Triple receptor agonists of glp-1, gip, gcg and methods of making and using the same

Abstract

The present application relates to the technical field of polypeptide drugs for metabolic diseases, and in particular relates to a triple receptor agonist (protein) of glucagon-like peptide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG). The triple receptor agonist protein provided in the technical solution achieves weight loss while exhibiting lower side effects and enhancing drug compliance. The triple receptor agonist developed in the technical solution can be applied to the preparation of a drug with weight loss, lipid-lowering or blood sugar-lowering effects, solving the technical problem that existing triple receptor agonists of GLP-1, GIP and GCG are difficult to reduce side effects while reducing body weight. The drug breaks through the traditional weight loss mode of appetite suppression, has a unique mechanism of action and good safety, provides a safer and more effective treatment for patients, and has a broad market prospect and social benefit.

Description

TECHNICAL FIELD

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

[0002] Glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon receptor (GCGR) have become very important drug targets for related enterocrines in the treatment of diabetes mellitus, obesity and other metabolic diseases. GLP-1R, GIPR and GCGR are all G protein-coupled receptors that regulate glucose, lipid and amino acid metabolism, and GLP-1R / GIPR / GCGR are important participants in maintaining blood glucose balance in the human body. GLP-1R has the effect of promoting insulin secretion, while GIPR increases insulin secretion in hyperglycemia and stimulates glucagon release in hypoglycemia; GCGR is involved in blood glucose and energy regulation. Glucagon-like peptide-1 (GLP-1) is an enterocrine secreted by L cells in the intestine, and GLP-1 receptor agonists can promote glucose-dependent insulin secretion, regulate appetite and energy metabolism, reduce blood glucose and body weight in vivo. Similar to GLP-1, glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) are also peptide hormones responsible for glucose homeostasis. GIP is a polypeptide hormone secreted by K cells in the intestine, which can promote insulin secretion to maintain glucose balance and promote the decomposition and deposition of white adipose tissue to supplement the effect of GLP-1 receptor agonists. GCG is secreted by alpha cells in the islets, which can stimulate energy consumption, reduce blood lipids, inhibit gastric emptying and appetite, thereby reducing body weight. In a hypoglycemic state, GIP can also promote the secretion of GCG to raise blood glucose and greatly reduce 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, particularly showing broad prospects in type 2 diabetes mellitus (T2DM), obesity, cardiovascular protection, and non-alcoholic fatty liver disease (NAFLD / NASH). Targeting intestinal hormone effects, GLP-1 receptor agonists such as liraglutide, dulaglutide, and smegglutide have been successfully developed for the treatment of type 2 diabetes. Eli Lilly's telpolide, the first GLP-1 / GIP dual receptor agonist developed in China, has been officially launched, synergistically regulating blood glucose and weight by activating two intestinal hormone receptors. While these peptide drugs have shown good efficacy in controlling blood glucose and reducing weight, existing GLP-1 receptor agonists (such as smegglutide) and GLP-1 / GIP dual receptor agonists (such as telpolide) are prone to gastrointestinal adverse reactions (such as nausea, vomiting, and diarrhea) and side effects such as increased heart rate (2-4 beats per minute) during clinical application, especially during dose escalation. Therefore, reducing the side effects of similar peptide drugs is a pressing issue that needs to be addressed in this field. For these reasons, it is essential to optimize GLP-1R, GIPR, and GCGR triple agonists to reduce their side effects while maintaining their therapeutic efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a GLP-1, GIP, GCG triple receptor agonist that exhibits the advantage of reducing weight while minimizing side effects, thereby solving the technical problem that existing GLP-1, GIP, GCG triple receptor agonists are unable to reduce side effects while simultaneously reducing weight.

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

[0006] GLP-1, GIP, and GCG triple receptor agonists, their amino acid sequences are as follows:

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

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

[0009] Furthermore, X 16 or / and X 33 It has a side chain group attached.

