WO2022247701A1 - 多激动剂及其应用 - Google Patents

多激动剂及其应用 Download PDF

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Publication number
WO2022247701A1
WO2022247701A1 PCT/CN2022/093577 CN2022093577W WO2022247701A1 WO 2022247701 A1 WO2022247701 A1 WO 2022247701A1 CN 2022093577 W CN2022093577 W CN 2022093577W WO 2022247701 A1 WO2022247701 A1 WO 2022247701A1
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Prior art keywords
compound
salt
pharmaceutical composition
solvate
glp
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PCT/CN2022/093577
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English (en)
French (fr)
Inventor
黄亮
曹春来
邓慧兴
周翠
何秀仪
刘筱潇
谢鑫
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United Bio Technology Hengqin Co Ltd
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United Bio Technology Hengqin Co Ltd
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Priority to EP22810428.7A priority Critical patent/EP4353249A4/en
Priority to US18/563,552 priority patent/US20250129135A1/en
Priority to KR1020237044842A priority patent/KR20240013798A/ko
Priority to CA3219647A priority patent/CA3219647A1/en
Priority to CN202511790403.5A priority patent/CN121574227A/zh
Priority to JP2023571479A priority patent/JP7803570B2/ja
Priority to CN202280024800.XA priority patent/CN117062618B/zh
Priority to AU2022280225A priority patent/AU2022280225B2/en
Application filed by United Bio Technology Hengqin Co Ltd filed Critical United Bio Technology Hengqin Co Ltd
Publication of WO2022247701A1 publication Critical patent/WO2022247701A1/zh
Priority to ZA2023/10867A priority patent/ZA202310867B/en
Anticipated expiration legal-status Critical
Priority to JP2025115133A priority patent/JP2025143457A/ja
Priority to AU2025279664A priority patent/AU2025279664A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a class of polypeptide compounds and their use in the field of medicine. More specifically, the present invention relates to glucagon-like peptide-1 receptor (GLP-1 R), glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon receptor (GCGR) A polypeptide compound with triple agonistic activity, and relates to its application in the treatment of metabolic syndrome.
  • GLP-1 R glucose-dependent insulinotropic polypeptide receptor
  • GCGR glucagon receptor
  • Type II diabetes and obesity are increasingly becoming global diseases affecting human health.
  • Obesity is the main cause of many chronic diseases such as diabetes, hypertension, heart disease, dyslipidemia, fatty liver disease, atherosclerosis, arthritis, stroke, neurodegenerative diseases, etc. Diabetes can also lead to cardiovascular and cerebrovascular diseases, etc. complication.
  • Most of the hypoglycemic drugs currently available on the market only have the effect of controlling blood sugar, but cannot improve the weight of obese patients, and some drugs even have side effects of weight gain. Therefore, there is still an urgent need to develop drugs that can lower blood sugar and improve body weight, and contain multiple beneficial effects to meet the needs of most obese and type 2 diabetic populations.
  • Incretin is a kind of polypeptide hormone secreted from the intestinal tract after eating stimulation under normal physiological conditions. Early studies have found that it can stimulate pancreatic ⁇ -cells to secrete insulin and regulate glucose levels as the glucose level rises after meals. Homeostasis, protect islet ⁇ -cells, reduce body weight by suppressing appetite and delaying gastric emptying. GLP-1 and GIP are two incretins discovered so far.
  • GLP-1 is expressed in intestinal mucosal L cells by the proglucagon gene, and has a polypeptide of 31 amino acids, which mainly acts on the GLP-1 receptor (GLP-1 R), stimulates insulin secretion, and inhibits glucagon secretion , protect pancreatic islet ⁇ -cells, have the physiological effect of regulating blood sugar homeostasis, and at the same time inhibit food intake and gastric emptying through the central nervous system signaling pathway, increase satiety, and reduce body weight.
  • Exendin-4 (Exendin-4) is a GLP-1 analogue extracted from the salivary gland of the African monster lizard, which has stronger GLP-1 receptor agonistic effect and similar GLP-1 effect. Compared with natural GLP-1, Exendin-4 has stronger resistance to DPP-4, and its plasma half-life in vivo is also longer.
  • GIP is a single-chain polypeptide of 42 amino acids, produced by K cells in the small intestinal mucosa, and mainly acts on the GIP receptor (GIPR) in islet cells and adipocytes.
  • GIP glucose-dependently promotes insulin secretion, enhances islet ⁇ -cell mass, stimulates insulin secretion, inhibits gastric acid secretion, and slows down gastric motility. In addition, it also stimulates the uptake and utilization of fatty acids by adipose tissue cells.
  • GIP also has the physiological effect of promoting osteoblast differentiation, inhibiting osteoblast apoptosis, inhibiting bone resorption, increasing bone mineral density, and protecting bone.
  • GCG is a polypeptide containing 29 amino acids, expressed and secreted by proglucagon gene in pancreatic ⁇ -cells, acting on glucagon receptors (GCG R) mainly distributed in liver and kidney, and stimulating liver glycogen decomposition , Elevate blood sugar, activate lipase, promote fat decomposition, inhibit liver fat synthesis, and strengthen fatty acid oxidation.
  • GCG R glucagon receptors
  • GLP-1 receptor agonists such as exenatide, lixisenatide, liraglutide, dulaglutide, and semaglutide have been successfully developed for the treatment of type II diabetes .
  • liraglutide has also been successfully developed for weight loss, and semaglutide is also undergoing clinical research for obesity indications.
  • the advantage of GLP-1 analogues is that they can lower blood sugar and also have cardiovascular benefits and weight management effects.
  • the current GLP-1 receptor agonists alone still reduce body weight by less than 10%, and there are obvious dose-related gastrointestinal effects.
  • GLP-1 variouslycemia, obesity and insulin resistance hinder the function of GIPR and its signaling pathway, but with the reduction of blood sugar and improvement of insulin resistance, the effect of GIP on stimulating insulin secretion and improving islet function can be improved.
  • GCG can also promote lipolysis, inhibit hepatic fat synthesis, and exert the effect of lowering blood fat and body weight, but it needs the synergy of GLP-1 to inhibit its effect of raising blood sugar.
  • lowering blood sugar and improving insulin resistance can further enhance the synergistic effect of GIP's insulin secretion-stimulating function and insulin sensitization, improve islet function, further enhance GCG's lipolysis and improve Lipid metabolism, enhance weight loss effect.
  • GCG has a strong effect of raising blood sugar
  • GLP-1/GCG receptor dual agonist MEDI0382 has relative activity EC 50 of GLP-1 and GCG receptors of 29% and 12.8%, respectively.
  • GLP-1/GIP/GCG triple receptor agonist molecule once a week, once every two weeks or once every three weeks should have as high as possible GLP-1R/GIPR activity and relatively controllable GCGR activity, In order to ensure the maximum effect of weight loss and hypoglycemia.
  • CN109071624A disclosed a series of ring-forming cyclic peptide molecules and long-acting Molecules composed of conjugates.
  • WO2015067716A1 WO2019125929A1 and WO2019125938A1 also disclose some polypeptides with fatty acid linked to the side chain of amino acid 17. These peptides all showed GLP-1/GIP/GCGR triple agonistic effect and had once-weekly long-acting potential.
  • the molecules disclosed in the above-mentioned patent applications cannot simultaneously have sufficiently high activity on GLP-1 R/GIP R/GCGR.
  • the present invention provides a novel polypeptide molecule with triple agonistic activity of GLP-1/GIP/GCGR, which can be used for type II diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and other related metabolic diseases.
  • the present invention provides GLP-1/GIP/GCGR triple agonist polypeptide compound or its salt or solvate having general formula (I):
  • X3 is Q or H
  • X6 is F, ⁇ MeF, or ⁇ MeF (2F);
  • X13 is ⁇ MeL, F, ⁇ MeF, or L;
  • X17 is ⁇
  • X25 is Y or F
  • X29 is T, S, G, or Aib
  • X30 is G, H, R, or Aib
  • X34 is G or Aib
  • X35 is A, Q, Aib, H;
  • X38 is Ac3c, P;
  • R 1 is NH 2 or OH, or a pharmaceutically acceptable salt and/or ester thereof;
  • ⁇ is a Lys whose side chain is modified by the following general formula (II), and the general formula (II) is:
  • Y is (AEEAc or Glu) a -(AEEAc or Glu) b -(AEEAc or Glu) c , wherein a, b, and c are each independently 0 or 1, and a, b, and c are not At the same time, it is 0 (as an example, it can be AEEAc-AEEAc- ⁇ Glu), the carboxyl terminal of Y is connected to the ⁇ -amino group of the side chain of Lys, and Z is -CO-(CH 2 ) m -R 2 , m is an integer between 6-24, R 2 is selected from -COOH.
  • the compound of general formula (I) contains at least two specific amino acids at the following positions:
  • X13 is F or ⁇ MeF
  • X25 is F
  • X29 is T or S
  • X30 is H, R or Aib
  • X35 is Q, Aib or H
  • X38 is Ac3c.
  • the general formula (II) is AEEAc-AEEAc- ⁇ Glu-CO(CH 2 ) 18 COOH.
  • the compound is selected from:
  • the compound is further selected from:
  • the relative activity of the compound for GLP-1 receptor agonism is at least 30%, preferably At least 60%, more preferably at least 80%, even more preferably at least 100%.
  • the relative activity of the compound for GIP receptor agonism is at least 100%, more preferably at least 150%.
  • the relative activity of the compound for GCG receptor agonism is at least 10%, more preferably at least 30%.
  • the relative activity of the compound on RIN-m5F cell agonism is at least 60%, preferably At least 80%, more preferably at least 100%.
  • the relative activity of the compound on 3T3-L1 cell agonism is at least 60%, preferably at least 100%.
