WO2021244391A1 - Small-molecule glp-1r agonist and use thereof - Google Patents

Small-molecule glp-1r agonist and use thereof Download PDF

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WO2021244391A1
WO2021244391A1 PCT/CN2021/096380 CN2021096380W WO2021244391A1 WO 2021244391 A1 WO2021244391 A1 WO 2021244391A1 CN 2021096380 W CN2021096380 W CN 2021096380W WO 2021244391 A1 WO2021244391 A1 WO 2021244391A1
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glp
compound
agonist
small molecule
glucose
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PCT/CN2021/096380
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French (fr)
Chinese (zh)
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章毅
张敏
杨晓华
刘云峰
刘涛
崔丽娟
薛欢
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山西医科大学
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D237/00Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/28Cinnolines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/08Antiepileptics; Anticonvulsants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/08Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D215/00Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20Oxygen atoms
    • C07D215/22Oxygen atoms attached in position 2 or 4
    • C07D215/233Oxygen atoms attached in position 2 or 4 only one oxygen atom which is attached in position 4
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D265/00Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/021,2-Oxazines; Hydrogenated 1,2-oxazines
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07Optical isomers

Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to a GLP-1R agonist and its application.
  • GLP-1(7-36) amide is the main active form of GLP-1. After GLP-1(7-36) amide enters the blood circulation, the dipeptidyl peptidase IV (DPP-IV) on the blood and cell membranes quickly It is cleaved into inactive GLP-1(9-36), and its half-life is only 1-2min, so it is not suitable for long-term treatment of T2DM. Further, scientific research has carried out corresponding transformations and modifications to the structure of GLP-1 to increase its half-life and thereby extend the biological effects in vivo. However, the long-acting GLP-1 analogs currently in clinical use, such as liraglutide, exenatide, etc., are all polypeptides. Frequent multiple injections and administration lead to poor patient compliance. Therefore, small molecule GLP-1R The development of agonists will start from the purpose of improving patient compliance, medication convenience and reducing drug side effects, and has broad clinical market prospects.
  • DPP-IV dipeptidyl peptidase
  • Diabetes is a chronic disease characterized by high blood sugar caused by insufficient human insulin secretion (relative or absolute) or impaired insulin action.
  • IDF International Diabetes Federation
  • Type 2 diabetes is the most common type of diabetes, accounting for approximately 90% of the total number of diabetic patients in the world. So far, the traditional drugs used clinically to treat T2DM, such as insulin and sulfonylurea drugs, can effectively reduce hyperglycemia, but after controlling the blood sugar within the normal range, they can still continue to play a hypoglycemic effect, which will lead to T2DM The patient is at risk of hypoglycemia. Therefore, there is an urgent need to develop a new therapeutic drug, which can not only effectively reduce the hyperglycemia of diabetic patients, but also reduce the risk of hypoglycemia.
  • T2DM Type 2 diabetes
  • Glucagon-like peptide-1 (GLP-1) is produced by prohormone converting enzyme PC1/3 after translation of the proglucagon gene expression product, and is secreted by gastrointestinal endocrine L cells , After the intake of glucose and other nutrients, it plays a hypoglycemic effect.
  • GLP-1 stimulates insulin secretion in a glucose concentration-dependent manner through the GLP-1 receptor (GLP-1R), which is the biggest advantage of GLP-1 over other hypoglycemic drugs.
  • GLP-1R GLP-1 receptor
  • GLP-1 can also increase insulin synthesis in the body, promote the proliferation and differentiation of ⁇ cells, and inhibit ⁇ cell apoptosis. These properties may help reverse or even prevent diabetes.
  • One object of the present invention is to provide a compound of general formula I, which is a small molecule GLP-1R agonist; another object of the present invention is to provide a compound of general formula I for preparing and treating diabetes Use in medicine.
  • the present invention provides a compound of general formula I, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof,
  • a and B are independently selected from C, N or O, between B and adjacent carbon atoms Represents a single bond or a double bond.
  • R 1 and R 2 are independently selected from: -H, -OH, -CF 3 , halogen, C 1-10 straight chain alkyl, C 3-10 branched chain alkyl, C 3-8 cycloalkyl, or R 1.
  • the carbon atoms between R 2 and R 1 and R 2 form a C 3-8 cycloalkyl group, an aromatic five-membered ring group or an aromatic six-membered ring group.
  • R 3 is selected from -H, C 1-5 straight chain alkyl, and C 3-8 cycloalkyl.
  • R 4 and R 5 are independently selected from -H, -OH, halogen, -CN, C 1-5 straight chain/branched chain alkyl, -N (C 0-10 alkyl) (C 0-10 alkyl) , -OC 0-10 alkyl, C 3-6 cycloalkyl.
  • said A is N
  • B is C, N or O
  • said R 1 and R 2 are independently selected from : -OH, -CH 3 , -Cl, -C 2 H 5 , or R 1 , R 2 and the carbon atoms between R 1 and R 2 form an aromatic six-membered ring group
  • R 3 is selected from -CH 3 , -C 2 H 5
  • R 4 and R 5 are independently selected from -H, -OH, halogen, and -CH 3 .
  • the compound of formula (I) has the following three-dimensional configuration:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of general formula I or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, and deuterated compound thereof, It also includes pharmaceutically acceptable excipients selected from the group consisting of carriers, diluents, binders, lubricants, and wetting agents.
  • the pharmaceutical composition contains a therapeutically effective amount of a compound of general formula I.
  • the pharmaceutical composition is suitable for gastrointestinal administration or parenteral administration, but the GLP-1R agonist provided by the present invention is a small molecule compound, which can be administered orally to reflect the relative advantages of macromolecular polypeptides.
  • the drug is only administered via intravenous and subcutaneous injections, which presents advantages.
  • the pharmaceutical composition can be prepared into pharmaceutical preparations in the following forms: injections, syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, creams, ointments, gels, emulsions and the like.
  • the present invention provides a compound of general formula I or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds for the preparation of small molecule GLP-1R agonists.
  • the small molecule GLP-1R agonist provided by the present invention can promote insulin secretion and reduce hyperglycemia. At the same time, due to the glucose concentration-dependent characteristic of promoting insulin secretion, it can also reduce the occurrence of hypoglycemia.
  • the present invention provides a compound of general formula I or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, and deuterated compound for preparing a medicine for preventing and/or treating diabetes. use.