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

[0011]

[0012]

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

[0014] Furthermore, Equation I can be any one of Equations II, III, IV, and VII;

[0015]

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

[0017] YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 The positional modification has a side chain as shown in Formula III;

[0018] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The positional modification has a side chain as shown in Formula III;

[0019] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 16 Position and X 33 The positional modification has a side chain as shown in Formula III;

[0020] Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The positional modification has a side chain as shown in Formula VII;

[0021] Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The positional modification has a side chain as shown in Formula III;

[0022] Or YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The positional modification has a side chain as shown in Formula IV;

[0023] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The positional modification has a side chain as shown in Formula III;

[0024] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The positional modification has a side chain as shown in Formula VI;

[0025] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 Positional modifications have side chains as shown in Formula V;

[0026] Or YAibQGT FTSDY SILLD KCAAQ AFIEY LLEGGPSCGA PPPS, and X 16 The positional modification has a side chain as shown in Formula II;

[0027] Or YAibQGT FTSDY SIα-MelLD KKAAQ AFIEY LLEGGPSCGAPPPS, and X 33 The positional modification has a side chain as shown in Formula VII;

[0028] Or YAibQGT FTSDY SILLD KIAQA AFIEY LLEGGPSCGAPPPS, and X 33 The positional modification has a side chain as shown in Formula VII;

[0029] Or YAibQGT FTSDY SILLD GIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The positional modification has a side chain as shown in Formula III;

[0030] Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 The positional modification has a side chain as shown in Formula VII;

[0031] Or YVQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGA PPPS, and X 33The positional modification has a side chain as shown in Formula III;

[0032] Or YVQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The positional modification has a side chain as shown in Formula III;

[0033] Or YGQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSCGA PPPS, and X 33 Positional modifications include side chains as shown in Formula VII.

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

[0035] YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGGPSKGA PPPS, and X 33 The modification has a side chain as shown in Formula III (analyte 2).

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

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

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

[0039] YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGGPSKGAPPPS, and X 33 The modification has a side chain as shown in Formula III (analyte 5).

[0040] This technical solution also provides a method for preparing a GLP-1, GIP, GCG triple receptor agonist, which includes the following steps: preparation of GLP-1, GIP, GCG triple receptor agonist precursors, modification of side chain groups, and chemical coupling of specific amino acids; modification of side chain groups in X 33 bit or X 16 One amino acid.

[0041] Furthermore, the nucleotide sequences for expressing the GLP-1, GIP, and GCG triple receptor agonist precursors were integrated into a vector to obtain an expression vector; engineered cells containing the expression vector were constructed; the engineered cells were induced to express the protein; the protein expressed by the engineered cells was subjected to inclusion body recovery, ion exchange chromatography, enzyme digestion, and reverse phase chromatography to obtain the GLP-1, GIP, and GCG triple receptor agonist precursors.

[0042] This technical solution also provides a method for preparing GLP-1, GIP, and GCG triple receptor agonists. The polypeptide sequences of GLP-1, GIP, and GCG triple receptor agonists are synthesized by solid-phase synthesis or recombinant expression of polypeptides to obtain GLP-1, GIP, and GCG triple receptor agonists.

[0043] This technical solution also provides the application of a GLP-1, GIP, 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 as follows:

[0045] This invention relates to the field of peptide drug technology for the treatment of metabolic diseases, and specifically targets three major metabolic regulatory targets: glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide receptor (GIPR), and glucagon receptor (GCGR). It provides a triple receptor agonist of glucagon-like peptide (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon (GCG), aiming to solve the problem of side effects of existing drugs while ensuring therapeutic efficacy.

[0046] The GLP-1, GIP, and GCG triple receptor agonist of this invention achieves weight loss while reducing side effects through structural optimization. In terms of preparation, this invention employs two methods: recombinant expression and solid-phase synthesis. The former involves constructing an expression vector and inducing expression in engineered cells, while the latter directly synthesizes the polypeptide sequence and modifies its side chains. Both methods yield high-purity, highly active triple receptor agonists.

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

[0048] (1) Excellent efficacy: Through in vitro and in vivo activity experiments, the triple receptor agonist provided by this invention has demonstrated efficacy comparable to or better than smegglutide and telposide in the treatment of metabolic diseases such as obesity. In particular, in multiple-dose experiments, analogues 4 and 2 both showed good weight-reduction effects, with a weight loss rate of about 30%, which is superior to smegglutide and telposide.

[0049] The triple receptor agonist provided by this invention not only has a good weight loss effect but also a good effect on blood glucose control. Animals in the experimental group were subcutaneously injected with agonist analog 4, fasted after administration, and injected with glucose at specified time points, with blood glucose levels measured. The results showed that the agonist in this regimen effectively enhanced glucose metabolism, had a significant hypoglycemic effect, and possessed potential application value.