  • the relative activity of the compound on hepatocyte stimulation is at least 60%, preferably at least 100%.
  • the present invention also provides a pharmaceutical composition, comprising an effective amount of the compound or its salt or solvate described in any aspect above, and a pharmaceutically acceptable adjuvant, diluent, carrier or vehicle.
  • the pharmaceutical composition is injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension or syrup; or the The pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles or liposomes.
  • the pharmaceutical composition is for oral administration, inhalation administration or parenteral administration selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection medicine.
  • the pharmaceutical composition is administered at least once a day, once a week, every two weeks or once a month.
  • the pharmaceutical composition can also be used in combination with at least one of the following therapeutically active substances, including antidiabetic active agents (such as: insulin and its analogs, biguanides, sulfonylureas, thiazolidinedione , ⁇ -glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, dual SGLT1/SGLT2 inhibitors, GLP-1 receptor agonists, amylin and its analogs), GIP receptor agonists, GCG Receptor agonist or antagonist, GLP-1/GIP receptor agonist, GLP-1/GCG receptor agonist, GIP/GCG receptor agonist, FGF-21 and its analogs, cholecystokinin B (CCKB ) and its analogs, PYY(3-36) and its analogs, leptin and its analogs, calcitonin and its analogs, blood lipid-regulating active drugs, PPAR- ⁇ , ⁇ , ⁇ agonists or modulators, Anti antidia
  • the anti-diabetic agents include insulin and its analogs, biguanides, sulfonylureas, thiazolidinediones, ⁇ -glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, Dual SGLT1/SGLT2 inhibitors, GLP-1 receptor agonists, or amylin and its analogs.
  • neurodegenerative diseases include Parkinson's disease, Alzheimer's disease.
  • Any of the aforementioned compounds of the present invention can be synthesized by solid phase.
  • GLP-1 R, GIP R and GCG R were found to be distributed and expressed in cells of many metabolic-related tissues and organs in the human body. For example, three receptors GLP-1 R, GIP R, and GCG R are simultaneously expressed in pancreatic islet tissue cells, and the receptor with the highest abundance is GLP-1 R; for example, GLP-1 R, GIP R, and For the expression of R, the abundance of GIP R was the highest; and the expression of GLP-1 R and GCG R was also reported in hepatocytes, and the abundance of GCG R was the highest.
  • the inventors have surprisingly found that although some compounds can have better EC 50 than wild-type polypeptides in single receptor stable cells, in the above-mentioned tissue cells, compared with wild-type polypeptides, even higher doses are required In order to fully stimulate the tissue cells. This is most likely related to the propensity for multi-agonist compounds to distribute to multiple receptors. Therefore, in order to achieve the maximum therapeutic effect, it is required that the three agonist molecules should be able to fully stimulate multiple receptors in tissue cells, at least they should be able to better stimulate high-abundance receptors in tissue cells. Then, the EC 50 of the three agonist molecules on each tissue cell should be at least better than that of the corresponding wild-type polypeptide GLP-1/GIP/GCG.
  • the compound of the present invention has strong relative activity EC 50 against three of GLP-1/GIP/GCGR at the same time, and can sufficiently stimulate corresponding target organ tissue cells.
  • the compound of the present invention can promote insulin secretion and lower blood sugar. Preferably, it can also inhibit food intake, delay gastric emptying, increase energy consumption, and finally observe the effect of reducing body weight.
  • the compound of the present invention can reduce the apoptosis of pancreatic islet ⁇ -cells, increase the number of pancreatic islet ⁇ -cells, and improve the function of pancreatic islet cells.
  • the compound of the present invention can also improve blood lipids, reduce liver fat accumulation, inhibit the development of liver inflammation, and prevent and treat non-alcoholic fatty liver disease.
  • the compound of the present invention is expected to promote the growth of brain neurons, remove neurotoxic substances, inhibit the development of inflammation, and play a role in neuroprotection.
  • the compounds or compositions of the present invention can be used to prevent and/or treat metabolic disorders and related complications. It is preferably used in the treatment of diabetes, obesity and non-alcoholic fatty liver disease.
  • the compound or composition of the present invention can be used to treat dyslipidemia and related diseases, neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease).
  • neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease.
  • the compound or composition of the present invention can be used to treat skeletal diseases related to endocrine diseases, metabolic disorders, kidney diseases, etc., such as osteoporosis and osteoarthritis.
  • the compound of the present invention has significant agonistic effects on GLP-1, GIP and GCG receptors.
  • polypeptide compounds described in the present invention can be synthesized and modified by those skilled in the art through known techniques.
  • the peptide sequence backbone of the polypeptide compound of the present invention can be prepared by methods such as synthesis.
  • the peptide backbone of the compound of the present invention is chemically modified at least at one site by a fatty acid side chain group.
  • the compound can have a stable peptide ⁇ -helical structure, can have enhanced albumin binding ability, that is, can achieve improved stability of the peptide compound and prolonged action time of the peptide.
  • the compound of the present invention has agonistic activity on GLP-1 receptor, GIP receptor and GCG receptor.
  • the "agonistic activity” means that the compound can stimulate specific receptor cells to produce cAMP, and the cells used can be host cells or pancreatic islets constructed by those skilled in the art to overexpress GLP-1 receptors or GIP receptors or GCG receptors Tissue cells, fat cells, liver cells, etc.
  • the receptor agonistic activity can be measured by using the EC 50 value of the compound to stimulate receptor cells to produce cAMP.
  • the EC 50 value refers to the drug concentration value required to achieve half of the maximum activity of the compound (50% activity) in a specific assay system.
  • the agonistic activity of a compound can be assessed by the relative activity of a particular natural compound. Relative activity is expressed as a percentage of the ratio of the EC50 value of a particular native compound to the EC50 value of the test compound.
  • the GLP-1/GIP/GCG R triple agonist polypeptide molecule provided by the present invention has better relative activity EC 50 of GLP-1 R, GIP R, GCG R and tissue cells.
  • the “relative activity EC 50” refers to the ratio of the EC 50 values of the corresponding wild-type positive peptides human GLP-1 (7-37), human GIP and human GCG to the EC 50 values of the compounds of the present invention.
  • amino acids in the compound sequences of the present invention are derived from natural amino acids or related amino acid variants and/or derivatives.
  • the abbreviations and codes of the natural amino acids adopt general rules well known to those skilled in the art.
  • the chemical structures of Aib, ⁇ -MeF, ⁇ -MeF(2F), ⁇ -MeL, Ac3c are as follows:
  • Aib ⁇ -amino isobutyric ( ⁇ -aminoisobutyric acid)
  • ⁇ -MeF ⁇ -methylphenylalanine ( ⁇ -methylphenylalanine)
  • ⁇ -MeF(2F) ⁇ -methyl-2-fluorophenylalanine ( ⁇ -methyl-2-fluorophenylalanine)
  • ⁇ -MeL ⁇ -methyl leucine ( ⁇ -methyl leucine)
  • AEEAc [2-(2-Amino-ethoxy)-ethoxy]-acetyl
  • cAMP cyclic adenosine monophosphate
  • HBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • Trt Trityl
  • DIPEA N,N-Diisopropylethylamine
  • HEK-293 Human embryonic kidney cells
  • GLP-1 Glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic peptide
  • GLP-1 R Glucagon-like peptide-1 receptor
  • GIPR Glucose-dependent insulinotropin receptor
  • GCG R Glucagon receptor
  • NAFLD Nonalcoholic fatty liver disease
  • Nonalcoholic steatohepatitis nonalcoholic steatohepatitis
  • DMEM Duchenne's Modified Eagle's Medium
  • FBS fetal bovine serum
  • FCS Fetal Calf Serum
  • PBS Phosphate Buffered Saline
  • HBSS Hank's buffered saline solution
  • IBMX 3-isobutyl-1-methylxanthine.
  • Embodiment 1 peptide compound is synthesized
  • the intermediates and compounds of the present invention can be prepared synthetically by various methods known in the art.
  • the following specific examples illustrate the preparation of the compounds of the present invention by chemical synthesis. Each specific synthesis step described can be combined with different materials and methods to synthesize a variety of corresponding compounds or salts thereof of the present invention.
  • the reagents and materials used are readily available to those of ordinary skill in the art. In particular, the following examples are only for illustrating the present invention and should not limit the scope of the present invention in any way.
  • the materials and reagents used in the present invention are all purchased from commercial goods, and the protected amino acids used in the whole synthesis process are as follows: Fmoc-Ser(tBu)-OH, Fmoc-Ac3c-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH , Fmoc-Lys(Boc)-OH, Fmoc-Lys(ivdde)-OH, Fmoc- ⁇ -MeLeu-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc- ⁇ -MePhe
  • the sequence of compound 10 main peptide sequence synthesis and amino acid coupling is Fmoc-Ser(tBu)-OH, Fmoc-Ac3c-OH, Fmoc-Pro-OH(2x), Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH(2x), Fmoc-Pro-OH, Fmoc-Gly-OH(2x), Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH(2x), Fmoc-Phe-OH , Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc -Lys(ivdde)-OH, Fm
  • Lys side chain modification Weigh AEEAc (2.0eq) and add it to the above resin, add DIEA (4.00eq) and add 5ml DMF to the reaction column, blow nitrogen, add HBTU (1.9eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly. Reaction at 25°C for 1h. The ninhydrin detection resin is colorless and transparent, that is, the reaction is complete. The reaction solution was sucked off, and washed with DMF (10ml) 6 times, 1min each time. Add 20% piperidine/DMF (10ml) to the reaction column, and blow nitrogen gas for 20min to remove the Fmoc group.
