Improved three-target agonists

Abstract

The present disclosure relates to a three-target agonist having activity on the GLP-1 receptor, the GCG receptor, and the GIP receptor, which has a significantly superior effect in the prevention / treatment of metabolic diseases and Alzheimer's disease than the GLP-1 agonist drug on the market.

Description

Technical Field

[0001] The present disclosure relates to a triple-target agonist that is active on the GLP-1 receptor, the GCG receptor, and the GIP receptor, and has significantly better effects than the GLP-1 agonist drugs already on the market in the prevention / treatment of metabolic diseases and Alzheimer's disease. Background Art

[0002] Chronic metabolic diseases, represented by type 2 diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD), have become a high-incidence global public health threat. By 2022, the number of people with diabetes will exceed 540 million, accounting for 10.6% of the global population. Meanwhile, obesity, as the primary cause of diabetes, poses a frequently overlooked and rapidly growing threat to public health. Currently, there are over 2 billion obese people worldwide, and this number is increasing. Although the pathogenesis of diabetes, particularly type 2 diabetes (T2D), and obesity differs, they are inherently related and often co-occur. Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease in the world, and increasing evidence indicates that it is closely associated with features of the metabolic syndrome, such as insulin resistance, hyperglycemia, obesity, and dyslipidemia. Compared with patients without metabolic abnormalities, patients with obesity, elevated fasting glucose, T2D, or other metabolic abnormalities are at increased risk of developing advanced liver fibrosis. Therefore, for the treatment of chronic metabolic diseases such as T2D, obesity, and NAFLD, controlling blood sugar, losing weight, and improving dyslipidemia are all important factors that must be considered. Because lifestyle changes are difficult and have been proven to be ineffective, therapeutic intervention remains the most effective way to correct these chronic metabolic diseases.

[0003] Alzheimer's disease (AD) is another rapidly growing public health issue, with significant detrimental consequences for patients and their families, and an increasingly severe impact on the global socio-economy. An estimated 70 to 100 million people worldwide suffer from early-stage Alzheimer's disease (mild cognitive impairment and mild dementia). The specific mechanism of action of GLP-1 against AD is currently unclear, but existing experimental results indicate that insulin resistance (IR), neuroinflammation, and oxidative stress all contribute to the development of AD, and GLP-1 analogs can improve these symptoms. Therefore, GLP-1 analogs have a positive effect on improving AD symptoms and delaying the progression of the disease.

[0004] Glucagon-like peptide-1 (GLP-1) and its analogs, such as liraglutide, semaglutide, and dulaglutide, are widely used non-insulin glucose-lowering drugs, all of which have demonstrated their value in the treatment of type 2 diabetes. GLP-1 stimulates the proliferation and differentiation of pancreatic β cells, inhibits apoptosis, and increases their number. GLP-1 also inhibits glucagon secretion, suppresses appetite and food intake, and delays gastric emptying. In addition to GLP-1, glucagon (GCG) and gastric inhibitory polypeptide (GIP) are also widely studied polypeptide hormones in the treatment of metabolic diseases. GLP-1 promotes insulin biosynthesis and secretion in a glucose-dependent manner, and elevated insulin levels have a significant hypoglycemic effect. Conversely, GCG has a strong effect on glycogenolysis and gluconeogenesis, significantly increasing blood glucose. It also activates lipase, promoting lipolysis and fatty acid oxidation, thereby increasing ketone body production. Therefore, while the effects of GCG conflict with the goal of glucose lowering, it does have significant fat-reducing and weight-loss effects. GIP, on the other hand, can inhibit gastric acid secretion, pepsinogen secretion, gastric motility and emptying, stimulate intestinal fluid secretion, and stimulate insulin or glucagon secretion in a glucose-dependent manner. Since the GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) all belong to the GPCR protein family and share similar structures and ligand binding mechanisms, this makes it possible to design dual- or triple-target GLP-1R / GCGR / GIPR agonists. In principle, these agents would simultaneously possess the functions of two or three hormones. GLP-1 can lower blood sugar and suppress appetite; GCG can reduce fat and weight, but can also increase blood sugar; and GIP balances insulin and glucagon secretion, maintaining stable blood sugar levels and minimizing the risk of hypoglycemia or hyperglycemia. These functions work together to form a feedback mechanism based on blood sugar levels, not only controlling blood sugar but also breaking down fat and reducing weight. For the treatment of the chronic metabolic diseases mentioned above, dual- or triple-target agonists theoretically offer significant advantages over single-target GLP-1 agonists. But in fact, the ratio of GLP-1, GCG and GIP activities of triple-target agonists needs to be carefully balanced, and improper ratios may lead to serious side effects. For example, excessive enhancement of GCG activity may provide more significant weight loss, but there is a risk of elevated glucose. Today, many dual agonists or triple agonists have proven their value in the treatment of T2D and obesity. Tirzeptide, a dual-target agonist of GLP-1R and GIPR developed by Eli Lilly and Company, has been shown to effectively control blood sugar and reduce weight in animal and clinical experiments. MEDI0382, another dual agonist of GLP-1R and GCGR designed by MedImmune Ltd, also obtained similar positive results.Compared with dual agonists, most triple agonists are still in preclinical or clinical development but have demonstrated equivalent or superior efficacy in correcting T2D and obesity in rodents or humans.