[0050] (2) Significantly Reduced Side Effects: Compared with existing GLP-1 receptor agonists and GLP-1 / GIP dual receptor agonists, the triple receptor agonist provided by this invention has a significant advantage in reducing gastrointestinal adverse reactions (such as nausea, vomiting, and diarrhea). In particular, in multiple-dose experiments in mice, analog 4 achieved a 29% weight loss while maintaining a high food intake, far exceeding smegglutinide and telposide, demonstrating that it breaks through the traditional drug-dependent appetite suppression weight loss model. In addition, in multiple-dose experiments in rats and beagle dogs, both analogs 2 and 4 showed significant effects in alleviating appetite-reducing side effects, with fewer side effects compared to LY3437943.

[0051] (3) Broad Application Prospects: The triple receptor agonist in this regimen has demonstrated significant weight loss effects as well as the ability to lower blood sugar and regulate glucose metabolism. Further applications are anticipated in the treatment of metabolic diseases such as diabetes and obesity, as well as various diseases including fatty liver disease, dyslipidemia, and non-alcoholic steatohepatitis. Its unique mechanism of action and good safety profile provide patients with a safer and more effective treatment option, possessing 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 Implementation

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

[0054] To make the invention easier to understand, certain technical and scientific terms are defined below.

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

[0056] The term "tirzepatide" refers to a novel injectable drug that acts as a dual incretin receptor agonist, simultaneously activating both the glucagon-like peptide-1 receptor (GLP-1R) and the glucose-dependent insulinotropic peptide receptor (GIPR). This dual mechanism of action makes tirzepatide highly effective in treating type 2 diabetes and also has a positive effect on weight loss. Side effects that may occur with this drug include gastrointestinal discomfort such as nausea, vomiting, diarrhea, constipation, indigestion, and stomach pain.

[0057] The term "semaglutide" refers to a 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 when using this medication.

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

[0059] The term "dulaglutide" refers to a GLP-1 receptor agonist that promotes insulin secretion and inhibits glucagon secretion, particularly at higher blood glucose levels. Gastrointestinal reactions such as nausea, vomiting, and diarrhea may occur when using this medication.

[0060] The term "glucagon-like peptide-1 receptor (GLP-1R)" refers to the fact that GLP-1R is mainly distributed on pancreatic β 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, which bind to and activate GLP-1R, thereby promoting insulin synthesis and release and inhibiting glucagon secretion. Furthermore, activation of GLP-1R can delay gastric emptying, increase satiety, and help control food intake.

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

[0062] The term "glucagon receptor (GCGR)" refers to glucagon receptors, which are primarily found in the liver and other target tissues. Their natural ligand is glucagon. Glucagon acts inversely to insulin, promoting glycogenolysis and gluconeogenesis, thus increasing blood glucose levels. Therefore, 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 triggering a specific biochemical reaction or physiological effect within the cell.

[0064] The term "engineered cell" refers to cells that have been modified through genetic engineering techniques. These cells can be bacteria, yeast, mammalian cells, or even insect cells, designed to achieve specific biological functions or produce specific biological products.

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

[0066] The term "competent cell" refers to a cell that, after special treatment, is able to absorb and integrate exogenous DNA under natural conditions.

[0067] The following detailed description illustrates the specific implementation method:

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

[0069] The amino acid sequences of GLP-1, GIP, and GCG triple receptor agonists are as follows:

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

[0071] Where X1 is H or Y; X2 is Aib or G or V; X 13 It is L or α-MeL (preferably L); X16 K, R, G, or S (preferably K); X 17 K, I, or C (preferably I); X 19 It is A or Q (preferably A); X 20 For A or Q (preferably Q); X 33 K or C (preferably K); the side chain is connected to X. 33 The amino or thiol group. The side chain is attached to X. 33 The amino or thiol group is in the conventional form of existing technology: after the carboxyl group at the end of the modifier is activated by succinimide (NHS) ester, it reacts with the amino group in the R group of K, releasing the leaving group NHS, forming an acylated product, and realizing the fatty acid side chain in X. 33 The linkage to the amino group; or, the double bond of maleimide with X. 33 The thiol group reacts to form a stable thioether bond, enabling the fatty acid side chain to be in X 33 The linker on the thiol group.

[0072] 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] Where a is 7-10, b is 1-4, and A is maleimide, succinimide, or a haloalkane;

[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] Among them, 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), and modifier g (HO-C20-Glu-AEEA-AEEA-Mal).