  • the diabetes is type 2 diabetes.
  • GLP-1R agonists that have been put into clinical use are peptide drugs, such as liraglutide and exenatide. Because peptide macromolecular drugs can only be administered by injection, but frequent multiple injections have led to poor patient compliance with medications, and because peptide drugs exhibit instability both in vivo and in vitro, they are administered in the form of injection It restricts its clinical application.
  • the GLP-1R agonist provided by the present invention is a small molecule drug, which can be administered through the gastrointestinal tract, and can significantly improve the medication compliance and medication convenience of patients.
  • the present invention provides the use of a compound of general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds in the preparation of Kv channel inhibitors.
  • the present invention provides a compound of general formula I or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds in the preparation of drugs for the treatment of diseases related to Kv channels the use of.
  • diseases related to Kv channels also include: epilepsy, cerebral ischemic diseases, and neurodegenerative diseases.
  • C 0-10 alkyl refers to H
  • C 0-10 alkyl includes H, linear or branched alkyl with 1-10 carbon atoms, The specific number of carbon atoms can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • branched alkyl groups examples include, but are not limited to, isopropyl, isobutyl, tert-butyl, and isopentyl.
  • C 3-8 cycloalkyl in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl base.
  • halogen in the present invention includes fluorine, chlorine, bromine and iodine.
  • Figure 1 The effect of S6 on rat pancreatic islet insulin secretion under different glucose conditions.
  • Figure 2 A shows the effect of S6 on [Ca 2+ ]i in the presence of 8.3 mM glucose in ⁇ cells; B shows the average value of F-F0 in response to S6.
  • Figure 3 A shows the recording current curve with or without S6; B shows the current-voltage curve of the voltage-gated Ca 2+ channel in the presence or absence of S6; C shows the average current density recorded at 0 mV .
  • Figure 4 A represents the cell current recorded with or without S6; B represents the current-voltage curve of the Kv channel; C represents the average current density of the Kv channel recorded at 80mV: D and E represent the absence or presence of S6 Sexual action potential waveform; F represents the average action potential duration (APD).
  • Figure 5 A shows the effect of 20mM tetraethylammonium chloride (TEA) on the [Ca 2+ ]i induced by S6 in the case of 8.3mM glucose; B shows the FF response to S6 in the presence or absence of 8.3mM TEA 0 mean value; C represents the effect of S6 on insulin secretion in the absence or presence of TEA with 8.3mM glucose.
  • TEA tetraethylammonium chloride
  • Figure 6 A shows the effect of L-type Ca 2+ channel antagonist Azelnidipine on S6-induced [Ca 2+ ]i in the presence of 8.3mM glucose; B shows the average value of FF 0 in response to S6 in the presence or absence of Azelnidipine ; C indicates that S6 induces a change in [Ca 2+ ]i in the absence of extracellular calcium; D indicates the average value of FF 0 in response to S6 in the absence of extracellular calcium.
  • Figure 7 A shows the SDS-PAGE result of cell-free protein expression
  • B shows the verification result of Western blot TOB cell-free protein expression of GLP-1R protein.
  • Figure 8 A shows the use of CETSA to detect changes in the expression of GLP-1R with increasing temperature after DMSO and S6 (10 ⁇ M) are used to treat cell lysates.
  • the quantitative data is shown in B.
  • Figure 10 A shows the effect of Exendin (9-39) on S6-induced [Ca 2+ ]i.
  • the change of [Ca 2+ ]i after different treatments is plotted with the ratio of F340/F380, and B is The average value of FF 0 for response S6 in the presence or absence of exendin (9-39).
  • Figure 11 A shows the current curve recorded by S6 with or without Exendin (9-39); B shows the current-voltage curve of the Kv channel; C shows the average current density of the Kv channel at 80 mV.
  • S4 Mix and add magnesium stearate to the granules prepared by S2, and control the hardness of the tablet machine within 4-5kgf method, and compress to obtain 1000 tablets containing compound S6.
  • step S2 Add a 3:1 mixture of sucrose:dextrin to the drug-lactose mixture prepared in step S1 (150g through an 80-mesh sieve, mix well, make soft materials, extrude, granulate, dry, sizing, and pass through a 12-mesh sieve , After sub-packaging, granules containing compound S6 are obtained.
  • step S3 Add 30 mL of an ethanol solution of povidone K30 with a mass concentration of 5% to the mixed fine powder obtained in step S2 to make a soft material, and pass through a 20-mesh screen to make wet granules;
  • step S4 After drying the wet granules obtained in step S3 at 50°C for 6 hours, they are passed through an 18-mesh sieve for granulation;
  • S5 Determine the content of compound S6 in the granules, calculate the filling amount, pack it, and package after inspection to obtain 1000 hard capsules containing compound S6.
  • the present invention will test the preferred compound S6 of the present invention according to the following effect examples to verify that compound S6 promotes insulin secretion in rats and its electrophysiological mechanism in a glucose concentration-dependent manner, and verify that compound S6 promotes insulin in rats by activating GLP-1R secretion.
  • the experimental animal used in the present invention is a male Wistar rat with a weight of 180-250g, purchased from the Experimental Animal Center of Shanxi Provincial People's Hospital, raised at a temperature of 20-22°C, and equipped with standard rodent food and drinking water. All operating procedures are in compliance with the management and use guidelines for laboratory animals of Shanxi Medical University.
  • the isolation and culture of rat pancreatic islet tissues and the isolation and culture of rat pancreatic islet ⁇ cells are routine experimental operations by those skilled in the art.
  • the data processing involved in the present invention uses SigmaPlot 12.5 and Igor 6.1 to process experimental data, and is represented by Mean ⁇ SEM.
  • G represents the glucose concentration in mmol/L, for example, 2.8G represents a glucose solution with a concentration of 2.8 mmol/L.