[0005] Semaglutide is a representative example of a GLP-1 analogue for the treatment of Alzheimer's disease (AD). Currently, a clinical trial of 14mg oral semaglutide for AD, led by Novo Nordisk, has entered Phase 3. Compared to single-target GLP-1 analogs, triple-target agonists offer superior efficacy against insulin resistance, are more effective in alleviating neuroinflammation and promoting neuroprotection, and consequently, are more effective in alleviating AD symptoms and slowing the progression of the disease.

[0006] The long-acting modification of GLP-1 analogs or dual agonists and tri-agonists is an important technical improvement in the clinical application of this type of compound. Currently marketed compounds of this type support a dosing regimen of once a day to a maximum of once a week. Exploring innovative compounds with longer-lasting in vivo activity is the key to whether the indications of this type of compound have clinical advantages and is also the focus of drug development. The modified polypeptide molecules designed by the present invention have a significantly longer in vivo half-life than the marketed products that are treated once a week, and the pharmacokinetic parameters show that they can support a lower frequency of dosing cycles, which will greatly improve the convenience of patient medication, increase drug compliance, and greatly reduce treatment costs, and have good clinical value and socioeconomic value. Summary of the Invention

[0007] One object of the present invention is to provide novel polypeptide compounds that have agonist activity against glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR), and gastric inhibitory peptide receptor (GIPR), as well as novel polypeptide compounds with ultra-long half-life by modifying specific amino acids with lipophilic groups.

[0008] The novel polypeptide compounds with triple-target agonist activity can be used to treat metabolic diseases and / or Alzheimer's disease. The inventors unexpectedly discovered that certain polypeptide molecules with specific sequences exhibit agonist activity at the GLP-1 receptor, GCG receptor, and GIP receptor. Furthermore, polypeptide molecules whose relative agonist activity at these three receptors conforms to a specific ratio exhibit significantly superior properties to single-target and dual-target agonists in terms of glucose reduction, weight loss, and reversal of fatty liver disease.

[0009] The inventors also unexpectedly discovered that amino acid substitutions of the non-natural amino acids aminoisobutyric acid Aib and norleucine Nle at specific sites can still maintain the triple-target agonist activity of the compound and greatly increase the stability, making the triple-target agonist compound of the present invention have a half-life length that is significantly better than that of single-target or dual-target drugs already on the market.

[0010] In one aspect, the present disclosure provides polypeptides having agonist activity at the glucagon-like peptide-1 receptor (GLP-1R), the glucagon receptor (GCGR), and the glucose-dependent insulin-releasing peptide receptor (GIPR), having the structure of Formula I:

[0011] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa1-Ala-Xaa2-Glu-Xaa3-Xaa4-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Ile-Xaa5-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa6

[0012] (Formula I)

[0013] in,

[0014] Xaa1 is arginine (Arg, R) or isoleucine (Ile, I),

[0015] Xaa2 is lysine (Lys, K), methionine (Met, M), leucine (Leu, L), or the unnatural amino acid norleucine (Nle),

[0016] Xaa3 is lysine (Lys, K),

[0017] Xaa4 is glutamic acid (Glu, E) or isoleucine (Ile, I),

[0018] Xaa5 is aspartic acid (Asp, D) or glutamic acid (Glu, E), and

[0019] Xaa6 is lysine (Lys, K) or serine (Ser, S).

[0020] In one embodiment, the polypeptide has a sequence selected from the group consisting of:

[0021]

[0022]

[0023] In another embodiment, at least one lysine in the sequence of the polypeptide is modified with a lipophilic group, for example, one, two or all lysines are modified with a lipophilic group, in particular at least 2 lysines are modified with a lipophilic group, wherein each of the lipophilic group modifications is the same or different.

[0024] In a specific embodiment, the lysine modified with a lipophilic group has a structure selected from the group consisting of:

[0025]

[0026] In each structure, L1 is an N-terminal linker connected to lysine, L2 is a C-terminal linker connected to lysine, and lysine forms a peptide bond with the adjacent amino acid through L1 and / or L2; when Xaa6 is a lysine modified with a lipophilic group, L2 is a hydroxyl group, an amino group or glycine (Gly, G).

[0027] In another specific embodiment, the polypeptide has a structure selected from the group consisting of:

[0028]

[0029]

[0030]

[0031] wherein X is the unnatural amino acid aminoisobutyric acid (Aib), X′ is the unnatural amino acid norleucine (Nle), and K(Rn) represents the lysine K-Rn modified with a lipophilic group, and n is selected from 1, 2, 3, 4 and 5.

[0032] In another embodiment, the relative agonist activity ratio of the polypeptide on the three receptors of GLP-1R, GCGR and GIPR is 100:(1-20):(1-20), 100:(1-10):(1-10) or 100:(1-10):(1-5), for example, 100:8:5, 100:5:5, 100:3:3, 100:5:2, 100:4:1, 100:3:1, 100:2:4 or 100:1:3.

[0033] In another aspect, the present disclosure provides the polypeptide according to the first aspect for use in preventing and / or treating a disease or condition selected from the group consisting of a metabolic disease and Alzheimer's disease.

[0034] In one embodiment, the metabolic disease is selected from diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic fatty liver disease, arteriosclerosis caused by hypercholesterolemia or hyperlipidemia, and coronary heart disease, and the diabetes is, for example, type II diabetes.

[0035] In another embodiment, the polypeptide is administered to a subject in need thereof once a week, once every two weeks, twice a month, once a month, once every two months, or less frequently, e.g., the polypeptide is not administered to the subject more frequently during a period of at least 1 month, 2 months, 3 months, 6 months, or 1 year.