[0082] For the structural formulas, please refer to Formulas II, III, IV, and 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, GCG triple receptor agonists

[0092] (i) Using biotechnology methods such as microbial fermentation

[0093] The synthesis of GLP-1, GIP, and GCG triple receptor agonists employs conventional methods and the general process is as follows:

[0094] Nucleotide sequences for expressing GLP-1, GIP, and GCG triple receptor agonist precursors were integrated into a vector to obtain an expression vector. Engineered cells (engineered bacteria) containing the expression vector were constructed. Protein expression was induced in the engineered cells. The expressed protein was then subjected to inclusion body recovery, ion exchange chromatography, enzyme digestion, and reverse-phase chromatography to obtain the GLP-1, GIP, and GCG triple receptor agonist precursors. Next, the GLP-1, GIP, and GCG triple receptor agonist precursors were... 33 Side chain modification is performed on the amino acids. A dipeptide is then coupled to the N-terminus of the modified GLP-1, GIP, GCG triple receptor agonist precursor. The dipeptide sequence is X1X2, where X1 is H or Y, and X2 is Aib. It is important to note that if X2 is not Aib but a natural amino acid (G or V), the entire polypeptide can be expressed using engineered bacteria; that is, the final N-terminal dipeptide coupling procedure can be omitted.

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

[0096] (1) Construction of engineered bacteria

[0097] Construction of fusion protein expression vectors: Fusion protein expression vectors were designed based on the agonist peptide sequence, and the cDNA sequence was optimized according to the codon preference of *E. coli*. Specific restriction enzyme sites (Nde I and HindIII) were added to 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 Escherichia coli BL21(DE3) cells. Positive clones were screened using kanamycin resistance, and strains with high expression levels were selected as engineered strains through induction experiments.

[0099] Fermentation and protein expression: Selected engineered bacteria were cultured on a large scale using conventional fermentation techniques. Conditions such as temperature, pH, and dissolved oxygen were adjusted to optimize the growth environment. Under appropriate conditions, the expression of the target protein was induced by IPTG.

[0100] The fermented cells were suspended, homogenized under high pressure, and the inclusion bodies were collected by centrifugation. The inclusion bodies were then obtained by enzyme digestion, modification, coupling, and purification to obtain the above-mentioned analogues.

[0101] (ii) Using chemical synthesis methods

[0102] In addition to the methods described above, the peptides and their modification sites can also be prepared using conventional solid-phase synthesis methods with standard side-chain protecting groups. The solid-phase synthesis method is as follows:

[0103] (1) Resin pretreatment

[0104] Weigh Rink Amide-MBHA Resin (loading 0.6 mmol / g, mass 1.0 g) 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 (10 mL each time) with N,N-dimethylformamide (DMF).

[0105] (2) Fmoc deprotection

[0106] Add 10 mL of DMF solution containing 20% ​​pyridine (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 equivalent) and 1-hydroxybenzotriazole (HoBt, 0.24 g, 1.8 mmol, 3.0 equivalent) were dissolved in 5 mL of DMF and activated by stirring for 5 minutes. N,N'-diisopropylcarbodiimide (DIC, 0.28 mL, 1.8 mmol, 3.0 equivalent) was added, and activation continued for 5 minutes. The activated solution was transferred to resin and reacted at 25 °C for 2 hours, with the reaction progress monitored using the ninhydrin assay (if incomplete, the reaction time was extended). After the reaction was complete, the solution was washed once with 10 mL of DMF containing 20% ​​pyridine, and then twice with 10 mL of DMF each time.

[0109] (4) Iteration

[0110] Repeat steps 2-3, sequentially coupling subsequent Fmoc protecting amino acids from the C-terminus to the N-terminus of the target sequence until the coupling of the last amino acid, Fmoc-Tyr(tBu)-OH (0.68 g, 1.8 mmol, 3.0 equivalence), is complete. Finally, remove the Fmoc protecting group (using the same method as step 2). Thoroughly wash the resin with DMF (10 mL × 3 times), ready for subsequent lysis and purification steps.

[0111] (5) Pyrolysis reaction

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

[0113] (6) Polypeptide sedimentation and washing

[0114] The lysis buffer was slowly added dropwise to 40 mL of methyl tert-butyl ether (MTBE), and the mixture was stirred in an ice bath for 10 minutes to precipitate the peptide. The precipitate was centrifuged at 4 °C (5000 rpm, 5 minutes), the supernatant was discarded, and the precipitate was collected. The precipitate was washed three times with 20 mL of MTBE, and the supernatant was discarded after each centrifugation. The precipitate was then vacuum dried for 2 hours to obtain a crude peptide white powder.