  • Peptide resin post-treatment Add the above-mentioned dried peptide resin to the prepared cleavage reagent (95% TFA: 2.5% Tis: 2.5% H2O), shake on a shaker for 2.5 hours to cleavage the resin. After filtration, the filtrate was added to 10 times the volume of ice isopropyl ether, centrifuged, and washed 5 times with isopropyl ether. The crude peptide was obtained by drying in vacuo for 2 h.
  • the resulting crude peptide powder was dissolved in 50% acetonitrile/H 2 O solution, purified by reverse phase C18 preparative chromatography column (Shimadzu, Inertsil ODS 20x 250mm 5um), and purified with 95% buffer A (0.1% TFA/ H2O) and 5% buffer B (0.075% TFA/acetonitrile) as the initial eluent, gradually increase the proportion of buffer B to 75%, run continuously for 30min for elution, and collect the target peptide components.
  • the purified peptide compounds were confirmed by analytical HPLC/MS method analysis. The purity of the obtained peptide compound is not lower than 95%.
  • Example 2 Peptide compound test for human GLP-1/GIP/GCG receptor stabilizing cell activity
  • HEK-293 cells stably overexpressing human GLP-1 or GCG receptors and CHO cells stably overexpressing GIP receptors were constructed respectively.
  • the agonistic activity of each compound on the corresponding receptor is determined by measuring the cAMP signal response level of the above cells.
  • Intracellular cAMP content was determined using a kit from Cisbio Corp. based on HTRF (homogeneous time-resolved fluorescence) technology.
  • 1 ⁇ HBSS containing 0.1% Casein, 250 ⁇ M IBMX
  • GLP-1 receptor cells Use natural wild-type human GLP-1(7-37), GIP, and GCG as positive controls for the receptor agonistic effect of the test compound.
  • GLP-1 receptor cells calculate the human GLP-1(7-37) The percentage of the ratio of the EC 50 value of the test compound to the EC 50 value of the test compound was used as the relative activity (%) to evaluate the GLP-1 receptor agonistic activity of the test compound.
  • the GIP receptor agonistic activity of the test compound was evaluated by calculating the percentage of the ratio of the EC 50 value of human GIP to the EC 50 value of the test compound as the relative activity (%).
  • the GCG receptor agonistic activity of the test compound was evaluated by calculating the percentage of the ratio of the EC 50 value of human GCG to the EC 50 value of the test compound as the relative activity (%).
  • Rel.A Relative Activity, relative activity
  • NT Not test, not tested
  • each group has at least 3 independent tests.
  • Example 3 Functional activity test of peptide compounds on rat insulinoma RIN-m5F cells
  • the rat insulinoma RIN-m5F cell line is derived from rat islet tissue and mainly expresses endogenous GLP-1/GIP/GCG receptors, among which GLP-1 receptor is the most abundant.
  • GLP-1 receptor is the most abundant.
  • 1 ⁇ HBSS containing 0.1% Casein , 250 ⁇ M IBMX
  • Rel.A Relative Activity, relative activity
  • NT Not test, not tested
  • each group has at least 3 independent tests.
  • All the compounds of the present invention in the table can exhibit relatively high relative activity in the insulinoma cell RIN-m5F.
  • Example 4 Test of the functional activity of peptide compounds on 3T3-L1 adipocytes
  • Mouse 3T3-L1 preadipocytes can be induced to differentiate into mature adipocytes.
  • 3T3-L1 cells were directly seeded in 96-well plates at a cell density of 4 ⁇ 10 4 /ml, cultured in DMEM medium containing 10% FCS and 1% P/S, and cultured at 37°C and 5% CO2 Cultivated in a box. After the cells were overgrown and contacted, the DMEM differentiation medium containing 20% FBS, 0.5mM IBMX, 0.4ug/mL dexamethasone, and 5ug/mL insulin was used to induce the differentiation of 3T3-L1 adipocytes.
  • the DMEM medium containing 20% FBS, 4ug/ml insulin and 10uM rosiglitazone continued to induce culture for 3 days, then changed to DMEM medium containing 10% FBS and continued to culture for 2-3 days to induce mature 3T3-L1 fat cells.
  • Induced and differentiated mature 3T3-L1 adipocytes are similar to adipose tissue cells and can express abundant GIP receptors.
  • the level of cAMP produced by the compound on 3T3-L1 adipocytes was determined.
  • the compound solution to be tested was diluted step by step according to a 3-fold concentration gradient, and a total of 10 serial concentration solutions were set up, added to the induced mature 3T3-L1 adipocytes, and incubated at room temperature for 60 min. After the drug incubation was completed, 10 ⁇ L of the detection reagent in the kit was added to each well, and incubated at room temperature for 60 min. Plates were placed in an EnVision multiplate reader (PerkinElmer) and read at 665/615 nm. Concentration-effect curves of compounds were prepared and calculated using GraphPad Prism 5 software.
  • Rel.A Relative Activity, relative activity
  • NT Not test, not tested
  • each group has at least 3 independent tests.
  • All the compounds of the present invention in the table can exhibit higher relative activity in adipocyte 3T3-L1.
  • Example 5 Functional activity test of peptide compounds on human primary hepatocytes
  • 1 ⁇ HBSS containing 0.1% Casein, 250 ⁇ M IBMX
  • Rel.A Relative Activity, relative activity
  • NT Not test, not tested
  • each group has at least 3 independent tests.
  • All the compounds of the present invention in the table can exhibit relatively high relative activity in hepatocytes.
  • mice fed a high-fat diet-induced obesity are characterized by obesity, elevated blood sugar, insulin resistance, and lipid abnormalities, all of which are very similar to the metabolic syndrome in humans.
  • DIO high-fat diet-induced obesity
  • mice 5-week-old male C57BL/6J mice (purchased from Shanghai Slack Experimental Animal Company) were raised in a clean environment without pathogens (controlled temperature 20-24°C, relative humidity 30-70%), 12 hours of light/12 hours Dark cycle, feeding with normal feed, 4 animals per cage, acclimatization for 2 weeks.
  • Mouse obesity was induced by feeding a high-fat diet (60kcal% of calories came from fat).
  • the body weight of DIO mice reached 41-55g, and the blood glucose range was 8-12mmol/L.
  • S.C. subcutaneous injection
  • the animals were injected subcutaneously with the vehicle control and the compound.
  • the compound was dissolved in 1 ⁇ PBS, and the dosage was 5ml/kg.
  • Q3D Once (Q3D), last for 15 days.
  • the animal body weight and food intake were measured before administration every day, and by comparing with the initial body weight and food intake before administration of the same animals, the percentage of change in animal body weight (%) and cumulative food intake were calculated to evaluate the effect of the compound on body weight and food intake. Effects of changes in food intake.
  • the body weight of the mice was measured, and then the fasting blood glucose of the animals was measured by blood sampling from the tip of the tail without anesthesia.
  • the animal was anesthetized with CO 2 and sacrificed, then blood was collected from the heart, and the plasma was centrifuged to separate plasma, which was used to determine plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), blood insulin levels.
  • TC total cholesterol
  • LDL-C low-density lipoprotein cholesterol
  • TG triglyceride
  • the liver was separated for homogenization, and the supernatant of the homogenate was obtained by centrifugation, which was used to determine the content of triglyceride in the liver.