[0036] In another aspect, the present disclosure provides a pharmaceutical composition comprising the polypeptide according to the first or second aspect above.

[0037] In yet another aspect, the present disclosure provides the use of the above-described polypeptide or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a metabolic disease or Alzheimer's disease. Specifically, the metabolic disease can be selected from diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic fatty liver disease, arteriosclerosis caused by hypercholesterolemia or hyperlipidemia, and coronary heart disease, and the diabetes is, for example, type II diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown is a preparative chromatogram of a polypeptide sample of SEQ ID NO. 61.

[0039] Figure 2 A chromatogram of UPLC analysis of a sample of the SEQ ID 61 polypeptide is shown.

[0040] Figure 3 A preparative chromatogram of a sample of the polypeptide of SEQ ID NO. 10 is shown.

[0041] Figure 4 The UPLC analysis chromatogram of the SEQ ID NO. 10 polypeptide sample is shown.

[0042] Figure 5 The preparation chromatogram of the SEQ ID NO. 14 polypeptide sample is shown.

[0043] Figure 6 The UPLC analysis chromatogram of the SEQ ID NO. 14 polypeptide sample is shown.

[0044] Figure 7 The preparation chromatogram of the SEQ ID NO. 18 polypeptide sample is shown.

[0045] Figure 8 The UPLC analysis chromatogram of the SEQ ID NO. 18 polypeptide sample is shown.

[0046] Figure 9 A preparative chromatogram of a sample of the polypeptide of SEQ ID NO. 35 is shown.

[0047] Figure 10 The UPLC analysis chromatogram of the SEQ ID NO. 35 polypeptide sample is shown.

[0048] Figure 11 Shown are the blood concentration curves of different compounds after subcutaneous administration to rats. Figure 11 A shows the plasma concentration-time curve of semaglutide. Figure 11B shows the plasma drug-dose curve of SEQ ID NO.5 polypeptide, Figure 11 C shows the plasma drug-dose curve of SEQ ID NO.6 polypeptide, Figure 11 D shows the plasma drug-dose curve of SEQ ID NO.7 polypeptide, Figure 11 E shows the plasma drug-dose curve of the polypeptide of SEQ ID NO.8, Figure 11 F shows the plasma drug-dose curve of SEQ ID NO.34 polypeptide, Figure 11 G shows the plasma drug-dose curve of the polypeptide of SEQ ID NO. 35, wherein the data are expressed as Mean±SD.

[0049] Figure 12 The percentage change in body weight of DIO obese mice over the course of the dosing period is shown. The black curve in each figure represents the body weight change of blank control mice. Figure 12 A shows semaglutide control drug (brown) and SEQ ID NO. 2 (green), Figure 12 B shows SEQ ID NO. 13 (blue) and 17 (purple), Figure 12 C shows SEQ ID NO. 21 (green) and 22 (brown), wherein the data are expressed as Mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 vs. solvent control by one-way ANOVA with Dunnett's post hoc test.

[0050] Figure 13 Figure 2 shows postprandial blood glucose changes in DIO mice with hyperglycemia. A shows blood glucose measurement results for a blank solvent, semaglutide, and SEQ ID NOs. 6, 10, and 18; B shows blood glucose measurement results for a blank solvent, semaglutide, and SEQ ID NOs. 9 and 10 at a low dose (100 μg / ml) and a high dose (300 μg / ml) of L (100 μg / ml). Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 vs. solvent control by one-way ANOVA with Dunnett's post hoc test.

[0051] Figure 14Figure 2 shows the results of IPGTT in DIO mice with hyperglycemia. Specifically, A shows the blood glucose curves for each group of mice after intraperitoneal glucose injection, and B shows the area under the curve for each group of mice after intraperitoneal glucose injection. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. vehicle control by one-way ANOVA with Dunnett's post hoc test.

[0052] Figure 15 shows the changes in fatty liver in DIO obese mice after administration. Specifically, A shows the changes in liver weight in each group of animals. Among them, semaglutide, SEQ ID NO.14, 18 and 22 significantly reduced the liver weight of animals after administration, and the fatty liver was visibly improved. The high dose of SEQ ID NO.22 H can completely restore the liver weight to normal mice, and the effect is better than semaglutide. B shows the changes in fat content in each group of animals. Among them, semaglutide, SEQ ID NO.14, 18 and 22 significantly reduced the fat content in animals after administration, while semaglutide did not change significantly. All groups of compounds are better than semaglutide. C shows oil red and H&E staining of animal livers after 4 weeks of administration. Among them, oil red staining shows that each group of compounds has a significant effect on alleviating liver inflammation, and the inflammation is basically relieved at high doses; H&E staining shows that each group of compounds significantly improves the abnormal structure of liver cells and the degree of liver fat vacuolization, and the liver cells are tightly arranged and have clear outlines.

[0053] Figure 16 The graph shows the changes in body weight of DIO obesity model mice after continuous administration of tilportide and SEQ ID NOs. 14, 18, 21 and 22.

[0054] Figure 17 The graph shows the effects of continuous administration of tilportide and SEQ ID NOs. 14, 18, 21, and 22 on mesenteric fat in DIO obesity model mice.

[0055] Figure 18 The curves showing changes in body weight within 7 days after a single administration of SEQ ID NO. 22 to mini pigs and beagle dogs are shown. DETAILED DESCRIPTION

[0056] Hereinafter, the present invention will be described in more detail.