  • Collagenase P comes from Roche Pharmaceuticals, Switzerland; DispaseII comes from Amresco, USA; Hanks buffer (HBSS) comes from Boster Bioengineering Co., Ltd.; Histopaque-1077 comes from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Albumin bovine serum (BSA) ) Comes from Beijing Soleibao Technology Co., Ltd.; RPMI 1640 medium comes from Hyclone, USA; TEA comes from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Fura 2-AM comes from Shanghai Dongren Chemical Technology Co., Ltd.; Fetal Bovine Serum (FBS) comes from American Thermo Fisher Technology; glucose comes from Beijing Soleibao Technology Co., Ltd.; analytical pure NaCl comes from BBI Life Science Co., Ltd.; analytical pure KCl comes from BBI Life Science Co., Ltd.; NaHCO 3 comes from BBI Life Science Co., Ltd.; HEPES comes from Beijing Soleib
  • the luxurious ultra-clean workbench comes from Shanghai Zhicheng Analytical Instrument Manufacturing Company; the constant temperature oscillator comes from Changzhou Guohua Electric Co., Ltd.; the automatic snowflake ice maker comes from Changshu Xueke Electric Co., Ltd.; the pressure steam sterilizer comes from Taiyuan Xingyu Science and Education Equipment Manufacturing Co., Ltd.; cell incubator from Eppendorf, Germany; inverted microscope from Olympus IX51, Japan; PP-380 vertical electrode drawing instrument from Narishige, Japan; MF-200 microelectrode polishing instrument from Japan It is from Narishige Company in Japan; the medical refrigerator is from Aucma Co., Ltd.; the digital speed type peristaltic pump is from Baoding Chuangrui Pump Co., Ltd.; the acidity meter is from Ohaus Instrument Co., Ltd.; the VORTEX vortex mixer is from Haimen City Qilin Bell Instrument Co., Ltd.
  • Figure A shows the effect of S6 on [Ca 2+ ]i in the presence of 8.3mM glucose in ⁇ cells.
  • the change in [Ca 2+ ]i is The ratio of F340/F380 is drawn;
  • B represents the average value of FF 0 in response to S6,
  • F represents the average value of 15s before and after the peak of different treatments, and
  • F 0 represents the average value of 15s before and after the minimum value of 2.8mM glucose concentration.
  • n 13, ***p ⁇ 0.001.
  • the intracellular Ca 2+ level is significantly increased; on this basis, by continuing to add S6, the intracellular Ca 2+ concentration can still increase further. Therefore, it can be inferred that the insulin secretion effect of S6 is achieved by increasing the intracellular Ca 2+ concentration, and the glucose concentration of 8.3G significantly promotes the increase of intracellular calcium ion concentration.
  • Effect Example 4 S6 inhibits the voltage-dependent potassium (Kv) channel of ⁇ cells and prolongs the action potential duration of ⁇ cells
  • the cell In the voltage clamp mode, the cell is clamped at -70mV for fast capacitance compensation; after the membrane is broken in the whole cell mode, slow capacitance compensation is performed, and the cell membrane capacitance is adjusted to ⁇ 7M ⁇ .
  • a current stimulation of 150pA and 4ms was given in the current clamp mode. Calculate the time from the start of cell depolarization to the time it takes for the cell to repolarize to 10 mV above the resting potential.
  • S6 and TEA significantly enhanced the secretion of insulin under the condition of 8.3mM glucose, respectively.
  • TEA and S6 were given at the same time, the ability of insulin secretion was further enhanced.
  • the calcium ion imaging experiments in Figure 6 A and B show that under the condition of 8.3G glucose, the intracellular calcium level increased significantly; then we found that when S6 and Azelnidipine were administered at the same time, [Ca 2+ ]i was still significantly increased.
  • CETSA Cellular Thermal Shift Assay
  • Tris is from Beijing Soleibao Technology Co., Ltd.
  • GLP-1R Antibody Rabbit Polyclonal is from Wuhan Sanying Biotechnology Co., Ltd.
  • HRP-conjugated Affinipure Rabbit&Anti-Goat IgG (H+L) is from Wuhan Sanying Biotechnology Co., Ltd.
  • SDS-PAGE gel preparation kit comes from Beijing Soleibao Technology Co., Ltd.
  • Glycine comes from Beijing Soleibao Technology Co., Ltd.
  • SDS comes from Beijing Soleibao Technology Co., Ltd.
  • Tween-20 comes from Beijing Soleibao Technology Co., Ltd.
  • the low-temperature high-speed centrifuge comes from Eppendorf, Germany; the multi-function microplate reader comes from BioTek, USA; the Western blot vertical electrophoresis system comes from BioTek, USA; The Western blot semi-dry transfer membrane system comes from BioTek, USA; Gel The imaging system is from BioTek, USA; the calcium imaging LAMBDA10-B system is from Beijing MDE; the PCR machine is from Bio-rad, the USA; the constant temperature oscillating incubator is from Taicang Huamei Biochemical Instrument Co., Ltd.; the constant temperature magnetic stirrer is from Taicang Huamei Biochemical Instrument Co., Ltd.
  • rat GLP-1R vector The sequence fragment of rat GLP-1R was obtained by PCR amplification technology, and the GLP-1R rat gene fragment was recovered from agarose gel, and the target gene GLP-1R was cloned into the vector pEX-3, pEX-3 was digested with EcoRI and BamHI, and the vector pEX-3 was recovered by electrophoresis; use Entry One Step Cloning Kit, recombinantly clone the amplified fragments into pEX-3 vector; transform the recombination ligation product into competent cells, pick the cloned colonies, extract a small amount of plasmids, use EcoRI and BamHI double enzyme digestion to identify, electrophoresis , Pick the positive clones; finally, after the recombinant plasmid is sequenced and verified, a large amount of extraction is carried out, and an appropriate amount of the bacterial solution corresponding to the
  • Experimental steps 1) Take out the total protein solution containing GLP-1R expressed by TOB expression system from the -80°C refrigerator, place it on ice, and take out the volume of total protein required for the experiment. The experiment is divided into 2 groups, and the experimental intervention group is added The final concentration was 10 ⁇ M S6. The control group was added with the same volume of DMSO and incubated in a 37°C incubator for 45 minutes.
  • the experimental intervention group and the control group were divided into 5 groups, and then heated by RT-PCR instrument at different temperatures (47 ⁇ 67°C) for 3min, cooled at room temperature for 3min, 4°C, 20,000g 20min centrifugation, and the Aspirate it to a 200 ⁇ L Ep tube, add 5 ⁇ Loading buffer, heat at 95°C for 5 minutes, and store at -80°C in a refrigerator.