  • Embodiment 7 the rat pharmacokinetic (PK) research of compound

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Abstract

涉及医药生物领域，具体涉及一种对胰高血糖素样肽-1受体(GLP-1R)，葡萄糖依赖性促胰岛素多肽受体(GIP R)和胰高血糖素受体(GCG R)具有三重激动活性的通式(I)的三激动剂多肽化合物或其盐或溶剂合物，并且涉及其在治疗代谢综合征中的应用。

Description

多激动剂及其应用 技术领域
本发明涉及一类多肽化合物及其在医药领域的用途。更具体地讲，本发明涉及对胰高血糖素样肽-1受体(GLP-1 R)，葡萄糖依赖性促胰岛素多肽受体(GIP R)和胰高血糖素受体(GCG R)具有三重激动活性的多肽化合物，并且涉及其在治疗代谢综合征中的应用。
背景技术
Ⅱ型糖尿病及肥胖症正日益成为影响人类健康的全球性疾病。肥胖是糖尿病、高血压、心脏病、血脂异常、脂肪性肝病、动脉粥样硬化、关节炎、中风、神经退行性疾病等众多慢性疾病的主要诱因，糖尿病也会导致心脑血管疾病等多种并发症。目前市售的降糖药物大多仅有控制血糖效果，无法改善肥胖患者体重，甚至有些药物还有增加体重的副作用。因此，仍然迫切需要开发出兼具降血糖和改善体重，同时包含多种获益效果的药物以满足大多数肥胖及Ⅱ型糖尿病人群的需求。
肠促胰素(Incretin)是一类在正常生理状态下进食刺激后从肠道中分泌的多肽类激素，早期研究发现其能够随进餐后葡萄糖水平上升而刺激胰岛β-细胞分泌胰岛素，调节葡萄糖水平内稳态，保护胰岛β-细胞，通过抑制食欲、延缓胃排空等途径降低体重。GLP-1和GIP是目前发现的两种肠促胰素。
GLP-1由胰高血糖素原基因在肠粘膜L细胞中表达，具有31个氨基酸的多肽，主要作用于GLP-1受体(GLP-1 R)，刺激胰岛素分泌，抑制胰高血糖素分泌，保护胰岛β-细胞，具有调节血糖稳态的生理作用，同时能够通过中枢神经系统信号通路抑制摄食和胃排空，增加饱腹感，从而降低体重。毒蜥外泌肽-4(Exendin-4)是从非洲毒蜥蜴唾液腺中提取的GLP-1类似物，具有更强的GLP-1受体激动效果，相似的GLP-1效应。相比于天然GLP-1，Exendin-4对DPP-4具有更强的抵抗性，其体内血浆 半衰期也更长。
GIP是42个氨基酸的单链多肽，由小肠粘膜K细胞产生，主要作用于胰岛细胞和脂肪细胞中的GIP受体(GIP R)。GIP葡萄糖依赖性促胰岛素分泌，增强胰岛β-细胞质量，刺激胰岛素分泌，抑制胃酸分泌，减缓胃蠕动。此外还刺激脂肪组织细胞摄取利用脂肪酸。GIP还具有促进成骨细胞分化，抑制成骨细胞凋亡，抑制骨吸收，增加骨矿物质密度的生理作用，起到保护骨的功能。
GCG是含29个氨基酸的多肽，由胰高血糖素原基因在胰岛α-细胞中表达分泌，作用于主要分布在肝脏和肾脏的胰高血糖素受体(GCG R)，刺激肝糖原分解，升高血糖，激活脂肪酶，促进脂肪分解，同时抑制肝脏脂肪合成，加强脂肪酸氧化。研究结果表明，GCG对于降低食物摄取、增加脂肪组织能量消耗，降低体脂量有一定的效果。GCG合适的升高血糖效果能反馈调节胰岛素的作用，降低低血糖事件发生。
针对肠促胰素效应，目前已成功开发出艾塞那肽、利司那肽、利拉鲁肽、杜拉鲁肽、索马鲁肽等GLP-1受体激动剂用于Ⅱ型糖尿病治疗。此外，利拉鲁肽还成功开发用于减肥，索马鲁肽也正在进行肥胖适应症的临床研究。GLP-1类似物的优势是降血糖的同时兼具心血管益处及体重管理效果，但目前单独的GLP-1受体激动剂降体重仍然不足10％，并且存在明显的剂量相关的胃肠道副反应(主要是恶心、呕吐、腹泻)，对于病情较复杂的代谢疾病，如肥胖型Ⅱ型糖尿病、非酒精性脂肪肝病/非酒精性脂肪肝炎(NAFLD/NASH)及伴随心血管风险的糖尿病及肥胖等人群仍然迫切需要降体重效果更强的治疗药物。
根据GLP-1、GIP和GCG的生理作用，目前多项研究证实，通过同时激动其中两者或三者的活性，比单独激动GLP-1 R能够实现更好的糖尿病及肥胖治疗效果。文献报道高血糖、肥胖及胰岛素抵抗阻碍GIP R及其信号通路的作用，但随着血糖降低和胰岛素抵抗改善，可改善GIP促胰岛素分泌和改善胰岛功能的作用。GCG除了促进糖原分解升高血糖外，还能促进脂肪分解、抑制肝脏脂肪合成，发挥降血脂和体重效果，但需要GLP-1的协同抑制其升血糖效应。因此，通过GLP-1的“排头兵” 效应，降低血糖、改善胰岛素抵抗，可实现进一步增强GIP的促胰岛素分泌功能和胰岛素增敏协同作用，改善胰岛功能，进一步增强GCG的脂质分解作用和改善脂质代谢，增强降体重效应。
随着GLP-1/GIP和GLP-1/GCG双激动剂临床数据的披露，GLP-1/GIP或者GLP-1/GCG双激动剂在各受体活力的分配对于药物临床治疗效果的影响也逐渐清晰。
比如，礼来的Tirzepatide(LY3298176)的Ⅱ期临床研究结果显示，Tirzepatide 1mg剂量几乎无降体重效果，降糖效果也显著低于度拉糖肽1.5mg。只有当Tirzepatide剂量升至5mg时，才能产生优于1.5mg度拉糖肽的降糖和减重效果。这很有可能就是由于Tirzepatide偏向于更强的GIP受体激动活性，而GLP-1受体活力EC 50仅为野生型GLP-1活力EC 50的大约15％所导致的。为了追求最大疗效，Tirzepatide剂量大幅提高(最大剂量达到15mg)，这将有可能会增加药物的安全风险。
另外一方面，由于GCG具有很强的升高血糖作用，因此在GLP-1/GCG受体双激动分子中，需要有足够的GLP-1活性才能平衡压制GCG的升血糖效应，进而保证降血糖效果。阿斯利康的GLP-1/GCG受体双激动分子MEDI0382(Cotadutide)的GLP-1和GCG受体的相对活力EC 50分别为29％，12.8％。其一项26周的临床试验结果显示，与利拉鲁肽相比，100～300ug Cotadutide呈剂量依赖性降低体重、血脂和ALT及AST水平，并且300μg剂量组的效果显著优于1.8mg利拉鲁肽。但是在糖化血红蛋白(HbA1c)的降低上，各剂量组与利拉鲁肽相比无显著优势。并且随着Cotadutide剂量的上升，300ug组的糖化血红蛋白降低效果反而劣于200ug组。这很有可能是由于随着剂量的增加，Cotadutide分子GLP-1受体活力偏低，不足以压制GCG的效应导致。
理想的一周一次、两周一次或三周一次的长效GLP-1/GIP/GCG三受体激动剂分子，应当是具有尽可能高的GLP-1R/GIPR活力和相对可控的GCGR活力，方可保证减重与降血糖效果的最大化。CN104902919A、CN111040022A披露了一系列基于毒蜥外泌肽-4(exendin-4)结构改造的GLP-1/GIP/GCG R三重激动剂分子；CN109071624A披露了一系列成环 的环肽分子与长效缀合物组合而成的分子。此外，WO2015067716A1、WO2019125929A1和WO2019125938A1也披露了一些在17位氨基酸的侧链链接脂肪酸的多肽。这些多肽均显示出了GLP-1/GIP/GCG R三重激动效果并具有一周一次的长效潜力。上述专利申请中所披露的分子，并不能在GLP-1 R/GIP R/GCG R上同时具有足够高的活力。
当前依然存在着进一步优化三激动剂分子的改进空间。
发明内容
针对以上技术现状，本发明提供一种新型的具有GLP-1/GIP/GCG R三重激动活性的多肽分子，可用于Ⅱ型糖尿病、肥胖、血脂异常、非酒精性脂肪肝病/非酒精性脂肪肝炎等相关代谢病领域的治疗。
本发明提供了具有通式(I)的GLP-1/GIP/GCG R三激动剂多肽化合物或其盐或溶剂合物：
Y-Aib-X3-GT-X6-TSDYSI-X13-LDK-X17-AQ-Aib-AFIE-X25-LLE-X29-X30-PSS-X34-X35-PP-X38-S-R 1 (I)，
其中，
X3为Q或H；
X6为F、αMeF、或αMeF(2F)；
X13为αMeL、F、αMeF、或L；
X17为ψ；
X25为Y或F；
X29为T、S、G、或Aib；
X30为G、H、R、或Aib；
X34为G或Aib；
X35为A、Q、Aib、H；
X38为Ac3c、P；
R 1为NH 2或OH，或其药学上可接受的盐和/或酯；
其中，ψ为侧链被具有下述通式(Ⅱ)结构修饰的Lys，所述通式(Ⅱ)为：
Y-Z(Ⅱ)，其中Y为(AEEAc或Glu) a-(AEEAc或Glu) b-(AEEAc或 Glu) c，其中a、b、c各自独立地为0或1，且a、b、c不同时为0(作为示例性说明，可以为AEEAc-AEEAc-γGlu)，所述Y的羧基端与Lys的侧链的ε-氨基相连，Z为-CO-(CH 2) m-R 2，m为6-24之间的整数，R 2选自-COOH。
本发明中，作为实施方案之一，所述通式(I)化合物中，含有至少两个以下位点的特定氨基酸：
X13为F或αMeF；
X25为F；
X29为T或S；
X30为H、R或Aib；
X35为Q、Aib或H；
X38为Ac3c。
本发明中，作为实施方案之一，所述通式(Ⅱ)为AEEAc-AEEAc-γGlu-CO(CH 2) 18COOH。
本发明中，作为实施方案之一，所述化合物选自：
Figure PCTCN2022093577-appb-000001
Figure PCTCN2022093577-appb-000002
Figure PCTCN2022093577-appb-000003
本发明中，作为实施方案之一，所述化合物进一步选自：
Figure PCTCN2022093577-appb-000004
Figure PCTCN2022093577-appb-000005
Figure PCTCN2022093577-appb-000006
本发明中，作为实施方案之一，在GLP-1受体稳转细胞的激动能力上，相较于天然GLP-1，所述化合物对GLP-1受体激动的相对活性至少30％，优选至少60％，更优选至少80％，更进一步优选至少100％。
本发明中，作为实施方案之一，在GIP受体稳转细胞的激动能力上，相较于天然GIP，所述化合物对GIP受体激动的相对活性至少100％，更优选至少150％。
本发明中，作为实施方案之一，在GCG受体稳转细胞的激动能力上，相较于天然GCG，所述化合物对GCG受体激动的相对活性至少10％，更优选至少30％。
本发明中，作为实施方案之一，在胰岛组织代表细胞RIN-m5F上，相较于天然GLP-1(7-37)，所述化合物对RIN-m5F细胞激动的相对活性至少60％，优选至少80％，更优选至少100％。
本发明中，作为实施方案之一，在脂肪组织代表细胞3T3-L1上，相较于天然GIP，所述化合物对3T3-L1细胞激动的相对活性至少60％，优选至少100％。