[0057] Unless otherwise defined herein, the scientific and technical terms used in this disclosure should have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and methods used in connection with chemistry, molecular biology, cell biology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0058] All combinations of the various elements disclosed herein belong to the scope of the present invention.In addition, the scope of the present invention should not be limited by the specific disclosure provided below.

[0059] In addition, the amino acids mentioned in the present invention are abbreviated as follows according to the IUPAC-IUB nomenclature rules:

[0060] Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine ​​(Cys, C), Glutamic acid (Glu, E), Glutamine (Gln, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), Valine (Val, V).

[0061] Specifically, all amino acid residues in the polypeptides described herein are in the L configuration.

[0062] According to peptide synthesis practice, the "-NH2" at the C-terminus of a peptide sequence can also usually be an amide group (-CONH2). Unless otherwise specified, the two can be used interchangeably.

[0063] In addition to natural amino acids, the polypeptide sequences described herein may also utilize unnatural amino acids, such as aminoisobutyric acid (Aib) and norleucine (Nle).

[0064] The triple-target agonists described in the present disclosure having significant agonist activity on GCG, GLP-1 and GIP receptors may exhibit in vitro activity of 2% or more, 5% or more, 1σ% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, and 100% or more, respectively, compared to the natural ligands (glucagon GCG, GLP-1 and GIP), but are not particularly limited thereto.

[0065] The method for measuring the in vitro activity of a triple-target agonist can refer to Example 3 of the present invention, but is not particularly limited thereto.

[0066] In addition, the triple-target agonist may be an agonist having an increased in vivo half-life relative to any one of native GLP-1, native glucagon GCG, and native GIP, but is not particularly limited thereto.

[0067] The method for measuring the pharmacokinetic parameters of the triple-target agonist in rats can refer to the embodiments of the present invention, such as Example 4, but is not particularly limited thereto.

[0068] The method for determining the biological activity of triple-target agonists in reducing animal weight can refer to the embodiments of the present invention such as Example 5, but is not particularly limited thereto.

[0069] The method for determining the blood glucose control biological activity of the triple-target agonist in animals can refer to the embodiment of the present invention such as Example 6, but is not particularly limited thereto.

[0070] The method for determining the biological activity of a triple-target agonist in treating non-alcoholic fatty liver disease in animals can refer to the embodiments of the present invention such as Example 7, but is not particularly limited thereto.

[0071] As used in the present disclosure, the term "polypeptide" means all forms of the polypeptide, its pharmaceutically acceptable salt, or its solvate. In addition, the polypeptide described herein may be in any pharmaceutically acceptable form. Specifically, the type of salt is not particularly limited, but is preferably a salt that is safe and effective for the subject (e.g., mammal). The term "pharmaceutically acceptable" refers to a substance that can be effectively used for the intended use within the scope determined by pharmacology (pharmaco-medica1) without causing excessive toxicity, irritation, allergic reaction, etc. The term "solvate" refers to a complex formed between the polypeptide or its salt described in the present disclosure and a solvent molecule.

[0072] Although described herein by “sequences of SEQ ID NOs. 1 to 48 and modified features”, as long as the polypeptide has an activity identical to or corresponding to the activity of a peptide consisting of the amino acid sequence of the corresponding SEQ ID NO, the polypeptide described herein does not exclude mutations that may be obtained by adding nonsense sequences upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO, or naturally occurring mutations within the sequence, or silent mutations, and even when sequence additions or mutations exist, they still fall within the scope of the present invention.

[0073] As used herein, the term "lipophilic group" refers to any chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic group is by the octanol-water partition coefficient, log Kow, where Kow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the structural components of the chemical, which are calculated using first principles or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety). In principle, a chemical is lipophilic when its log Kow exceeds 0. Typically, the log Kow of a lipophilic group exceeds 1, exceeds 1.5, exceeds 2, exceeds 3, exceeds 4, exceeds 5, or exceeds 10. For example, the log Kow of 6-aminohexanol is predicted to be approximately 0.7. Using the same method, the logKow of cholesteryl N-(hexan-6-ol)carbamate was predicted to be 10.7.

[0074] In some embodiments, the lipophilic group can be an aliphatic, cyclic, such as alicyclic, or polycyclic structure such as a polyester ring, such as a steroid (such as a sterol) or a straight or branched aliphatic hydrocarbon group. The lipophilic group can generally include a hydrocarbon chain, which can be cyclic or acyclic. The hydrocarbon chain can include various substituents and / or one or more heteroatoms, such as oxygen or nitrogen atoms. Such lipophilic aliphatic groups include but are not limited to saturated or unsaturated C4-C30 hydrocarbon groups (such as C6-C18 hydrocarbons), saturated or unsaturated fatty acids, waxes (such as monohydric alcohol esters of fatty acids and fatty diamides), terpenes (for example, C10 terpenes, C15 sesquiterpenes, C20 diterpenes, C30 triterpenes and C40 tetraterpenes) and other polyester ring hydrocarbon structures. For example, the lipophilic group can contain a C4-C30 hydrocarbon chain (such as a C4-C30 alkyl or alkenyl) or a C4-C30 fatty acid. In some embodiments, the lipophilic group contains a saturated or unsaturated C6-C18 hydrocarbon chain (such as a straight chain C6-C18 alkyl or alkenyl) or a C6-C18 fatty acid. In one embodiment, the lipophilic group contains a saturated or unsaturated C16 hydrocarbon chain (such as a straight chain C16 alkyl or alkenyl).