  • the plasmids were tested as follows: 1) Gene sequence detection using sequence alignment: the sequencing result was consistent with the target sequence; 2) Vector sequence detection using sequence alignment: the flanking 20bp sequence was consistent with the vector; 3) using agar Carbohydrate electrophoresis method to detect the fragment size: the plasmid has no contamination band, the fragment size is correct; 4) The concentration is detected by ultraviolet spectrophotometry: OD260/280 is 1.8-2.0.
  • GLP-1R is a specific target of S6
  • S6 can play a role in promoting insulin secretion in rats.
  • the present invention also verified that S6 is an effective small molecule GLP-1R agonist.
  • CETSA technology uses CETSA technology to observe the relationship between GLP-1R and S6, detect protein samples through Western-Blot, and observe the changes in GLP-1R expression.
  • Figure A shows the CETSA of cell lysates treated with DMSO and S6 (10 ⁇ M). Western blotting was used to detect the interaction between S6 and GLP-1R.
  • Figure 11 shows the cell current curve recorded in the presence or absence of Exendin (9-39);
  • Figure B shows the current-voltage curve of the Kv channel;
  • S6 significantly reduced the Kv channel current, and after using Exendin (9-39), the effect of S6 on the Kv channel current was reversed.
  • GLP-1R belongs to the B subclass of G protein-coupled receptors. When GLP-1 and its analogues bind to GLP-1R, they can trigger a series of signal responses and ultimately regulate the secretion of insulin. In the insulin secretion experiment, we observed that in the presence of the GLP-1R blocker Exendin (9-39), S6 attenuated the effect of insulin secretion in rats, indicating that S6 can promote insulin secretion through GLP-1R, again confirming S6 acts on GLP-1R.
  • mice Male mice were randomly divided into 5 groups with 10 mice in each group. Compound S6 was dissolved in physiological saline for oral administration. The dosages of groups 1-6 were 1.5g/kg, 3.0g/kg, 5.0g/kg, 7.5g/kg, 15.0g/kg. After the administration, the mice were reared according to conventional standards. After 10 days, the death of each group was recorded, and SPSS13.0 software was used to calculate the median lethal dose (LD50) of the drug. The results of the acute toxicity test of the drug are shown in the following table. The results show that the LD50 value of the compound provided by the present invention is 6.2 g/kg, the 95% confidence limit is 4.3-7.9 g/kg, and the toxicity is low.
  • LD50 median lethal dose

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Abstract

Provided in the present invention is a small-molecule GLP-1R agonist, which is a compound of general formula (I), or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate and deuterated compound thereof. The compound is a small-molecule GLP-1R agonist, and can promote insulin secretion, thus can reduce hyperglycemia; in addition, the compound can also reduce the occurrence of hypoglycemia due to the fact that the compound has a glucose concentration dependent characteristic when promoting insulin secretion. Furthermore, the compound of the general formula (I) provided in the present invention can be administrated orally when same is a small-molecule GLP-1R agonist, can improve the medication compliance and medication convenience of patients, and has wide prospects in the clinical market.

Description

一种小分子GLP-1R激动剂及其应用A small molecule GLP-1R agonist and its application 技术领域Technical field
本发明属于医药技术领域,具体涉及一种GLP-1R激动剂及其应用。The invention belongs to the technical field of medicine, and specifically relates to a GLP-1R agonist and its application.
背景技术Background technique
GLP-1(7-36)酰胺作为GLP-1的主要活性形式,GLP-1(7-36)酰胺进入血液循环后,血液和细胞膜上的二肽基肽酶IV(DPP-IV)迅速将其裂解为无活性的GLP-1(9-36),其半衰期仅1-2min,因此不适用于T2DM的长期治疗。进一步的,科学研究已经对GLP-1的结构进行相应的改造和修饰,以增加其半衰期进而延长体内生物学效应。然而,目前临床上投入使用的长效GLP-1类似物如利拉鲁肽、艾塞那肽等均为多肽,频繁多次注射给药导致患者的依从性较差,因此小分子GLP-1R激动剂的开发将从提高患者的依从性、用药方便性以及降低药物副作用的目的出发,具有广阔的临床市场前景。GLP-1(7-36) amide is the main active form of GLP-1. After GLP-1(7-36) amide enters the blood circulation, the dipeptidyl peptidase IV (DPP-IV) on the blood and cell membranes quickly It is cleaved into inactive GLP-1(9-36), and its half-life is only 1-2min, so it is not suitable for long-term treatment of T2DM. Further, scientific research has carried out corresponding transformations and modifications to the structure of GLP-1 to increase its half-life and thereby extend the biological effects in vivo. However, the long-acting GLP-1 analogs currently in clinical use, such as liraglutide, exenatide, etc., are all polypeptides. Frequent multiple injections and administration lead to poor patient compliance. Therefore, small molecule GLP-1R The development of agonists will start from the purpose of improving patient compliance, medication convenience and reducing drug side effects, and has broad clinical market prospects.
糖尿病是由于人体胰岛素分泌(相对或绝对)不足或者胰岛素作用障碍导致的以高血糖为特征的慢性疾病。根据国际糖尿病-联盟(International Diabetes Federation,IDF)最新颁布的第九版世界糖尿病地图显示,2019年全球约有4.63亿成人(20-79岁)患糖尿病,预计到2030年糖尿病患病人数将达到5.78亿。若按照此趋势持续下去,2045年全球将有7亿糖尿病患者。因此,糖尿病已成为21世纪全球范围内面临的最为严峻的社会健康问题之一。Diabetes is a chronic disease characterized by high blood sugar caused by insufficient human insulin secretion (relative or absolute) or impaired insulin action. According to the latest ninth edition of the World Diabetes Map issued by the International Diabetes Federation (IDF), approximately 463 million adults (20-79 years old) worldwide will suffer from diabetes in 2019, and the number of diabetic patients is expected to reach by 2030 578 million. If this trend continues, there will be 700 million diabetic patients worldwide in 2045. Therefore, diabetes has become one of the most severe social health problems facing the world in the 21st century.