本发明中，作为实施方案之一，在肝脏组织代表细胞人原代肝细胞上，相较于天然GCG，所述化合物对肝细胞激动的相对活性至少60％，优选至少100％。
本发明还提供了一种药物组合物，包括有效量的前述任一方面所述化合物或其盐或溶剂合物，和药学上可接受的辅料、稀释剂、载剂或赋形剂。
在一个方面，所述药物组合物为注射剂或冻干粉、片剂、丸剂、锭剂、软胶囊剂、硬胶囊剂、颗粒剂、散剂、溶液剂、混悬剂或糖浆剂；或者所述药物组合物为微囊、微球、纳米粒或脂质体形式。
在一个方面，所述药物组合物用于口服给药、吸入给药或肠胃外给药，所述肠胃外给药选自腹膜内、肌内、动脉内、静脉内、皮下或皮内注射给药。
在一个方面，所述药物组合物以至少一天给药一次，一周给药一次，两周给药一次或一个月给药一次的频率给药。
在一个方面，所述药物组合物还可与至少一种下述治疗用活性物质联合使用，包括抗糖尿病活性剂(如：胰岛素及其类似物、双胍类、磺脲类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、DPP-4抑制剂、SGLT2抑制剂、双重SGLT1/SGLT2抑制剂、GLP-1受体激动剂、胰淀素及其类似物)、GIP受体激动剂、GCG受体激动剂或拮抗剂、GLP-1/GIP受体激动剂、GLP-1/GCG受体激动剂、GIP/GCG受体激动剂、FGF-21及其类似物、缩胆囊素B(CCKB)及其类似物、PYY(3-36)及其类似物、瘦素及其类似物、降钙素及其类似物、调血脂活性药物、PPAR-α、β、δ激动剂或调节剂、抗血小板聚集活性剂、PCSK9抑制剂、脂肪酶抑制剂、抗肝纤维化或肝硬化活性剂、抗炎活性剂。作为实施方式之一，所述抗糖尿病活性剂包括胰岛素及其类似物、双胍类、磺脲类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、DPP-4抑制剂、SGLT2抑制剂、双重SGLT1/SGLT2抑制剂、GLP-1受体激动剂、或胰淀素及其类似物。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备促进胰岛素分泌，降低血糖的药物中的应用。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备抑制摄食，延缓胃排空，增加能量消耗，降低体重的药物中的应用。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在 制备减少胰岛β-细胞凋亡，增加胰岛β-细胞数量，改善胰岛细胞功能的药物中的应用。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备改善血脂，减少肝脏脂肪累积，抑制肝脏炎症发展，预防及治疗非酒精性脂肪肝病的药物中的应用。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备促进大脑神经元生长、清除神经毒性物质，抑制炎症发展，起到神经保护作用的药物中的应用。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备用于预防及/或治疗代谢紊乱疾病及其相关并发症的药物中的应用，优选用于治疗糖尿病、肥胖症或非酒精性脂肪肝病的药物。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备用于治疗血脂代谢紊乱及其相关疾病、神经退行性疾病的药物中的应用，所述神经退行性疾病包括帕金森病、阿尔茨海默症。
本发明前述任一化合物或其盐或溶剂合物或前述任一药物组合物在制备用于治疗内分泌疾病、代谢紊乱、肾病等原因相关的骨骼疾病的药物中的应用，所述骨骼疾病包括骨质疏松症、骨关节炎。
本发明前述任一化合物均可采用固相合成。
在一个方面，提供了具有如下序列的化合物或其盐或溶剂合物：
化合物1(SEQ ID NO:1)
Figure PCTCN2022093577-appb-000007
化合物2(SEQ ID NO:2)
Figure PCTCN2022093577-appb-000008
化合物3(SEQ ID NO:3)
Figure PCTCN2022093577-appb-000009
化合物4(SEQ ID NO:4)
Figure PCTCN2022093577-appb-000010
化合物5(SEQ ID NO:5)
Figure PCTCN2022093577-appb-000011
化合物6(SEQ ID NO:6)
Figure PCTCN2022093577-appb-000012
化合物7(SEQ ID NO:7)
Figure PCTCN2022093577-appb-000013
化合物8(SEQ ID NO:8)
Figure PCTCN2022093577-appb-000014
化合物9(SEQ ID NO:9)
Figure PCTCN2022093577-appb-000015
化合物10(SEQ ID NO:10)
Figure PCTCN2022093577-appb-000016
化合物11(SEQ ID NO:11)
Figure PCTCN2022093577-appb-000017
化合物12(SEQ ID NO:12)
Figure PCTCN2022093577-appb-000018
化合物13(SEQ ID NO:13)
Figure PCTCN2022093577-appb-000019
化合物14(SEQ ID NO:14)
Figure PCTCN2022093577-appb-000020
化合物15(SEQ ID NO:15)
Figure PCTCN2022093577-appb-000021
化合物16(SEQ ID NO:16)
Figure PCTCN2022093577-appb-000022
化合物17(SEQ ID NO:17)
Figure PCTCN2022093577-appb-000023
化合物18(SEQ ID NO:18)
Figure PCTCN2022093577-appb-000024
化合物19(SEQ ID NO:19)
Figure PCTCN2022093577-appb-000025
化合物20(SEQ ID NO:20)
Figure PCTCN2022093577-appb-000026
化合物21(SEQ ID NO:21)
Figure PCTCN2022093577-appb-000027
化合物22(SEQ ID NO:22)
Figure PCTCN2022093577-appb-000028
化合物23(SEQ ID NO:23)
Figure PCTCN2022093577-appb-000029
Figure PCTCN2022093577-appb-000030
化合物24(SEQ ID NO:24)
Figure PCTCN2022093577-appb-000031
化合物25(SEQ ID NO:25)
Figure PCTCN2022093577-appb-000032
化合物26(SEQ ID NO:26)
Figure PCTCN2022093577-appb-000033
化合物27(SEQ ID NO:27)
Figure PCTCN2022093577-appb-000034
化合物28(SEQ ID NO:28)
Figure PCTCN2022093577-appb-000035
化合物29(SEQ ID NO:29)
Figure PCTCN2022093577-appb-000036
化合物30(SEQ ID NO:30)
Figure PCTCN2022093577-appb-000037
化合物31(SEQ ID NO:31)
Figure PCTCN2022093577-appb-000038
化合物32(SEQ ID NO:32)
Figure PCTCN2022093577-appb-000039
化合物33(SEQ ID NO:33)
Figure PCTCN2022093577-appb-000040
化合物34(SEQ ID NO:34)
Figure PCTCN2022093577-appb-000041
化合物35(SEQ ID NO:35)
Figure PCTCN2022093577-appb-000042
化合物36(SEQ ID NO:36)
Figure PCTCN2022093577-appb-000043
化合物37(SEQ ID NO:37)
Figure PCTCN2022093577-appb-000044
化合物38(SEQ ID NO:38)
Figure PCTCN2022093577-appb-000045
化合物39(SEQ ID NO:39)
Figure PCTCN2022093577-appb-000046
化合物40(SEQ ID NO:40)
Figure PCTCN2022093577-appb-000047
化合物41(SEQ ID NO:41)
Figure PCTCN2022093577-appb-000048
化合物42(SEQ ID NO:42)
Figure PCTCN2022093577-appb-000049
化合物43(SEQ ID NO:43)
Figure PCTCN2022093577-appb-000050
GLP-1 R、GIP R和GCG R被发现在人体多个代谢相关组织器官细胞中均有分布表达。比如，胰岛组织细胞中同时表达GLP-1 R、GIP R、GCG R三个受体，其中丰度最高的受体是GLP-1 R；又比如，脂肪细胞中发现有GLP-1 R、GIP R的表达，GIP R丰度最高；而肝细胞中也有报道发现GLP-1 R、GCG R的表达，GCG R丰度最高。同时发明人惊奇地发现，有些化合物虽然能够在单个受体稳转细胞中具有比野生型多肽更好的EC 50，但是在上述组织细胞中，相较于野生型多肽，甚至需要更高的剂量才能充分激动组织细胞。这很有可能与多激动剂化合物在多个受体的分布倾向有关。因此，为达到最大的疗效，这就要求三激动剂分子应当能够充分激动组织细胞中的多个受体，至少应当能够较好地激动组织细胞中的高丰度受体。那么，三激动剂分子对各组织细胞的EC 50，应当至少比对应的野生型多肽GLP-1/GIP/GCG更好。
本发明化合物同时对GLP-1/GIP/GCG R中的三者具有较强的相对活性EC 50，并且能够充分激动相应的靶器官组织细胞。
优选地，本发明所述化合物可以促进胰岛素分泌，降低血糖。优选地，还可以抑制摄食，延缓胃排空，增加能量消耗，最终可观察到体重降低效果。
优选地，本发明所述化合物可以减少胰岛β-细胞凋亡，增加胰岛β-细胞数量，改善胰岛细胞功能。
优选地，本发明所述化合物还可以改善血脂，减少肝脏脂肪累积，抑制肝脏炎症发展，预防及治疗非酒精性脂肪肝病。
