randomly divided into 3 groups (negative control group, high-dose experimental group, low-dose experimental group), with 5 mice in each group. The animals in the low-dose experimental group and the high-dose experimental group were subcutaneously injected with analog 4, with doses of 5nmol / kg and 20nmol / kg, respectively, and the injection volume was 0.1ml. The control group was subcutaneously injected with the corresponding solvent. After a single dose, the animals were fasted and the blood glucose levels were measured at 0h, 3h, 6h, 12h, 24h, and 36h. The measurement method was to inject 50% glucose intraperitoneally for 10 minutes, and then draw blood from the tail vein for blood glucose detection. The experimental results are shown in Table 10. The blood glucose levels of all dosage groups were significantly lower than those of the control group, confirming that the agonist of the present invention can effectively enhance the body's glucose metabolism ability and has a hypoglycemic effect.

[0170] Table 10: Blood glucose results after intraperitoneal injection of glucose in mice

[0171]

[0172] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the structure of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A triple receptor agonist of GLP-1, GIP and GCG, characterized in that: Its amino acid sequence is: X1X2QGT FTSDY SIX 13 LD X 16 X 17 AX 19 X 20 AFIEY LLEGGPSX 33 GA PPPS; Wherein, X1 is H or Y; X2 is Aib or G or V; X 13 is L or α-MeL; X 16 K or R or G or S; X 17 K or I or C; X 19 A or Q; X 20 A or Q; X 33 K or C.

2. The GLP-1, GIP, and GCG triple receptor agonist according to claim 1, characterized in that: X 16 or / and X 33 There are side chain groups attached.

3. The GLP-1, GIP, and GCG triple receptor agonist according to claim 2, characterized in that: The side chain group is as shown in Formula I, or as shown in Formula V, or as shown in Formula VI: wherein a is 7-10, b is 1-4, and A is maleimide, succinimide, or halogenated hydrocarbon.

4. The GLP-1, GIP, and GCG triple receptor agonist according to claim 3, characterized in that: Formula I is any one of Formula II, Formula III, Formula IV and Formula VII; 5. The GLP-1, GIP, and GCG triple receptor agonist according to claim 4, characterized in that: Its amino acid sequence and side chain modifications are: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 The position is modified with a side chain as shown in formula III; Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula III; Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 Bit and X 33 The position is modified with a side chain as shown in formula III; Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII; Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula III; Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula IV; Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula III; Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula VI; Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula V; Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The position is modified with a side chain as shown in formula II; Or YAibQGT FTSDY SIα-MelLD KKAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII; Or YAibQGT FTSDY SILLD KIAQA AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII; Or YAibQGT FTSDY SILLD GIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula III; Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The position is modified with a side chain as shown in formula VII; Or YVQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula III; Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The position is modified with a side chain as shown in formula III; Or YGQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The modified position has a side chain as shown in formula VII.

6. The GLP-1, GIP, and GCG triple receptor agonist according to claim 5, characterized in that: Its amino acid sequence is: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The modified position has a side chain as shown in formula III.

7. The GLP-1, GIP, and GCG triple receptor agonist according to claim 5, characterized in that: Its amino acid sequence is: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The modified position has a side chain as shown in formula VII.

8. The GLP-1, GIP, and GCG triple receptor agonist according to claim 5, characterized in that: Its amino acid sequence is: YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The modified position has a side chain as shown in formula III.

9. The method for preparing a triple receptor agonist of GLP-1, GIP and GCG according to any one of claims 1 to 8, characterized in that: Preparation of GLP-1, GIP, GCG triple receptor agonist precursors, modification of side chain groups and chemical coupling of special amino acids; side chain group modification in X 33 Bit or X 16 amino acid; Alternatively, the polypeptide sequence of the GLP-1, GIP, and GCG triple receptor agonist is synthesized by solid phase polypeptide synthesis or recombinantly expressed to obtain the GLP-1, GIP, and GCG triple receptor agonist.

10. Use of the GLP-1, GIP, GCG triple receptor agonist according to any one of claims 1 to 8 in the preparation of a drug for weight loss, lipid reduction or blood sugar reduction.

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