2型糖尿病(T2DM)是最常见的糖尿病类型,约占全球糖尿病患者总数的90%。迄今为止,临床上用于治疗T2DM的传统药物,如胰岛素、磺酰脲类药物,虽然能够有效降低高血糖,但将血糖控制在正常范围后,仍能继续发挥降糖作用,这会导致T2DM患者出现低血糖的风险。因此,迫切需要开发一种新的治疗药物,所述药物不仅能有效降低糖尿病患者的高血糖,同时还能降低出现低血糖的风险。 Type 2 diabetes (T2DM) is the most common type of diabetes, accounting for approximately 90% of the total number of diabetic patients in the world. So far, the traditional drugs used clinically to treat T2DM, such as insulin and sulfonylurea drugs, can effectively reduce hyperglycemia, but after controlling the blood sugar within the normal range, they can still continue to play a hypoglycemic effect, which will lead to T2DM The patient is at risk of hypoglycemia. Therefore, there is an urgent need to develop a new therapeutic drug, which can not only effectively reduce the hyperglycemia of diabetic patients, but also reduce the risk of hypoglycemia.
胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是激素原转化酶PC1/3对胰高血糖素原基因表达产物翻译后切割而成,通过胃肠内分泌L细胞分泌,在葡萄糖和其他营养物质的摄取后发挥降糖作用。研究报道GLP-1通过GLP-1受体(GLP-1R)以葡萄糖浓度依赖性的刺激胰岛素分泌,这是GLP-1相对于其他降糖药物的最大优点。除此之外,GLP-1还可增加体内胰岛素合成,促进β细胞增值分化,抑制β细胞凋亡,这些特性可能有助于逆转甚至预防糖尿病。Glucagon-like peptide-1 (GLP-1) is produced by prohormone converting enzyme PC1/3 after translation of the proglucagon gene expression product, and is secreted by gastrointestinal endocrine L cells , After the intake of glucose and other nutrients, it plays a hypoglycemic effect. Studies have reported that GLP-1 stimulates insulin secretion in a glucose concentration-dependent manner through the GLP-1 receptor (GLP-1R), which is the biggest advantage of GLP-1 over other hypoglycemic drugs. In addition, GLP-1 can also increase insulin synthesis in the body, promote the proliferation and differentiation of β cells, and inhibit β cell apoptosis. These properties may help reverse or even prevent diabetes.
发明内容Summary of the invention
本发明的一个目的是提供一种通式为Ⅰ的化合物,所述化合物为一种小分子GLP-1R激动剂;本发明的另一个目的是提供一种通式为Ⅰ的化合物在制备治疗糖尿病药物中的用途。One object of the present invention is to provide a compound of general formula I, which is a small molecule GLP-1R agonist; another object of the present invention is to provide a compound of general formula I for preparing and treating diabetes Use in medicine.
第一方面,本发明提供一种通式为Ⅰ的化合物,或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物,In the first aspect, the present invention provides a compound of general formula I, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, or deuterated compound thereof,
Figure PCTCN2021096380-appb-000001
Figure PCTCN2021096380-appb-000001
其中,A和B独立的选自C、N或O,B与相邻碳原子之间的
Figure PCTCN2021096380-appb-000002
表示单键或双键。
Among them, A and B are independently selected from C, N or O, between B and adjacent carbon atoms
Figure PCTCN2021096380-appb-000002
Represents a single bond or a double bond.
R 1、R 2独立的选自:-H、-OH、-CF 3、卤素、C 1-10直链烷基、C 3-10支链烷基、C 3-8环烷基,或R 1、R 2与R 1和R 2之间的碳原子形成C 3-8环烷基、芳香五元环基团或芳香六元环基团。 R 1 and R 2 are independently selected from: -H, -OH, -CF 3 , halogen, C 1-10 straight chain alkyl, C 3-10 branched chain alkyl, C 3-8 cycloalkyl, or R 1. The carbon atoms between R 2 and R 1 and R 2 form a C 3-8 cycloalkyl group, an aromatic five-membered ring group or an aromatic six-membered ring group.
R 3选自-H、C 1-5直链烷基、C 3-8环烷基。 R 3 is selected from -H, C 1-5 straight chain alkyl, and C 3-8 cycloalkyl.
R 4、R 5独立的选自-H、-OH、卤素、-CN、C 1-5直链/支链烷基、-N(C 0-10烷基)(C 0-10烷基)、-OC 0-10烷基、C 3-6环烷基。 R 4 and R 5 are independently selected from -H, -OH, halogen, -CN, C 1-5 straight chain/branched chain alkyl, -N (C 0-10 alkyl) (C 0-10 alkyl) , -OC 0-10 alkyl, C 3-6 cycloalkyl.
优选的,所述A为N,B为C、N或O,且当B为O时,B与相邻的碳原子之间是碳氧单键;所述R 1、R 2独立的选自:-OH、-CH 3、-Cl、-C 2H 5,或R 1、R 2与R 1和R 2之间的碳原子形成芳香六元环基团;R 3选自-CH 3、-C 2H 5;R 4、R 5独立的选自-H、-OH、卤素、-CH 3Preferably, said A is N, B is C, N or O, and when B is O, there is a carbon-oxygen single bond between B and the adjacent carbon atom; said R 1 and R 2 are independently selected from : -OH, -CH 3 , -Cl, -C 2 H 5 , or R 1 , R 2 and the carbon atoms between R 1 and R 2 form an aromatic six-membered ring group; R 3 is selected from -CH 3 , -C 2 H 5 ; R 4 and R 5 are independently selected from -H, -OH, halogen, and -CH 3 .
优选地,所述式(Ⅰ)化合物具有如下立体构型:Preferably, the compound of formula (I) has the following three-dimensional configuration:
Figure PCTCN2021096380-appb-000003
Figure PCTCN2021096380-appb-000003
第二方面,本发明提供一种药物组合物,所述药物组合物包含通式Ⅰ的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物,还包括药剂学上可接受的辅料,所述辅料选自:载体、稀释剂、粘合剂、润滑剂、润湿剂。In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of general formula I or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, and deuterated compound thereof, It also includes pharmaceutically acceptable excipients selected from the group consisting of carriers, diluents, binders, lubricants, and wetting agents.
优选的,所述药物组合物包含治疗有效量的通式Ⅰ的化合物。Preferably, the pharmaceutical composition contains a therapeutically effective amount of a compound of general formula I.
所述药物组合物适于胃肠给药或非胃肠给药,但本发明提供的GLP-1R激动剂为小分子化合物,可以通过口服给药的方式,体现出相对于大分子多肽类的药物仅通过静脉内和皮下注射给药体现出优势。The pharmaceutical composition is suitable for gastrointestinal administration or parenteral administration, but the GLP-1R agonist provided by the present invention is a small molecule compound, which can be administered orally to reflect the relative advantages of macromolecular polypeptides. The drug is only administered via intravenous and subcutaneous injections, which presents advantages.