优选地，本发明所述化合物预期可以促进大脑神经元生长、清除神经毒性物质，抑制炎症发展，起到神经保护作用。
优选地，本发明所述化合物或组合物可以用于预防及/或治疗代谢紊乱疾病及其相关并发症。优选用于治疗糖尿病、肥胖症及非酒精性脂肪肝病。
优选地，本发明所述化合物或组合物可用于治疗血脂代谢紊乱及其相关疾病、神经退行性疾病(例如帕金森病、阿尔茨海默症)。
优选地，本发明所述化合物或组合物可用于治疗内分泌疾病、代谢紊乱、肾病等原因相关的骨骼疾病，如骨质疏松症、骨关节炎。
本发明化合物对GLP-1、GIP、GCG受体均具有显著的激动效果。
本发明所述的多肽化合物均可由本领域技术人员通过已知技术方法合成和修饰得到。例如，本发明所述的多肽化合物的肽序列骨架可通过合成法等方法制备。
本发明所述化合物的肽骨架上至少在一个位点处通过脂肪酸侧链基团化学修饰。优选地，所述化合物可具有稳定的肽α-螺旋结构，可具有增强的白蛋白结合力，即可实现提高的肽化合物的稳定性及延长的肽作用时间。
本发明所述化合物对GLP-1受体、GIP受体、GCG受体均具有激动活性。所述的“激动活性”是指化合物可激动特定受体细胞产生cAMP，所使用细胞可为由本领域技术人员构建的过表达GLP-1受体或GIP受体或GCG受体的宿主细胞或胰岛组织细胞、脂肪细胞、肝细胞等。所述受体激动活性可采用化合物激动受体细胞产生cAMP的EC 50值作为衡量数值。EC 50值是指在特定的测定体系中达到化合物最大活性一半(50％活性)时所需要的药物浓度值。
具体实施方案中，化合物的激动活性可通过对特定天然化合物的相对活性进行评估。相对活性为特定天然化合物的EC 50值与测试化合物的EC 50值比值的百分比。
相对于现有化合物，本发明提供的GLP-1/GIP/GCG R三激动剂多肽分子，具有更好的GLP-1 R、GIP R、GCG R和组织细胞相对活性EC 50。
定义
所述的“相对活性EC 50”是指相应的野生型阳性肽人GLP-1(7-37)、人GIP和人GCG的EC 50值与本发明化合物的EC 50值的比值。
本发明所述化合物序列中的氨基酸来源于天然氨基酸或相关的氨基 酸变构体和/或衍生物。所述天然氨基酸的缩写和代码采用本行业内人员熟知的通用规则。Aib,α-MeF,α-MeF(2F),α-MeL,Ac3c的化学结构式如下：
Figure PCTCN2022093577-appb-000051
除非另外定义，本文所用的所有技术和科学术语具有本领域技术人员通常理解的相同含义。在冲突的情况下，以包括定义在内的本文件为准。下面描述优选的方法和材料，但是与本文所述那些类似或等同的方法和材料可用于实施或测试本发明。本文公开的材料、方法和实例仅是说明性的，而非旨在限制。
本发明所使用的缩写具体含义如下：
Aib：α-amino isobutyric(α-氨基异丁酸)
α-MeF：α-methyl phenylalanine(α-甲基苯丙氨酸)
α-MeF(2F)：α-methyl-2-fluorophenylalanine(α-甲基-2-氟苯丙氨酸)
α-MeL：α-methyl leucine(α-甲基亮氨酸)
Ac3c：1-aminocyclopropanecarboxylic acid(1-氨基环丙烷羧酸)
AEEAc：[2-(2-氨基-乙氧基)-乙氧基]-乙酰基
cAMP：环磷酸腺苷
Fmoc：芴甲氧羰基
Boc：叔丁氧羰基
DMF：二甲基甲酰胺
HBTU：苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
Trt：三苯甲基
ivdde：1-(4,4-二甲基-2,6-二氧代亚环己基)-3-甲基-丁基
tBu：叔丁基
OtBu：氧叔丁基
TFA：三氟乙酸
Tis：三异丙基硅烷
HPLC/MS：高效液相色谱/质谱
HPLC-UV：高效液相色谱-紫外
IPTG：异丙基-β-D-硫代半乳糖苷
Tris：三羟甲基氨基甲烷
DCM：二氯甲烷
THF：四氢呋喃
DIPEA：N，N-二异丙基乙胺
NMP：N-甲基吡咯烷酮
HEK-293：人胚肾细胞
CHO：中国仓鼠卵巢细胞
GLP-1：胰高血糖素样肽-1
GIP：葡萄糖依赖性促胰岛素释放肽
GCG：胰高血糖素
GLP-1 R：胰高血糖素样肽-1受体
GIP R：葡萄糖依赖性促胰岛素释放肽受体
GCG R：胰高血糖素受体
NAFLD：Nonalcoholic fatty liver disease(非酒精性脂肪肝病)
NASH：Nonalcoholic steatohepatitis(非酒精性脂肪肝炎)
DMEM：杜氏改良Eagle培养基
FBS：胎牛血清
FCS：小牛血清
P/S：青霉素/链霉素
PBS：磷酸盐缓冲溶液
HBSS：Hank’s缓冲盐溶液
EC 50：半数效应浓度
IBMX：3-异丁基-1-甲基黄嘌呤。
具体实施方式
以下实施例和试验例用于进一步阐述本发明，但不以任何的方式限制本发明的有效范围。
实施例1：肽化合物合成
本发明的中间体和化合物均可通过本领域已知的多种方法合成制备。下面具体实施例中举例说明了本发明化合物采用化学合成方法制备。所描述的每一具体合成步骤可以采用不同的材料和方式组合，以合成多种对应的本发明所述化合物或其盐。所使用的试剂和原料为本领域普通技术人员容易获得。特别地，下面实施例仅用于说明本发明，不应以任何方式限制本发明的范围。
材料：
本发明所用材料及试剂均购买自商业化商品，整个合成过程所使用的保护氨基酸如下：Fmoc-Ser(tBu)-OH、Fmoc-Ac3c-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(ivdde)-OH、Fmoc-α-MeLeu-OH、Fmoc-Thr(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-α-MePhe-OH、Fmoc-α-MePhe(2F)-OH、Fmoc-Aib-OH、Boc-Tyr(tBu)-OH。
下面以化合物10为例，说明本发明化合物的合成制备方法(其中其余化合物的制备仅需替换氨基酸原料合成顺序即可)。
(1)Rink氨基树脂前处理：称取1g干燥Rink Amide MBHA树脂(替代度S＝0.28mmol/g)至反应柱中，加入10ml DMF，氮气鼓气30min，抽 去溶剂。再加入10ml DMF洗涤3次，每次1min，排去溶剂。
(2)脱除保护基Fmoc：往上述处理好的Rink氨基树脂中加入20％哌啶/DMF溶液(10ml)，氮气鼓起反应20min。反应过程中使用茚三酮显色法监测反应程度，如果树脂颜色显蓝色，表示Fmoc脱除成功。反应结束后过滤除去溶剂，向反应体系中加入DMF洗涤树脂1min，重复洗涤6次。
(3)偶联反应(肽键形成)：将配置好的相应Fmoc保护的氨基酸溶液(3.0eq)加入到反应器中，然后加入DIEA(6.0eq)，补加5ml DMF至反应柱中，鼓氮气。待氨基酸溶解后HBTU(2.85eq)。调节好氮气使树脂均匀鼓起，25℃反应30min，反应过程中使用茚三酮显色法监测反应进行程度，如果树脂无色透明，说明偶联成功。反应完成后过滤除去溶剂，向反应体系中加入DMF搅拌洗涤树脂1min，重复洗涤6次。重复以上操作，依次加入相应的氨基酸溶液直至肽链合成完毕。最后一个氨基酸用Boc-Tyr(tBu)-OH进行偶联，用四氯苯醌检测树脂无色透明即为偶联完成。侧链修饰位点的Lys用Fmoc-Lys(ivdde)-OH代替。化合物10主肽序列合成加入氨基酸偶联的顺序依次为Fmoc-Ser(tBu)-OH、Fmoc-Ac3c-OH、Fmoc-Pro-OH(2x)、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH(2x)、Fmoc-Pro-OH、Fmoc-Gly-OH(2x)、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH(2x)、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Ala-OH、Fmoc-Aib-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ala-OH、Fmoc-Lys(ivdde)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Ile-OH、Fmoc-Ser(tBu)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-α-MeF(2F)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Gln(Trt)-OH、Fmoc-Aib-OH、Boc-Tyr(tBu)-OH。
(4)Lys的侧链保护基ivdde脱除：向反应柱中加入3％水合肼/DMF溶液(10ml)，氮气鼓起反应20min，脱除修饰位点Lys的侧链保护基ivdde，茚三酮检测树脂颜色显蓝色表明脱除完成。脱除成功后除去溶剂，向体 系中加入DMF，洗涤1min，排干溶剂，重复洗涤6次。
(5)Lys侧链修饰：称取AEEAc(2.0eq)加入到上述树脂中，加入DIEA(4.00eq)补加5mlDMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(1.9eq)。调节好氮气使树脂均匀鼓起。25℃反应1h。茚三酮检测树脂无色透明即反应完全。抽掉反应液，用DMF(10ml)洗涤6次，每次1min。加入20％的哌啶/DMF(10ml)至反应柱中，氮气鼓吹反应20min，脱除Fmoc基团。加入DMF(10ml)洗涤6次，每次1min，抽干溶剂。依次按照上述方法步骤加入AEEAc、Fmoc-Glu(OtBu)-OH、C 20单叔丁基酯进行偶联，完成侧链修饰。最后用MeOH(10ml)收缩树脂，每次3min，抽干溶剂，将树脂倒出干燥，备用。
(6)肽树脂后处理：将上述干燥的肽树脂加入配制好的切割试剂(95％TFA：2.5％Tis：2.5％H2O)中，摇床震荡2.5h进行切割树脂。过滤，滤液加入到10倍体积冰异丙醚中，离心，再用异丙醚洗涤5次。在真空干燥2h得到粗肽。
(7)肽化合物粗品纯化：
将所得的肽粗品粉末溶于50％乙腈/H 2O溶液中，通过反相C18制备型色谱柱(岛津，Inertsil ODS 20x 250mm 5um)进行纯化，以95％缓冲液A(0.1％TFA/H2O)和5％缓冲液B(0.075％TFA/乙腈)为起始洗脱液，逐渐增加缓冲液B的比例至75％，连续运行30min进行洗脱，收集目标肽组分。通过分析型HPLC/MS方法分析确证纯化的肽化合物。所得肽化合物纯度不低于95％。
表1.合成肽化合物列表及分子量
Figure PCTCN2022093577-appb-000052