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

[0116] Mobile phases: 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 (maintenance).

[0118] Detection wavelength: 220 nm, target peak collected. Fractions containing the target peptide were combined, acetonitrile removed by rotary evaporation, and the remaining aqueous phase was lyophilized. Through the above process, high-purity target peptide (white powder) was obtained. Its structure was confirmed by high-performance liquid chromatography-mass spectrometry analysis. It should be noted that during the solid-phase synthesis of the peptide sequence, X... 16 or X 33 The amino acid has already been pre-attached with side-chain groups using conventional methods, so the final product obtained is an analog with side chains. The existing process for attaching side chains to amino acids 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, thus achieving the attachment of the fatty acid side chain to the amino acid. 16 or X 33 The linkage to the amino group; or, the double bond of maleimide with X. 16 or X 33 The thiol group reacts to form a stable thioether bond, enabling the fatty acid side chain to be in X 16 or X 33 The linker on the thiol group.

[0119] The following examples demonstrate the experimental 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: Synthesis information of the peptides to be tested (analytes 1-17, double underlines 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 were named analogues 1-17 according to their serial numbers.

[0121]

[0122]

[0123] Example 3: In vitro activity study

[0124] The effects of the analytes on different target sites were analyzed using HTRF cAMP assay (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: Results of in vitro activity studies (EC) 50 (nM)

[0126]

[0127] The experimental data above show that analog 2 has the most significant effect on all three receptor targets, followed by analog 4 and analog 5. Although all three are at X... 33 While the modified side chains were added at the same positions and the non-natural amino acid sites were identical, minor differences in the peptide sequences led to significant variations in their effects on the three targets. Based on the data presented later, analog 2 showed the most ideal weight loss effect with a single dose, making it the most potent agonist, far exceeding other candidate analog agonists. The weight loss effects of multiple doses of analog 2 and analog 4 were essentially the same, and analog 4 had the least impact on mouse feed intake, allowing for safe and effective weight reduction in mice, and thus also serving 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 an acclimatization period of 5 days were implemented before drug administration, during which time they were provided with ample food and water. Individual mice were marked and weighed before the start of the formal experiment and randomly assigned to groups. Mice were administered a single subcutaneous injection according to their experimental groups. Body weight and food intake were measured 72 hours after administration (measured at the same time daily). The mean weight loss ratio and mean Δ body weight were calculated for each mouse: 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. Six mice were used in each group. Experimental data are expressed as mean weight loss ratio ± standard deviation and mean Δ body weight ± standard deviation.

[0131] Specifically, the single-dose dosage was as follows: 30 nmol / kg for all analogues (test peptide drugs); 0.9% NaCl was used as the negative control. Experimental results are detailed in Table 3.

[0132] Table 3: Results of Single-Dose Experiments

[0133] Group Mean Δ Body Weight (g) Mean 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 existing technologies for designing peptides with similar functions (treating diabetes, fatty liver disease, dyslipidemia, non-alcoholic steatohepatitis, and obesity, etc.), non-natural amino acids, such as Aib and α-MeL, are introduced into the peptide sequence to enhance their effectiveness. For example: X 20 Using Aib can optimize GIP activity, pharmacokinetics (PK), and X13 Using α-MeL13 (α-methyl-L-leucine) can enhance the activity of GCG and GIP. However, the introduction of these non-natural amino acids can pose challenges to sequence synthesis and hinder large-scale industrial synthesis. The peptides in this scheme differ from existing technologies by minimizing the use of non-natural amino acids while ensuring peptide efficacy.

[0135] In the process of studying peptide sequences and side chain modifications, the inventors tried X 16 The method of using amino acids as side chain modification sites. In order to... 16 The amino acid residue at this position is modified to K to connect to the side chain, forming the sequence design shown in SEQ ID NO.4, as detailed in analogues 7 / 8 / 9 / 10. Experimental results show that the sequence design of SEQ ID NO.4, in conjunction with X... 16 Side-chain modifications at specific sites cannot effectively guarantee the weight-reducing effect of the product. According to the experimental results in Table 3, analogs 7-10 basically did not have a significant weight-reducing effect, and some analogs even led to weight gain in experimental mice. Therefore, designing amino acid sequences and side-chain modifications to effectively ensure the efficacy of agonists is a challenging problem.