所述药物组合物可以制备成以下形式的药物制剂:针剂、糖浆剂、酏剂、悬浮剂、 粉剂、颗粒剂、片剂、胶囊、锭剂、霜剂、膏剂、凝胶剂、乳剂等。The pharmaceutical composition can be prepared into pharmaceutical preparations in the following forms: injections, syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, creams, ointments, gels, emulsions and the like.
第三方面,本发明提供一种通式Ⅰ的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物在制备小分子GLP-1R激动剂用途。In the third aspect, the present invention provides a compound of general formula I or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds for the preparation of small molecule GLP-1R agonists.
本发明提供的小分子GLP-1R激动剂能促进胰岛素分泌,降低高血糖,同时由于促进胰岛素分泌具有葡萄糖浓度依赖特性,还能减少低血糖现象的发生。The small molecule GLP-1R agonist provided by the present invention can promote insulin secretion and reduce hyperglycemia. At the same time, due to the glucose concentration-dependent characteristic of promoting insulin secretion, it can also reduce the occurrence of hypoglycemia.
第四方面,本发明提供一种通式Ⅰ的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物在制备预防和/或治疗糖尿病药物中的用途。In a fourth aspect, the present invention provides a compound of general formula I or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, and deuterated compound for preparing a medicine for preventing and/or treating diabetes. use.
优选的,所述糖尿病为二型糖尿病。Preferably, the diabetes is type 2 diabetes.
目前临床上已投入使用的GLP-1R激动剂大多数是多肽类药物,如利拉鲁肽、艾塞那肽等。由于多肽类大分子药物只能通过注射途径给药,但是频繁多次注射给药导致患者用药依从性较差,而且由于肽类药物在体内外均表现出不稳定性,以注射给药的形式制约着其临床应用。本发明提供的GLP-1R激动剂属于小分子药物,可通过胃肠给药,能显著提高患者用药依从性、用药方便性。At present, most of the GLP-1R agonists that have been put into clinical use are peptide drugs, such as liraglutide and exenatide. Because peptide macromolecular drugs can only be administered by injection, but frequent multiple injections have led to poor patient compliance with medications, and because peptide drugs exhibit instability both in vivo and in vitro, they are administered in the form of injection It restricts its clinical application. The GLP-1R agonist provided by the present invention is a small molecule drug, which can be administered through the gastrointestinal tract, and can significantly improve the medication compliance and medication convenience of patients.
第五方面,本发明提供一种通式Ⅰ的化合物及其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物在制备Kv通道抑制剂中的用途。In the fifth aspect, the present invention provides the use of a compound of general formula I and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds in the preparation of Kv channel inhibitors.
第六方面,本发明提供一种通式Ⅰ的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物在制备治疗与Kv通道相关疾病的药物中的用途。In the sixth aspect, the present invention provides a compound of general formula I or its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds in the preparation of drugs for the treatment of diseases related to Kv channels the use of.
除了本发明前述的糖尿病,与Kv通道相关的疾病还包括:癫痫、脑缺血性疾病、神经退行性疾病。In addition to the aforementioned diabetes in the present invention, diseases related to Kv channels also include: epilepsy, cerebral ischemic diseases, and neurodegenerative diseases.
本发明中所述的术语C 0-10烷基,C 0烷基是指H,因此,C 0-10烷基包括H、碳原子为1-10的直链烷基或支链烷基,具体碳原子数可以为1,2,3,4,5,6,7,8,9,10。 In the present invention, the term C 0-10 alkyl, C 0 alkyl refers to H, therefore, C 0-10 alkyl includes H, linear or branched alkyl with 1-10 carbon atoms, The specific number of carbon atoms can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
可以举出的支链烷基的例子包括但不限于异丙基、异丁基、叔丁基、异戊基。Examples of branched alkyl groups that may be cited include, but are not limited to, isopropyl, isobutyl, tert-butyl, and isopentyl.
本发明中所述的术语C 3-8环烷基,包括C 3环烷基、C 4环烷基、C 5环烷基、C 6环烷基、C 7环烷基、C 8环烷基。 The term C 3-8 cycloalkyl in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl base.
本发明所述的术语卤素,包括氟、氯、溴、碘。The term halogen in the present invention includes fluorine, chlorine, bromine and iodine.
附图说明Description of the drawings
图1 S6对不同葡萄糖条件下大鼠胰岛胰岛素分泌的影响。Figure 1 The effect of S6 on rat pancreatic islet insulin secretion under different glucose conditions.
图2 A表示在β细胞中存在8.3mM葡萄糖的情况下,S6对[Ca 2+]i的影响;B表示响应于S6的F-F0的平均值。 Figure 2 A shows the effect of S6 on [Ca 2+ ]i in the presence of 8.3 mM glucose in β cells; B shows the average value of F-F0 in response to S6.
图3 A表示在有或没有S6的情况下记录电流曲线;B表示存在或不存在S6时,电压门控的Ca 2+通道的电流-电压关系曲线;C表示在0mV处记录的平均电流密度。 Figure 3 A shows the recording current curve with or without S6; B shows the current-voltage curve of the voltage-gated Ca 2+ channel in the presence or absence of S6; C shows the average current density recorded at 0 mV .
图4 A表示使用或不使用S6记录的细胞电流;B表示Kv通道的电流-电压曲线;C表示以80mV记录的Kv通道的平均电流密度:D和E表示不存在S6或存在S6时的代表性动作电位波形;F表示平均动作电位时程(APD)。Figure 4 A represents the cell current recorded with or without S6; B represents the current-voltage curve of the Kv channel; C represents the average current density of the Kv channel recorded at 80mV: D and E represent the absence or presence of S6 Sexual action potential waveform; F represents the average action potential duration (APD).
图5 A表示在8.3mM葡萄糖的情况下,20mM氯化四乙铵(TEA)对S6诱导的[Ca 2+]i影响;B表示在存在或不存在8.3mM的TEA时,响应S6的F-F 0平均值;C表示在不存在 或存在8.3mM葡萄糖的TEA下,S6对胰岛素分泌的作用。 Figure 5 A shows the effect of 20mM tetraethylammonium chloride (TEA) on the [Ca 2+ ]i induced by S6 in the case of 8.3mM glucose; B shows the FF response to S6 in the presence or absence of 8.3mM TEA 0 mean value; C represents the effect of S6 on insulin secretion in the absence or presence of TEA with 8.3mM glucose.