[0075] The polypeptides disclosed herein can be synthesized by solid-phase or liquid-phase methods or produced by genetic engineering techniques. Specifically, the polypeptides of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other methods known in the art.

[0076] As used herein, the term "metabolic disease" refers to symptoms of various diseases occurring alone or in combination due to metabolic disorders. In particular, examples of diseases that belong to metabolic diseases may include glucose intolerance, diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic fatty liver disease, and arteriosclerosis, atherosclerosis, and coronary heart disease caused by hypercholesterolemia or hyperlipidemia, but are not limited thereto.

[0077] Example

[0078] Example 1: Synthesis and Preparation of Triple-Target Agonist Peptides

[0079] Triple-target agonists showing activity against all of the GLP-1, GIP, and GCG receptors were prepared using a peptide synthesizer. Their amino acid sequences are shown in Table 2 below.

[0080]

Table 2

[0081]

[0082] *Note: In the sequences described in Table 2, X represents the unnatural amino acid aminoisobutyric acid (Aib); X' represents the unnatural amino acid norleucine (Nle).

[0083] Peptide Synthesis: Starting from Fmoc-AA-Wang resin with various amino acids, deprotection coupling was performed according to the synthetic sequence. The coupling materials were prepared in a molar ratio of Fmoc-AA-Wang resin / Fmoc-AA-OH / HOBT / DIC = 1 / 5 / 6 / 6, with DMF as the activation solvent. The coupling time was 1.5 h. Deprotection was performed using 20% ​​pip / DMF for 5 min + 15 min, with both reactions and deprotection performed at room temperature. Peptide synthesis was completed according to the peptide sequence shown in Table 2, and the peptide resin was dried to obtain the desired product.

[0084] Prepare a lysis buffer with a ratio of TFA / PhSMe / PhOMe / EDT = 90 / 5 / 2 / 3. Use 10 ml of lysis buffer per 1 g of peptide resin. Add the peptide resin and react for 10 minutes at low temperature, then transfer to room temperature and react for 2 hours. Filter the filtrate and add glacial methyl tert-butyl ether to precipitate. Precipitate once with 10 ml of glacial methyl tert-butyl ether per 1 ml of lysis buffer. Wash five times with the same amount of glacial methyl tert-butyl ether. Centrifuge to obtain the crude peptide.

[0085] Preparation of synthetic samples: The crude peptide was vacuum dried and dissolved in triethylamine phosphate buffer (pH 1.8-3.0) to a solution of approximately 5 mg / ml. The sample was filtered through a 0.45 μm filter. The filtrate was adjusted to the isoelectric point of the sample with dilute ammonia solution, centrifuged, and the precipitate collected. The precipitate was then reconstituted in triethylamine phosphate buffer, filtered through a 0.45 μm filter, and set aside.

[0086] Peptide purification: Use 90% 20mM triethylamine phosphate buffer (pH = 1.80) + 10% acetonitrile as phase A and 90% acetonitrile + 10% 20m triethylamine phosphate buffer as phase B. Use a 20*250mm, C18 reverse-phase column at a flow rate of 10ml / min for gradient elution. Collect the target peak and combine qualified fractions. Remove the acetonitrile from the collected fractions by rotary evaporation. Then adjust the pH to the isoelectric point of the sample with dilute ammonia water. Centrifuge and collect the precipitate. Wash with water, dissolve in 0.01M dilute acetic acid aqueous solution, filter, and freeze-dry the filtrate. Calculate the yield by weighing ≥40%. Aliquot and freeze for storage. Analyze the corresponding mass spectrometry data based on the molecular weight of the target polypeptide. The theoretical value and the measured value should be within ±1.

[0087] The reverse chromatograms of some peptide preparations and the UPLC analysis chromatograms of the pure product after freeze-drying are as follows: Figure 1 and 2 shown.

[0088] Example 2: Synthesis of a triple-target agonist peptide with chemically modified lysine residues

[0089] The amino acid sequences of triple-target agonist peptides with R1, R2, R3, R4, and R5 chemical modifications on lysine side chains are shown in the table below:

[0090]

Table 3

[0091]

[0092]

[0093] *Note: In the sequences described in Table 3, X represents the unnatural amino acid aminoisobutyric acid (Aib); X′ represents the unnatural amino acid norleucine (Nle); -NH2 represents the amidated C-terminus of the polypeptide (-CONH2); K(R1) is K-R1, K(R2) is K-R2, K(R3) is K-R3, K(R4) is K-R4, and K(R5) is K-R5, indicating that the lysine side chain is chemically modified with a lipophilic group.

[0094] Synthesis of lipophilic group-modified peptides: Starting from Rink Amide-MBHA resin, deprotection coupling was performed according to the synthetic sequence. The coupling materials were prepared in a molar ratio of: Rink Amide-MBHA resin / Fmoc-AA-OH / HOBT / DIC = 1 / 5 / 6 / 6, with DMF as the activation solvent. The coupling time was 1.5 h. Deprotection was performed using 20% ​​pip / DMF for 5 min + 15 min, with both reaction and deprotection performed at room temperature. Synthesis was completed according to the peptide sequence in Table 3, and the peptide resin was dried. Cleavage and washing were performed as described in Example 1, and the crude peptide was dried.