Figure PCTCN2022093577-appb-000053
Figure PCTCN2022093577-appb-000054
Figure PCTCN2022093577-appb-000055
Figure PCTCN2022093577-appb-000056
Figure PCTCN2022093577-appb-000057
实施例2：肽化合物对人源GLP-1/GIP/GCG受体稳转细胞活性测试
首先分别构建稳定过表达人源GLP-1或GCG受体的HEK-293细胞和GIP受体的CHO细胞。再通过测定上述细胞的cAMP信号响应水平来确定各化合物对相应受体的激动活性。细胞内cAMP含量使用Cisbio Corp.的试剂盒基于HTRF(均相时间分辨荧光)技术测定。
将冻存的稳定过表达人源GLP-1或GIP或GCG受体的细胞置于37℃恒温水浴锅中快速解冻复苏，将细胞液转移至10ml HBSS中重悬，室温1000rpm离心5min，弃去上清，用1×HBSS(含0.1％Casein，250μΜIBMX)重悬细胞，调整细胞密度为1.0×10 5/mL。
384孔板中每孔加入10μL细胞悬液。待测化合物溶于1×PBS缓冲液中配制成一定浓度母液，按3倍逐级稀释，共配制12个浓度点的化合物溶液。利用ECHO液体转移系统将100nL配置好的化合物溶液分别加入384孔板对应细胞悬液中，1000rpm旋转摇晃1min使混合均匀，随后室温孵育60min。药物孵育完成后各孔加入10μL试剂盒中的检测试剂，再室温孵育60min。将板置于EnVision多功能酶标仪(PerkinElmer)中测定665/615nm处读数。利用GraphPad Prism 5作图软件制作化合物浓度-效应曲线并计算EC 50(nM)值。
使用天然野生型人源GLP-1(7-37)、GIP、GCG作为待测化合物受体激动效果的阳性对照，对于GLP-1受体细胞，通过计算人源GLP-1(7-37)的EC 50值与待测化合物EC 50值比值的百分比作为相对活性(％)评估待测化合物的GLP-1受体激动活性。
对于GIP受体细胞，通过计算人源GIP的EC 50值与待测化合物EC 50值比值的百分比作为相对活性(％)评估待测化合物的GIP受体激动活性。
对于GCG受体细胞，通过计算人源GCG的EC 50值与待测化合物EC 50值比值的百分比作为相对活性(％)评估待测化合物的GCG受体激动活性。
表2.肽化合物的人GLP-1/GIP/GCG受体稳转细胞活性
Figure PCTCN2022093577-appb-000058
Figure PCTCN2022093577-appb-000059
Rel.A:Relative Activity，相对活性；NT:Not test，未进行检测；各组均具有至少3次独立的检测。
表中数据显示，各化合物均能够在人GLP-1/GIP/GCG受体稳转细胞中展现出较高的相对活力。
实施例3：肽化合物对大鼠胰岛瘤RIN-m5F细胞功能活性测试
大鼠胰岛瘤RIN-m5F细胞系源自大鼠胰岛组织，主要表达内源性GLP-1/GIP/GCG受体，其中GLP-1受体最为丰富。本实验测定化合物对RIN-m5F细胞作用产生的cAMP水平。
将冻存的RIN-m5F细胞置于37℃恒温水浴锅中快速解冻复苏，将细胞液转移至10ml HBSS中重悬，室温1000rpm离心5min，弃去上清，用1×HBSS(含0.1％Casein，250μΜIBMX)重悬细胞，调整细胞密度为1.0×10 5/mL。
384孔板中每孔加入10μL细胞悬液。待测化合物溶于1×PBS缓冲液中配制成一定浓度母液，按3倍逐级稀释，共配制12个浓度点的化合物溶液。利用ECHO液体转移系统将100nL配置好的化合物溶液分别加入384孔板对应细胞悬液中，1000rpm旋转摇晃1min使混合均匀，随后室温孵育60min。药物孵育完成后各孔加入10μL试剂盒中的检测试剂，再室温孵育60min。将板置于EnVision多功能酶标仪(PerkinElmer)中测定665/615nm处读数。利用GraphPad Prism 5作图软件制作化合物浓度-效应曲线并计算。
表3.肽化合物的RIN-m5F大鼠胰岛瘤细胞活性
Figure PCTCN2022093577-appb-000060
Figure PCTCN2022093577-appb-000061
Rel.A:Relative Activity，相对活性；NT:Not test，未进行检测；各组均具有至少3次独立的检测。
表中本发明化合物均能够在胰岛瘤细胞RIN-m5F中展现出较高的相对活力。
实施例4：肽化合物对3T3-L1脂肪细胞功能活性测试
小鼠3T3-L1前体脂肪细胞可被诱导分化为成熟脂肪细胞。将3T3-L1细胞按4×10 4/ml的细胞密度直接接种于96孔板中，培养于含10％FCS和1％P/S的DMEM培养基中，置于37℃，5％CO2培养箱中培养。待细胞长满接触后换用含20％FBS，0.5mM IBMX，0.4ug/mL地塞米松，5ug/mL胰岛素的DMEM分化培养基开始诱导3T3-L1脂肪细胞分化，诱导培养5天后换用含20％FBS,4ug/ml胰岛素和10uM罗格列酮的DMEM培养基继续诱导培养3天，然后换用含10％FBS的DMEM培养基继续培养2～3天即可诱导为成熟的3T3-L1脂肪细胞。
诱导分化成熟的3T3-L1脂肪细胞内类似于脂肪组织细胞，可以表达丰富的GIP受体。本实验测定化合物对3T3-L1脂肪细胞作用产生的cAMP水平。将待测化合物溶液按3倍浓度梯度逐级稀释，共设置10个系列浓度溶液，加入诱导成熟的3T3-L1脂肪细胞中，室温孵育60min。药物孵育完成后各孔加入10μL试剂盒中的检测试剂，再室温孵育60min。将板置于EnVision多功能酶标仪(PerkinElmer)中测定665/615nm处读数。利用GraphPad Prism 5作图软件制作化合物浓度-效应曲线并计算。
表4.肽化合物的小鼠3T3-L1脂肪细胞活性
Figure PCTCN2022093577-appb-000062
Figure PCTCN2022093577-appb-000063
Rel.A:Relative Activity，相对活性；NT:Not test，未进行检测；各组均具有至少3次独立的检测。
表中本发明化合物均能够在脂肪细胞3T3-L1中展现出较高的相对活力。
实施例5：肽化合物对人原代肝细胞功能活性测试
本实验测定化合物对人原代肝细胞作用产生的cAMP水平。人原代肝细胞采购自Lonza(细胞货号HUCPG)。
将冻存的人原代肝细胞置于37℃恒温水浴锅中快速解冻复苏，将细胞液转移至10ml HBSS中重悬，室温1000rpm离心5min，弃去上清，用1×HBSS(含0.1％Casein，250μΜIBMX)重悬细胞，调整细胞密度为1.0×10 5/mL。
384孔板中每孔加入10μL细胞悬液。待测化合物溶于1×PBS缓冲液中配制成一定浓度母液，按3倍逐级稀释，共配制12个浓度点的化合物溶液。利用ECHO液体转移系统将100nL配置好的化合物溶液分别加入384孔板对应细胞悬液中，1000rpm旋转摇晃1min使混合均匀，随后室温孵育60min。药物孵育完成后各孔加入10μL试剂盒中的检测试剂，再室温孵育60min。将板置于EnVision多功能酶标仪(PerkinElmer)中测 定665/615nm处读数。利用GraphPad Prism 5作图软件制作化合物浓度-效应曲线并计算。
表5.肽化合物的人原代肝细胞活性
Figure PCTCN2022093577-appb-000064
Figure PCTCN2022093577-appb-000065
Rel.A:Relative Activity，相对活性；NT:Not test，未进行检测；各组均具有至少3次独立的检测。
表中本发明化合物均能够在肝细胞中展现出较高的相对活力。
实施例6：体内药效学
采用高脂饮食诱导肥胖(DIO)的小鼠具有肥胖、血糖升高、胰岛素抵抗及脂质异常等特点，这些都是与人体的相似非常显著的代谢综合征。在C57 BL/6J DIO小鼠中研究本发明化合物对DIO小鼠体重、摄食、血糖和脂质的影响。
5周龄的雄性C57BL/6J小鼠(购自上海斯莱克实验动物公司)饲养于无病原体的洁净环境中(控制温度20-24℃，相对湿度30-70％)，12小时光照/12小时黑暗循环，用正常饲料饲养,每笼4只动物，适应2周。通过喂食高脂饮食(60kcal％热量来自脂肪)诱导小鼠肥胖，高脂饮食饲养16周后，DIO小鼠体重达41～55g，血糖范围8～12mmol/L，根据体重和空腹血糖将DIO小鼠进行随机分组(n＝6)，使得各组动物具有接近的平均体重和血糖。分组后以每笼一只动物进行饲养一周，期间对每只动物采用皮下注射(S.C.)方式施用溶媒(1×PBS，5ml/kg)以使动物预适应操作过程。
动物预适应结束后，根据实验分组皮下注射给予动物溶媒对照、化合物，化合物溶解于1×PBS中，给药剂量为5ml/kg，给药操作在早上9:00开始进行，每三天给药一次(Q3D)，持续15天。在整个试验研究过程中每天给药前测量动物体重和摄食量，通过与相同动物给药前的初始体重和摄食量比较，计算动物体重变化百分比(％)和累积摄食量，评估化合物对体重和摄食量变化的影响。
到达实验终点(Day 15)，测定小鼠体重，然后通过无麻醉尾尖断尾 采血测定动物禁食血糖。采血完成后使用CO 2麻醉动物并处死，然后通过心脏取血，离心分离血浆，血浆用于测定血浆总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、血胰岛素含量。分离肝脏进行匀浆，离心取匀浆上清，用于测定肝脏甘油三酯含量。
所有结果数值以Mean±SEM表示，结果使用GraphPad Prism 5软件的单因素方差分析(One-way ANOVA)及随后与Vehicle对照组比较的Dunnett’s事后检验。差异在p＜0.05水平被认为是具有显著统计学意义的。
表6-1.化合物对DIO小鼠体重变化的影响
Figure PCTCN2022093577-appb-000066
表6-2.化合物对DIO小鼠体重变化的影响
Figure PCTCN2022093577-appb-000067
从表6-1、表6-2的结果可以看出，本发明化合物有明显的减重效果。
表7-1.化合物对DIO小鼠血脂的影响
Figure PCTCN2022093577-appb-000068
表7-2.化合物对DIO小鼠血脂的影响
Figure PCTCN2022093577-appb-000069
Figure PCTCN2022093577-appb-000070
从表7-1、表7-2结果可以看出，本发明化合物有明显的降低血脂疗效。
表8-1. 3nM化合物对DIO小鼠血糖和血胰岛素的影响