图6 A表示在8.3mM葡萄糖存在下,L型Ca 2+通道拮抗剂Azelnidipine对S6诱导的[Ca 2+]i的影响;B表示在存在或不存在Azelnidipine时,响应S6的F-F 0平均值;C表示在没有细胞外钙的情况下,S6诱导[Ca 2+]i的变化;D表示在不存在细胞外钙的情况下,响应S6的F-F 0平均值。 Figure 6 A shows the effect of L-type Ca 2+ channel antagonist Azelnidipine on S6-induced [Ca 2+ ]i in the presence of 8.3mM glucose; B shows the average value of FF 0 in response to S6 in the presence or absence of Azelnidipine ; C indicates that S6 induces a change in [Ca 2+ ]i in the absence of extracellular calcium; D indicates the average value of FF 0 in response to S6 in the absence of extracellular calcium.
图7 A表示无细胞蛋白表达SDS-PAGE结果;B表示Western blot TOB透析式无细胞蛋白表达GLP-1R蛋白的验证结果。Figure 7 A shows the SDS-PAGE result of cell-free protein expression; B shows the verification result of Western blot TOB cell-free protein expression of GLP-1R protein.
图8 A表示使用CETSA检测在DMSO和S6(10μM)处理细胞裂解物后,GLP-1R的表达随温度升高的变化,定量数据如B中显示。Figure 8 A shows the use of CETSA to detect changes in the expression of GLP-1R with increasing temperature after DMSO and S6 (10 μM) are used to treat cell lysates. The quantitative data is shown in B.
图9在存在或不存在Exendin(9-39)的情况下,S6对胰岛素分泌的影响。Figure 9 The effect of S6 on insulin secretion in the presence or absence of Exendin (9-39).
图10 A表示Exendin(9-39)对S6诱导[Ca 2+]i的影响,在8.3mM葡萄糖存在下,不同处理后[Ca 2+]i的变化以F340/F380的比例绘制,B表示在存在或不存在exendin(9-39)时对响应S6的F-F 0平均值。 Figure 10 A shows the effect of Exendin (9-39) on S6-induced [Ca 2+ ]i. In the presence of 8.3mM glucose, the change of [Ca 2+ ]i after different treatments is plotted with the ratio of F340/F380, and B is The average value of FF 0 for response S6 in the presence or absence of exendin (9-39).
图11 A表示S6在有或无Exendin(9-39)的情况下记录的电流曲线;B表示Kv通道的电流-电压曲线;C表示80mV下Kv通道的平均电流密度。Figure 11 A shows the current curve recorded by S6 with or without Exendin (9-39); B shows the current-voltage curve of the Kv channel; C shows the average current density of the Kv channel at 80 mV.
具体实施方式detailed description
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的部分实施例,而不是全部。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
实施例1 含有化合物S6的片剂Example 1 Tablets containing compound S6
S1:将化合物S6 50g按递增法添加乳糖220g的方法混合研磨80min,然后混入微晶纤维素35g和山梨醇10g再次研磨30min,研磨温度低于30℃,得到药物-乳糖混合物;S1: Mix 50g of compound S6 with 220g of lactose in an incremental method, mix and grind for 80 minutes, then mix with 35g of microcrystalline cellulose and 10g of sorbitol and grind again for 30 minutes at a grinding temperature below 30°C to obtain a drug-lactose mixture;
S2:将混合物投入到湿法制粒机混合5min,按制剂重量每千克4mL的比例取吐温-80(溶于95%乙醇中),然后与0.5%聚维酮K30水溶液按1:20的混合作为黏合剂加到湿法颗粒机混合物中进行制粒;S2: Put the mixture into a wet granulator and mix for 5 minutes, take Tween-80 (dissolved in 95% ethanol) at a ratio of 4 mL per kilogram of the preparation weight, and then mix it with 0.5% povidone K30 aqueous solution at a ratio of 1:20 As a binder, it is added to the wet granulator mixture for granulation;
S4:向S2制得的颗粒中混合加入硬脂酸镁,压片机硬度控制在4-5kgf法以内,压片得到含有化合物S6的片剂1000片。S4: Mix and add magnesium stearate to the granules prepared by S2, and control the hardness of the tablet machine within 4-5kgf method, and compress to obtain 1000 tablets containing compound S6.
实施例2 含有化合物S6的颗粒剂Example 2 Granules containing compound S6
S1:将化合物S6 50g按递增法添加乳糖220g的方法混合研磨80min,然后混入微晶纤维素35g和山梨醇10g再次研磨30min,研磨温度低于30℃,得到药物-乳糖混合物;S1: Mix 50g of compound S6 with 220g of lactose in an incremental method, mix and grind for 80 minutes, then mix with 35g of microcrystalline cellulose and 10g of sorbitol and grind again for 30 minutes at a grinding temperature below 30°C to obtain a drug-lactose mixture;
S2:向步骤S1制备的药物-乳糖混合物中加入蔗糖:糊精3:1的混合物(过80目筛150g,混匀,制软材,挤压制粒,干燥,整粒,过12目筛,分装后即得含有化合物S6的颗粒剂。S2: Add a 3:1 mixture of sucrose:dextrin to the drug-lactose mixture prepared in step S1 (150g through an 80-mesh sieve, mix well, make soft materials, extrude, granulate, dry, sizing, and pass through a 12-mesh sieve , After sub-packaging, granules containing compound S6 are obtained.