[0095] The crude peptide was added to a 0.25 M triethylamine phosphate buffer solution (pH 11.65) and stirred to dissolve (concentration: 10 mg peptide / ml buffer). The mixture was cooled to 0-5°C in an ice bath. 3.0 eq of a lipophilic group (100 mg / ml) dissolved in N-methylpyrrolidone was added and reacted at 0-5°C for 10 min to obtain the crude peptide. The reaction method for linking different lipophilic groups to the peptide was the same.

[0096] Treatment of synthetic samples: The crude peptide was vacuum dried and then dissolved in 0.01 M dilute ammonium bicarbonate solution to prepare a 5 mg / ml sample solution. The solution was filtered through a 0.45 μm filter membrane and set aside.

[0097] Peptide purification: Phase A is an aqueous solution containing 1% acetic acid and 20% ethanol, and Phase B is an aqueous solution containing 1% acetic acid and 80% ethanol. Gradient elution is performed on a 20x250 mm PS reverse-phase column at a flow rate of 10 ml / min. The target peak is collected and qualified fractions are combined. The organic solvent is removed by rotary evaporation from the collected fractions. The pH is then adjusted to the isoelectric point with 0.1 M dilute acetic acid. The precipitate is collected by centrifugation, washed, reconstituted, filtered, and lyophilized. The yield is calculated to be ≥70% by weight. Aliquot and store frozen. Analyze the corresponding mass spectrometry data based on the molecular weight of the target peptide. The theoretical value and the measured value should be within ±1.

[0098] The reverse chromatograms of some peptide preparations and the UPLC analysis chromatograms of the pure product after freeze-drying are as follows: Figures 3 to 10 shown.

[0099] Example 3: In vitro activity assay of triple-target agonists

[0100] The activity of the lipophilic group-modified triple-target agonist polypeptide prepared in Example 2 was measured using a method for measuring in vitro cell activity using cell lines transformed with the GLP-1 receptor, GCG receptor, and GIP receptor, respectively. The GLP-1 receptor, GCG receptor, and GIP receptor were expressed in HEK293 cells, and in vitro activity was measured using the transformed cell lines. This was performed using the cAMP-GS Dynamic Kit from Cisbio, using HTRF (Homogeneous Time-Resolved Fluorescence) as the assay method.

[0101] To measure the GLP-1 activity of the triple-target agonist prepared in Example 2, a three-fold serial dilution from 3000 nM to 1 nM was performed. As a positive control, native GLP-1 was also diluted three-fold from 3000 nM to 1 nM. Adherently cultured HEK293 cells expressing the GLP-1R were digested, centrifuged, and harvested. The cells were diluted to a concentration of 600,000 cells / ml and added to the assay wells of a 384-well plate at a concentration of 5 μL per well. Subsequently, 5 μL of the diluted triple-target agonist was added to each well, mixed, and incubated at 37°C for 90 minutes. Following the instructions for the CAMP-GS Dynamic Kit, the diluted cAMP-d2 reagent and cAMP-Cryptate reagent were added to the assay wells of the 384-well plate, mixed thoroughly, and incubated at room temperature in the dark for 60 minutes. The 384-well plate was placed in a microplate reader equipped with an HTRF module, and the EC50 value was calculated by measuring the cAMP value. The EC50 value was compared with the relative potency of human GLP-1 and is shown in Table 4 below.

[0102] In order to measure the GCG activity of the triple-target agonist prepared in Example 2, the triple-target agonist prepared in Example 2 was serially diluted 3-fold from 3000nM to 1nM; as a positive control, natural GCG was serially diluted 3-fold from 1000nM to 0.5nM. The adherently cultured HEK293 cells expressing GCGR were digested, centrifuged, and collected, diluted to a concentration of 600,000 cells / ml, and added to the detection wells of a 384-well plate, with 5μL added to each well. Subsequently, the diluted triple-target agonist was added, 5μL per well, mixed, and incubated at 37°C for 90min. The EC50 value and relative potency were calculated by referring to the GLP-1 kit assay method. The relative potency to human GCG is shown in Table 4 below.

[0103] To measure the GIP activity of the triple-target agonist prepared in Example 2, the triple-target agonist prepared in Example 2 was serially diluted 3-fold from 1000 nM to 0.1 nM; as a positive control, native GIP was serially diluted 3-fold from 30 nM to 0.25 nM. Adherently cultured HEK293 cells expressing GIPR were digested, centrifuged, and collected, then diluted to a concentration of 600,000 cells / ml and added to the assay wells of a 384-well plate, 5 μL per well. Subsequently, 5 μL of the diluted triple-target agonist was added to each well, mixed, and incubated at 37°C for 90 minutes. The EC50 value and relative potency were calculated using the GLP-1 assay method. The relative potency relative to human GIP is shown in Table 4 below.

[0104]

Table 4

[0105]

[0106]

[0107] Example 4: Pharmacokinetic study of triple-target agonists in rats

[0108] To investigate the in vivo metabolism of triple-target agonists, we conducted a pharmacokinetic study in rats. Six male and female SD rats (three per sex) were used for each drug. The test article was administered by single subcutaneous injection at a dose of 0.36 (SEQ ID NOs. 34 and 35) or 3.6 mg / kg (SEQ ID NOs. 9, 10, 11, and 12). Plasma samples were collected before, and 0.5, 1, 3, 6, 12, 18, 24, 36, 48, 72, 120, 168, and 216 hours after administration. The concentrations of the test articles in the plasma samples were determined by LC-MS / MS. A total of six triple-target agonist molecules, identified as SEQ ID NOs. 9, 10, 11, 12, 34, and 35, were validated in this study.