Figure PCTCN2022093577-appb-000071
表8-2. 3nM化合物对DIO小鼠血糖和血胰岛素的影响
Figure PCTCN2022093577-appb-000072
从表8-1、表8-2结果可以看出，本发明化合物有明显的降低血糖和血胰岛素的疗效。
表9-1. 3nM化合物对DIO小鼠肝的影响
化合物 剂量(nmol/kg) Liver TG(mg)
Vehicle - 181.5±33.45
3 3 32.2±2.99
10 3 81.6±8.50
20 3 26.7±2.81
24 3 30.7±1.31
表9-2. 3nM化合物对DIO小鼠肝的影响
化合物 剂量(nmol/kg) Liver TG(mg)
Vehicle - 129.8±20.21
19 3 38.0±3.37
21 3 41.4±6.33
40 3 42.1±5.22
从表9-1、表9-2结果可以看出，本发明化合物有明显降低肝脏甘油三酯的疗效。
实施例7：化合物的大鼠药代动力学(PK)研究
7-9周龄雄性SD大鼠(220～250g，3只/组)接受30nmol/kg化合物皮下注射给药，给药后在0.25、2、4、8、12、16、24、48、96、120、144h时间点通过颈静脉采集血液，血液经处理得到血浆样品后采用LC-MS/MS分析样品，使用Phoenix WinNonlin 6.3版本软件(非房室模型)分析血药浓度-时间曲线，计算PK参数和半衰期。
使用上述方法计算的PK参数见表10。
表10化合物的大鼠药代动力学(PK)参数
Figure PCTCN2022093577-appb-000073
表10结果显示，各化合物均表现出延长的药代动力学分布。

Claims (25)

  1. 具有通式(I)的GLP-1/GIP/GCG R三激动剂多肽化合物或其盐或溶剂合物：
    Y-Aib-X3-GT-X6-TSDYSI-X13-LDK-X17-AQ-Aib-AFIE-X25-LLE-X29-X30-PSS-X34-X35-PP-X38-S-R 1(I)，
    其中，
    X3为Q或H；
    X6为F、αMeF、或αMeF(2F)；
    X13为αMeL、F、αMeF、或L；
    X17为ψ；
    X25为Y或F；
    X29为T、S、G、或Aib；
    X30为G、H、R、或Aib；
    X34为G或Aib；
    X35为A、Q、Aib、H；
    X38为Ac3c、P；
    R 1为NH 2或OH，或其药学上可接受的盐和/或酯；
    其中，ψ为侧链被具有下述通式(II)结构修饰的Lys，所述通式(II)为：
    Y-Z(II)，其中
    Y为(AEEAc或Glu) a-(AEEAc或Glu) b-(AEEAc或Glu) c，其中a、b、c各自独立地为0或1，且a、b、c不同时为零，所述Y的羧基端与Lys的侧链的ε-氨基相连；
    Z为-CO-(CH 2) m-R 2，m为6-24之间的整数，R 2选自-COOH。
  2. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，所述通式(I)化合物中，含有至少两个以下位点的特定氨基酸：
    X13为F或αMeF；
    X25为F；
    X29为T或S；
    X30为H、R或Aib；
    X35为Q、Aib或H；
    X38为Ac3c。
  3. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，所述通式(II)为AEEAc-AEEAc-γGlu-CO(CH 2) 18COOH。
  4. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，所述化合物选自：
    Figure PCTCN2022093577-appb-100001
    Figure PCTCN2022093577-appb-100002
    Figure PCTCN2022093577-appb-100003
  5. 根据权利要求4所述的多肽化合物或其盐或溶剂合物，其特征在于，所述化合物进一步选自：
    Figure PCTCN2022093577-appb-100004
    Figure PCTCN2022093577-appb-100005
    Figure PCTCN2022093577-appb-100006
  6. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在GLP-1受体稳转细胞的激动能力上，相较于天然GLP-1，所述化合物对GLP-1受体激动的相对活性至少30％，优选至少60％，更优选至少80％，更进一步优选至少100％。
  7. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在GIP受体稳转细胞的激动能力上，相较于天然GIP，所述化合物对GIP受体激动的相对活性至少100％，更优选至少150％。
  8. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在GCG受体稳转细胞的激动能力上，相较于天然GCG，所述化合物对GCG受体激动的相对活性至少10％，更优选至少30％。
  9. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在胰岛组织代表细胞RIN-m5F上，相较于天然GLP-1(7-37)，所述化合物对RIN-m5F细胞激动的相对活性至少60％，优选至少80％，更优选至少100％。
  10. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在脂肪组织代表细胞3T3-L1上，相较于天然GIP，所述化合物对3T3-L1细胞激动的相对活性至少60％，优选至少100％。
  11. 根据权利要求1所述的多肽化合物或其盐或溶剂合物，其特征在于，在肝脏组织代表细胞人原代肝细胞上，相较于天然GCG，所述化合物对肝细胞激动的相对活性至少60％，优选至少100％。
  12. 一种药物组合物，包括有效量的权利要求1～11任一所述化合物或其盐或溶剂合物，和药学上可接受的辅料、稀释剂、载剂或赋形剂。
  13. 根据权利要求12所述的药物组合物，其特征在于，所述药物组合物为注射剂或冻干粉、片剂、丸剂、锭剂、软胶囊剂、硬胶囊剂、颗粒剂、散剂、溶液剂、混悬剂或糖浆剂；或者所述药物组合物为微囊、微球、纳米粒或脂质体形式。
  14. 根据权利要求12所述的药物组合物，其特征在于，所述药物组合物用于口服给药、吸入给药或肠胃外给药，所述肠胃外给药选自腹膜内、肌内、动脉内、静脉内、皮下或皮内注射给药。
  15. 根据权利要求12所述的药物组合物，其特征在于，所述药物组合物以至少一天给药一次，一周给药一次，两周给药一次或一个月给药一次的频率给药。
  16. 根据权利要求12所述的药物组合物，其特征在于，所述药物组合物与至少一种治疗用活性物质联合使用，所述治疗用活性物质包括抗糖尿病活性剂、GIP受体激动剂、GCG受体激动剂或拮抗剂、GLP-1/GIP受体激动剂、GLP-1/GCG受体激动剂、GIP/GCG受体激动剂、FGF-21及其类似物、缩胆囊素B(CCKB)及其类似物、PYY(3-36)及其类似物、瘦素及其类似物、降钙素及其类似物、调血脂活性药物、PPAR-α、β、δ激动剂或调节剂、抗血小板聚集活性剂、PCSK9抑制剂、脂肪酶抑制剂、抗肝纤维化或肝硬化活性剂、或抗炎活性剂。
  17. 根据权利要求16所述的药物组合物，其特征在于，所述抗糖尿病活性剂包括胰岛素及其类似物、双胍类、磺脲类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、DPP-4抑制剂、SGLT2抑制剂、双重SGLT1/SGLT2抑制剂、GLP-1受体激动剂、或胰淀素及其类似物。
  18. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备促进胰岛素分泌，降低血糖的药物中的应用。
  19. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备抑制摄食，延缓胃排空，增加能量消耗，降低体重的药物中的应用。
  20. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备减少胰岛β-细胞凋亡，增加胰岛β-细胞数量，改善胰岛细胞功能的药物中的应用。
  21. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备改善血脂，减少肝脏脂肪累积，抑制肝脏 炎症发展，预防及治疗非酒精性脂肪肝病的药物中的应用。
  22. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备促进大脑神经元生长、清除神经毒性物质，抑制炎症发展，起到神经保护作用的药物中的应用。
  23. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备用于预防及/或治疗代谢紊乱疾病及其相关并发症的药物中的应用，优选用于治疗糖尿病、肥胖症或非酒精性脂肪肝病的药物。
  24. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备用于治疗血脂代谢紊乱及其相关疾病、神经退行性疾病的药物中的应用，所述神经退行性疾病包括帕金森病、阿尔茨海默症。
  25. 权利要求1～11中任一所述的化合物或其盐或溶剂合物或12～17任一所述的药物组合物在制备用于治疗内分泌疾病、代谢紊乱、肾病等原因相关的骨骼疾病的药物中的应用，所述骨骼疾病包括骨质疏松症、骨关节炎。
PCT/CN2022/093577 2021-05-26 2022-05-18 多激动剂及其应用 Ceased WO2022247701A1 (zh)

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CN116832141B (zh) * 2023-06-06 2024-02-09 诺博泰科(成都)生物科技有限公司 一种用于治疗糖尿病的glp-1、gip和gcg受体三激动多肽化合物
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CN119241684A (zh) * 2024-11-22 2025-01-03 徐州医科大学 一种肠促胰岛素类似物及其用途

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