[0136] Analogs 5 and 6 form the sequence shown in SEQ ID NO. 3 and are coupled to X. 33 Side chain modification at the site yielded agonists that were not very effective in reducing mouse weight; they had virtually no weight-reducing effect and could even lead to an increase in mouse weight.

[0137] The inventors then adjusted the polypeptide sequence, connecting the adjusted polypeptide sequence with X. 33 Site-specific side chain modifications are combined. See analogs 2 (SEQ ID NO.1), 4 (SEQ ID NO.2), 12 (SEQ ID NO.6), and 13 (SEQ ID NO.7) for details. Results showed that analog 2 was superior to analogs 4 and 12, achieving a weight reduction of approximately 18%, which was better than the approximately 17% of analog 4, approximately 2% of analog 12, and approximately 4% of analog 13. Specific polypeptide sequences and X... 33 Modifications and combinations are necessary to effectively ensure the agonist's effect. In terms of the effect of a single dose, SEQ ID NO.1 and SEQ ID NO.2 are the optimal choices.

[0138] In addition, the inventors also studied the weight reduction effect of the optimal sequence SEQ ID NO.1 under different modification methods (different modification sites), as detailed in the experimental data of analogues 1-3. Regarding X of SEQ ID NO.1... 16 Site side chain modification and X 33 Site-specific side chain modifications can reduce mouse body weight, but in X... 33 Side chain modification at two sites yields superior results and represents a more optimized approach. Since side chain modification at both sites can enhance drug efficacy to varying degrees, the inventors also attempted to modify the peptide at two sites to improve drug action. Data from analogue 3 showed that dual modification did not effectively enhance efficacy and even increased mouse weight after administration. Therefore, it is evident that at X... 33 Modifying fatty acid sites is key to maximizing the weight-loss effect of agonists. The inventors experimented with different side-chain modification sites, and after numerous trials, finally determined X to be the optimal site. 33 Side chain modification of an amino acid at position X is a novel modification site not previously reported in existing technologies. 33 Side chain modifications at specific sites, combined with specific peptide sequences, can effectively enhance the weight-reducing effect of agonists.

[0139] The polypeptide sequences of analogs 14-17 contain no non-natural amino acids, yet they still exhibit some weight-loss effects. Analog 11 incorporates the non-natural amino acid α-Mel into its sequence, but its weight-loss effect is not as good as that of analogs 2 or 4, further demonstrating that SEQ ID NO.1 and SEQ ID NO.2 are the optimal choices for single-dose efficacy.

[0140] (2) Multiple-dose experiment

[0141] In addition to single-dose experiments, multiple-dose experiments were conducted. The experimental groups involved were: a negative control (0.9% NaCl) and a peptide drug treatment group, with administration every 72 hours for a total of three doses; the single dose in the peptide drug treatment group was 30 nmol / kg; the peptide drugs included: smegglutide (batch number 202202AMV1), telpolide (batch number D792710D), analog 2, and analog 4. During the experiment, the mean weight loss ratio and mean Δ body weight were recorded for each group. Five mice were included in each group. Experimental data are expressed as mean weight loss ratio ± standard deviation and mean Δ body weight ± standard deviation. The average daily food intake of each group of experimental animals was also recorded.

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

[0143] Table 4: Results of multiple-dose trials (mean weight loss ratio and mean Δbody weight)

[0144]

[0145] Table 5: Average food intake of laboratory 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 Tirzepatide 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 analogues tested above, analogues 4 and 2 showed good weight-loss effects after multiple administrations, with a weight loss rate of approximately 30%, which is superior to smegglutide and telpolide, and also superior to analogues 11 and 12 (see Table 4 for details). It is worth noting that analogue 11 showed relatively ideal results in single-dose experiments. However, with multiple administrations, the weight-loss effect of analogue 11 deteriorated, far less than that of analogues 2 and 4. The inventors speculate that this is because the experimental animals may have developed resistance to analogue 11, mainly manifested as a significant decrease in food intake and weight after the initial injection, with subsequent injections showing even worse results. For analogue 11, the food intake on D1, D4, and D7 represents 24 hours after injection, but it already showed an increasing trend. This phenomenon may be due to the animals developing resistance to analogue 11. In contrast, the food intake of analogues 2 and 4 was not significantly different on D1, D4, and D7 immediately after injection, indicating that analogues 2 and 4 are less likely to develop resistance. The weight loss effect of analogue 12 was not ideal in a single experiment, and its effect did not improve significantly in multiple experiments.