[0109] At the same time, the marketed drug semaglutide was used as a positive control. After research, the drug-time curve of each molecule was shown in Figure 11 .

[0110] The pharmacokinetic parameters of various molecules were analyzed, and the resulting pharmacokinetic parameters are listed in Table 5 below:

[0111]

Table 5

[0112]

[0113] As can be seen from the data in Table 5, the elimination phase half-life (HL_Lambda_z(h)) of SEQ ID NOs. 9, 10, 11, 12, 34, and 35 molecules was significantly prolonged relative to the positive control semaglutide, ranging from a minimum of 120% to a maximum of 270% of the in vivo half-life of semaglutide. SEQ ID NOs. 9, 10, 11, and 12 molecules were modified with different lipophilic groups attached to the same peptide chain. The inventors unexpectedly discovered that compounds modified with lipophilic groups with fewer than 20 carbon atoms significantly increased their in vivo half-life (SEQ ID NOs. 9 and 10 molecules), while further extension of the lipophilic group's carbon chain (SEQ ID NO. 12 molecule) or the ethylene glycol linker (SEQ ID NO. 11 molecule) resulted in a shortened in vivo half-life. The optimal lipophilic modification herein is preferably a lipophilic molecule (R2) containing a 20-carbon fatty acid chain. The in vivo half-life of the dual-modified molecules (SEQ ID NOs. 34 and 35) is extended by more than 2.5 times compared to once-weekly semaglutide. This half-life extension supports extending the dosing frequency of the new molecules to once every two weeks to once a month, achieving an ultra-long-lasting effect.

[0114] Example 5: Weight loss pharmacodynamics study in DIO obesity model mice using semaglutide as a positive control In order to study the therapeutic effect of new compounds on obesity, DIO obese model mice with hyperglycemia characteristics were selected for in vivo experimental verification. The DIO model mice with hyperglycemia characteristics are obese mouse models constructed by using a high-fat diet to induce C57BL / 6J mice. Compared with normal mice, their body weight has increased significantly, body fat accumulation is obvious, and blood sugar levels are higher than normal mice. This model mainly examines the efficacy of drugs in weight loss and lipid reduction, and also examines the hypoglycemic efficacy. We examined five compounds SEQ ID NO. 2, 13, 17, 21 and 21. Semaglutide is a representative drug with a single target of the GLP-1 receptor. Semaglutide was used as a control drug, and the dose was 100 μg / kg, subcutaneously injected once every three days, and the drug was continuously administered for more than four weeks to observe changes in body weight. The results are shown in Figure 12 .

[0115] Compared to the saline blank control, all treatment groups demonstrated significant weight loss, with the compound SEQ ID NO. 22 showing the strongest weight loss effect, with a nearly 20% weight loss after four weeks of treatment. This weight loss was significantly different from the blank control, and significantly outperformed the single-target GLP-1 receptor positive control drug, semaglutide.

[0116] Example 6 Pharmacodynamics of blood glucose control in hyperglycemic DIO obesity model mice using semaglutide as a positive control Research

[0117] To investigate the therapeutic efficacy of the new compounds for type 2 diabetes, in vivo experiments were conducted on the hyperglycemic DIO model mice described in Example 5. The hyperglycemic DIO model is an obese mouse model established by inducing a high-fat diet in C57BL / 6J mice, resulting in significantly elevated blood glucose levels compared to normal mice. For the four compounds described in SEQ ID NOs. 6, 9, 10, and 18, semaglutide was used as a control drug, with a dose of 100 μg / kg administered subcutaneously every three days for at least four weeks.

[0118] The changes in blood glucose were monitored after 4 weeks of administration. The blood glucose levels of DIO model mice increased only slightly, and different doses of SEQ ID NO. 6, 9, 10 and 18 compounds still showed significant hypoglycemic effects. Figure 13 The results showed that after one day of administration, the postprandial blood glucose levels of each treatment group decreased significantly compared to the blank control group, and all returned to normal blood glucose levels. At the same time, when SEQ ID NO. 10 was administered at a high dose three times the concentration, blood glucose levels remained stable and no hypoglycemia occurred, indicating that the new compound has good safety.

[0119] At the same time, the animals' glucose tolerance (IPGTT) was examined. After 4 weeks of administration, the animals in the SEQ ID NO.14, 18 and 22 groups and the semaglutide positive control group were fasted for 12 hours without water withdrawal. Blood glucose levels were measured by tail tip sampling. After intraperitoneal injection of 1g / kg glucose solution, blood glucose levels were measured at 15, 30, 60 and 120 minutes. The blood glucose curve was drawn and the area under the curve was calculated. The results are shown in Figure 14 It can be seen that each drug-treated group showed significant effects in controlling glucose tolerance, and the results were better than the glucose tolerance control of the positive control drug semaglutide.

[0120] Example 7: Study on the efficacy of semaglutide in treating fatty liver in DIO obese mice using semaglutide as a positive control

[0121] In order to study the therapeutic effect of the new compound on fatty liver, the DIO obese model mice in Example 5 were selected for in vivo experimental verification. For the four compounds SEQ ID NO.10, 18, 21 and 22, the single target agonist representative drug semaglutide was used as a control drug, and the experiment was the same as in Example 5. After the administration, the mice were euthanized and dissected, and the liver tissue was weighed, the fat content was determined, and pathological sections were made. The results of oil red and H&E staining are shown in Figure 15. The results showed that the three compounds had a significant effect on the reversal of liver fat in obese mice, and the SEQ ID NO.10 compound was better than semaglutide, and the consumption of whole body fat was significantly better than semaglutide. The staining of liver pathological sections showed that it could significantly improve the fat vacuoles and inflammatory aggregation in the liver, showing a significant effect on improving fatty liver.