[0148] To investigate the food intake of experimental animals (as a side effect of appetite suppression), the average daily food intake of mice was recorded simultaneously during multiple dosing experiments to study the drug's side effects. The experimental data in Table 5 show that the food intake of experimental animals decreased significantly on the day of each administration of analog 2 (D1, D4, D7), and gradually increased over time after administration. Overall, the appetite-suppressing side effect of analog 2 is less than that of the existing technology telpolide and similar to that of smegglutide (but with significantly superior weight loss). Analog 2, with less side effect (appetite suppression), effectively reduces the weight of experimental animals compared to these existing drugs. Therefore, analog 2 is a relatively ideal peptide drug for reducing side effects and effectively controlling weight. Analog 4 has a smaller effect on food intake than analog 2, especially in the early stages (24 hours after injection on D1, D4, and D7), with significantly lower suppression of food intake compared to smegglutide and telpolide. Furthermore, combining the data in Table 4, analog 4 showed a significantly higher weight loss effect than smegglutide and telpolide, demonstrating that it can still achieve unexpected weight loss while maintaining a suitable diet. More specifically, compared to telpolide, which reduced food intake to extremely low levels after each injection, analog 4 achieved a weight loss of approximately 30% while maintaining a relatively high food intake, far exceeding smegglutide (-5.27%) and telpolide (-14.04%). This proves that it breaks through the traditional drug-dependent appetite-suppressing weight loss model and can still achieve unexpected weight loss while maintaining a suitable diet. The structural differences between analog 2 and analog 4 mainly lie in the amino acid residue types of X33 (K and C, respectively) and the side chain modification types on X33 (Formula III and Formula VII, respectively). These structural differences lead to differences in efficacy and mode of action. However, overall, analog 2 and analog 4 are superior to similar peptide products in the prior art in improving weight loss and reducing side effects affecting appetite.

[0149] In addition to analogues 2 and 4, the inventors have also conducted extensive research on other sequence structure analogues. For example, analogue 11 differs from analogue 4 primarily in that X... 13 Position (L for analogue 4, α-Mel for analogue 11) and X 17 The amino acid residues at positions 1 (I for analog 4 and K for analog 11) are different. Although the differences in amino acid residues are relatively small, the weight loss effect (with repeated administration) and the effect of reducing appetite suppression side effects of analog 11 are far inferior to those of analog 4. Furthermore, the use of analog 11 may even lead to drug resistance in experimental animals. Therefore, the appropriate design of the amino acid sequence is crucial for ensuring the efficacy of peptide drugs. The superior effects achieved by analogs 2 and 4 are an unexpected technological achievement.

[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 position Q (for analog 4 and A for analog 12) were compared. Analog 12 was inferior to analog 11 in several aspects, including single-dose and multiple-dose experiments, and its effect on suppressing appetite in mice. These experimental results further demonstrate that analogs 2 and 4 can achieve the aforementioned superior effects, representing an unexpected technological advantage.

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

[0152] Multiple administration experiments (n=3) were conducted on rats. The dosage of the polypeptide drug was 200 nmol / kg (0.9% NaCl for the negative control group). Administration was performed subcutaneously every 48 hours, starting from day 1, for a total of 5 administrations. The average daily food intake of each group of animals was recorded daily. The results are detailed in Table 6. The experimental data showed that analogs 2 and 4 reduced the food intake of the experimental animals to some extent compared to the negative control. However, compared to the existing technology LY3437943, analogs 2 and 4 significantly reduced the appetite-suppressing effect in mice, indicating that the analogs 2 and 4 developed in this protocol have fewer side effects and achieved unexpected technical results in reducing appetite suppression.

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

[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: Effect of drugs on the food intake of gorillas

[0156] Multiple dosing experiments (n=1) were conducted on beagle dogs. The peptide drug was administered at a dose of 100 nmol / kg (subcutaneous injection), starting on day 1, every 72 hours for a total of 5 doses. The experimental results are detailed in Table 7. The results showed that analogs 2 and 4 had less appetite-reducing side effects than LY3437943, consistent with the trend observed in Example 5. The total food intake of analogs 2 and 4 was 1.6-1.7 times that of the prior art LY3437943.

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

[0158]

[0159]

[0160] Meanwhile, the metabolic parameters of the experimental animals were measured. The levels of total cholesterol (CHO), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured 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: Levels of total cholesterol (CHO), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in experimental animals before and after 5 treatments.

[0162]

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

[0164] Example 7: Rat pharmacokinetic (PK) study of the drug

[0165] Male rats were administered a subcutaneous injection of 400 nmol / kg. Following administration, 0.2 ml of whole blood (EDTA anticoagulant tube) was collected from each rat at time points of 0 h, 2 h, 6 h, 10 h, 24 h, 34 h, 48 h, 72 h, 96 h, 120 h, and 144 h. After processing, plasma samples were analyzed using LC-MS / MS. Graphpad Pism 9.5 software was used to analyze the blood drug concentration-time curves, and pharmacokinetic (PK) parameters and half-life were calculated. The PK parameters calculated using the above method are shown in Table 9, illustrating the maximum plasma concentrations (C0) of analogs 2 and 4. max Peak time (T) max ), half-life (T) 1 / 2 ), Area under the curve (AUC) 0-last All of the above analogues exhibited prolonged pharmacokinetic distribution.

[0166] Table 9: Rat pharmacokinetic (PK) parameters of the drug

[0167]

[0168] Example 8: Assessment of glucose tolerance in normal KM mice

[0169] Blood glucose and body weight were measured in KM mice, which were randomly divided into three groups (negative control group, high-dose experimental group, and low-dose experimental group), with five mice in each group. The low-dose and high-dose experimental groups were subcutaneously injected with analogue 4 at doses of 5 nmol / kg and 20 nmol / kg, respectively, with an injection volume of 0.1 ml. The control group was subcutaneously injected with the corresponding solvent. After a single administration, the mice were fasted, and blood glucose levels were measured at 0h, 3h, 6h, 12h, 24h, and 36h. Measurement was performed by intraperitoneal injection of 50% glucose for 10 minutes, followed by blood collection from the tail vein. The experimental results are detailed in Table 10. Blood glucose levels in all dose groups were significantly lower than those in the control group, confirming that the agonist of this invention can effectively enhance the body's glucose metabolism capacity and has a hypoglycemic effect.

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

[0171]

[0172] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A GLP-1, GIP, GCG triple receptor agonist, characterized in that: Its amino acid sequence and side chain modifications are as follows: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGG PSKGA PPPS, and the 16th amino acid is modified with a side chain as shown in Formula III; Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGG PSKGA PPPS, with the 33rd amino acid modified with a side chain as shown in Formula III; Or YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGG PSCGA PPPS, with the 33rd amino acid modified with a side chain as shown in Formula VII; Formula III Formula VII.

2. The GLP-1, GIP, and GCG triple receptor agonist according to claim 1, characterized in that: Its amino acid sequence is as follows: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGG PSKGA PPPS, and the 33rd amino acid is modified with a side chain as shown in Formula III.

3. The GLP-1, GIP, and GCG triple receptor agonist according to claim 1, characterized in that: Its amino acid sequence is as follows: YAibQGT FTSDY SILLD KIAAQ AFIEY LLEGG PSCGA PPPS, and the 33rd amino acid is modified with a side chain as shown in Formula VII.

4. The GLP-1, GIP, and GCG triple receptor agonist according to claim 1, characterized in that: Its amino acid sequence is as follows: YAibQGT FTSDY SILLD RIAAQ AFIEY LLEGG PSKGA PPPS, and the 16th amino acid is modified with a side chain as shown in Formula III.

5. The method for preparing the GLP-1, GIP, and GCG triple receptor agonist according to any one of claims 1-4, characterized in that: Preparation of GLP-1, GIP, GCG triple receptor agonist precursors, modification of side chain groups, and chemical coupling of specific amino acids; side chain group modification at amino acid position 16 or amino acid position 33; Alternatively, the polypeptide sequences of GLP-1, GIP, and GCG triple receptor agonists can be synthesized by solid-phase synthesis or recombinant expression of the polypeptides to obtain GLP-1, GIP, and GCG triple receptor agonists.

6. The use of the GLP-1, GIP, GCG triple receptor agonist according to any one of claims 1-4 in the preparation of drugs for weight loss, lipid reduction, or hypoglycemia.

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