[0122] Example 8 Weight Loss Pharmacodynamic Study in DIO Obese Model Mice Using Telportide as Positive Control

[0123] Tirzepatide is a representative drug that is a dual-target agonist of GLP-1 receptor and GIP receptor. The four compounds of SEQ ID NO. 14, 18, 21 and 22 were investigated. The experiment was carried out according to the steps in Example 5. The results are shown in Figure 5. Figure 16 The inventors unexpectedly discovered that the weight loss of animals after 21 days of administration of the three compounds SEQ ID NO. 18, 21 and 22 exceeded 20%, and SEQ ID NO. 21 and 22 even approached 30%, which were significantly better than the dual-target agonist tilportide (15%).

[0124] Example 9 Study on the efficacy of tilportide as a positive control in reducing organ fat in DIO obese mice Research

[0125] Refer to the protocol of Example 8. Telportide was still used as the control drug, and the effects of SEQ ID NOs. 14, 18, 21, and 22 on reducing organ fat in the DIO obese mouse model were investigated. After administration, the animals were euthanized and dissected, and the mesenteric fat was isolated. Blood and tissue fluid were removed with absorbent paper, and the isolated mesenteric fat was weighed. The results are shown in Figure 17 The inventors unexpectedly discovered that SEQ ID 18, 21 and 22 have significant advantages over the dual-target agonist telpotide in reversing and inhibiting mesenteric fat in organs, and the extent of reduction in mesenteric fat is particularly superior to telpotide.

[0126] Example 10 Pharmacodynamic Study of Weight Loss by Three Agonists in Large Animals

[0127] This example aims to observe the weight changes of non-rodent beagle dogs and mini pigs after a single high-dose administration. Male beagle dogs and mini pigs were selected, with 6 animals in each group. The polypeptide of SEQ ID NO. 22 was dissolved in physiological saline to prepare a 1 mg / ml concentration solution. 0.1 mg / kg of the compound was injected subcutaneously into the mini pigs and beagle dogs, respectively. The animals were observed and their weight changes were measured daily for 7 consecutive days. The weight changes of the animals were recorded. The results are shown in Figure 18 The results showed that after a single large dose of the drug was administered to mini pigs and beagles, the body weight of the animals decreased significantly, especially in mini pigs.

Claims

1. A polypeptide having agonist activity on glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulin-releasing peptide receptor (GIPR), having the structure of formula I: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa1-Ala-Xaa2-Glu-Xaa3-Xaa4-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Ile-Xaa5-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa6 (Formula I) in, Xaa1 is arginine (Arg, R) or isoleucine (Ile, I), Xaa2 is lysine (Lys, K), methionine (Met, M), leucine (Leu, L), or the unnatural amino acid norleucine (Nle), Xaa3 is lysine (Lys, K), Xaa4 is glutamic acid (Glu, E) or isoleucine (Ile, I), Xaa5 is aspartic acid (Asp, D) or glutamic acid (Glu, E), and Xaa6 is lysine (Lys, K) or serine (Ser, S).

2. The polypeptide according to claim 1, having a sequence selected from the group consisting of:

3. The polypeptide according to claim 1 or 2, wherein at least one lysine in the sequence is modified with a lipophilic group, for example one, two or all lysines are modified with a lipophilic group, in particular at least two lysines are modified with a lipophilic group, wherein each of the lipophilic group modifications is the same or different.

4. The polypeptide according to claim 3, wherein the lysine modified with a lipophilic group has a structure selected from the group consisting of: In each structure, L1 is an N-terminal linker connected to lysine, L2 is a C-terminal linker connected to lysine, and lysine forms a peptide bond with the adjacent amino acid through L1 and / or L2; when Xaa6 is a lysine modified with a lipophilic group, L2 is a hydroxyl group, an amino group or glycine (Gly, G).

5. The polypeptide according to claim 3 or 4, which has a structure selected from the group consisting of: wherein X is the unnatural amino acid aminoisobutyric acid (Aib), X′ is the unnatural amino acid norleucine (Nle), and K(Rn) represents the lysine K-Rn modified with a lipophilic group, and n is selected from 1, 2, 3, 4 and 5.

6. The polypeptide according to any one of claims 1 to 5, for use in preventing and / or treating a disease or condition selected from the group consisting of metabolic diseases and Alzheimer's disease.

7. The polypeptide according to claim 6, wherein the metabolic disease is selected from the group consisting of diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic fatty liver disease, arteriosclerosis caused by hypercholesterolemia or hyperlipidemia, and coronary heart disease, and the diabetes is, for example, type II diabetes.

8. The polypeptide of any one of claims 1 to 7, wherein the polypeptide is administered to a subject in need thereof once a week, once every two weeks, twice a month, once a month, once every two months, or less frequently, such as during a period of at least 1 month, 2 months, 3 months, 6 months, or 1 year, the polypeptide is not administered to the subject more frequently.

9. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 8.

10. Use of the polypeptide according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating metabolic diseases or Alzheimer's disease.

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