实施例3 含有化合物S6的胶囊剂Example 3 Capsules containing compound S6
S1:将3.5g的氢氧化钠用少量质量浓度为50%的乙醇水溶液溶解,加入化合物S6 50g,混合并研磨,得到混合物;S1: Dissolve 3.5 g of sodium hydroxide in a small amount of 50% ethanol aqueous solution by mass concentration, add 50 g of compound S6, mix and grind to obtain a mixture;
S2:向混合物中加入220g乳糖,混合后进行在55℃条件下干燥80min,粉碎、过40目筛网,得到混合细粉;S2: Add 220g of lactose to the mixture, dry it at 55°C for 80 minutes after mixing, pulverize and pass through a 40-mesh screen to obtain mixed fine powder;
S3:将30mL质量浓度为5%的聚维酮K30的乙醇溶液加入步骤S2所得混合细粉中制软材,过20目筛网制湿颗粒;S3: Add 30 mL of an ethanol solution of povidone K30 with a mass concentration of 5% to the mixed fine powder obtained in step S2 to make a soft material, and pass through a 20-mesh screen to make wet granules;
S4:将步骤S3制得湿颗粒在50℃条件下干燥6h后,过18目筛网进行整粒;S4: After drying the wet granules obtained in step S3 at 50°C for 6 hours, they are passed through an 18-mesh sieve for granulation;
S5:测定颗粒中化合物S6的含量,计算装量,装囊,检查合格后包装,即得含有化合物S6的硬胶囊1000粒。S5: Determine the content of compound S6 in the granules, calculate the filling amount, pack it, and package after inspection to obtain 1000 hard capsules containing compound S6.
实施例4 含有化合物S6的注射剂Example 4 Injection containing compound S6
S1:配液,取20mg化合物S6溶于1000mL磷酸盐缓冲溶液中,配成药物浓度为0.02mg/mL的药液;S1: Preparation: Dissolve 20 mg of compound S6 in 1000 mL of phosphate buffer solution to prepare a drug solution with a drug concentration of 0.02 mg/mL;
S2:活性炭处理,向药液中加入溶液总量0.2%的针用活性炭吸附处理0.5小时;S2: Activated carbon treatment, add 0.2% of the total solution to the drug solution and treat it with activated carbon for 0.5 hours;
S3:初滤,用滤纸将药液进行过滤;S3: Preliminary filtration, filter the liquid medicine with filter paper;
S4:过滤除菌,用孔径为0.22μm的微孔滤膜对药液进行除菌,除热源;S4: Sterilize by filtration, use a microporous filter membrane with a pore size of 0.22μm to sterilize the liquid medicine and remove heat sources;
S5:以2mL为单位对药液进行分装,灌封,得到注射液。S5: Divide the medicinal solution in units of 2 mL, fill and seal, to obtain the injection solution.
接下来,本发明将按照以下效果例对本发明优选化合物S6进行测试,验证化合物S6呈葡萄糖浓度依赖性促进大鼠胰岛素分泌及其电生理机制,及验证化合物S6通过激活GLP-1R促进大鼠胰岛素分泌。Next, the present invention will test the preferred compound S6 of the present invention according to the following effect examples to verify that compound S6 promotes insulin secretion in rats and its electrophysiological mechanism in a glucose concentration-dependent manner, and verify that compound S6 promotes insulin in rats by activating GLP-1R secretion.
本发明使用的实验动物为雄性Wistar大鼠,体重为180~250g,购自于山西省人民医院实验动物中心,饲养于温度为20~22℃,并配有标准啮齿动物食物及饮用水。所有操作流程均符合山西医科大学实验动物的管理和使用指南。The experimental animal used in the present invention is a male Wistar rat with a weight of 180-250g, purchased from the Experimental Animal Center of Shanxi Provincial People's Hospital, raised at a temperature of 20-22°C, and equipped with standard rodent food and drinking water. All operating procedures are in compliance with the management and use guidelines for laboratory animals of Shanxi Medical University.
大鼠胰岛组织的分离与培养、大鼠胰岛β细胞的分离与培养均是本领域技术人员常规的实验操作。本发明涉及的数据处理用SigmaPlot 12.5和Igor 6.1处理实验数据,并以Mean±SEM表示。使用Student’s t-test,one-way ANOVA或paired t-test进行统计分析,当P<0.05时被认为具有统计学意义,使用Image LabTM软件对蛋白条带进行定量。The isolation and culture of rat pancreatic islet tissues and the isolation and culture of rat pancreatic islet β cells are routine experimental operations by those skilled in the art. The data processing involved in the present invention uses SigmaPlot 12.5 and Igor 6.1 to process experimental data, and is represented by Mean±SEM. Use Student’s t-test, one-way ANOVA or paired t-test for statistical analysis. When P<0.05, it is considered statistically significant. Use Image LabTM software to quantify protein bands.
G表示葡萄糖浓度mmol/L,比如2.8G表示浓度为2.8mmol/L的葡萄糖溶液。G represents the glucose concentration in mmol/L, for example, 2.8G represents a glucose solution with a concentration of 2.8 mmol/L.
一、验证S6呈葡萄糖浓度依赖性促进大鼠胰岛素分泌及其电生理机制1. Verify that S6 promotes insulin secretion in rats in a glucose concentration-dependent manner and its electrophysiological mechanism
主要药品与试剂Main drugs and reagents
Collagenase P来自于瑞士Roche制药;DispaseII来自于美国Amresco公司;Hanks缓冲液(HBSS)来自于博士德生物工程有限公司;Histopaque-1077来自于Sigma-Aldrich(上海)贸易有限公司;Albumin bovine serum(BSA)来自于北京索莱宝科技有限公司;RPMI 1640培养基来自于美国Hyclone公司;TEA来自于Sigma-Aldrich(上海)贸易有限公司;Fura 2-AM来自于上海东仁化学科技公司;胎牛血清(FBS)来自于美国Theromo Fisher科技;葡萄糖来自于北京索莱宝科技有限公司;分析纯NaCl来自于BBI生命科学有限公司;分析纯KCl来自于BBI生命科学有限公司;NaHCO 3来自于BBI 生命科学有限公司;HEPES来自于北京索莱宝科技有限公司;CaCl 2来自于BBI生命科学有限公司;分析纯MgCl 2来自于BBI生命科学有限公司;KH 2PO 4来自于BBI生命科学有限公司;MgSO 4来自于BBI生命科学有限公司;100×青链霉素混合液来自于北京索莱宝科技有限公司。 Collagenase P comes from Roche Pharmaceuticals, Switzerland; DispaseII comes from Amresco, USA; Hanks buffer (HBSS) comes from Boster Bioengineering Co., Ltd.; Histopaque-1077 comes from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Albumin bovine serum (BSA) ) Comes from Beijing Soleibao Technology Co., Ltd.; RPMI 1640 medium comes from Hyclone, USA; TEA comes from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Fura 2-AM comes from Shanghai Dongren Chemical Technology Co., Ltd.; Fetal Bovine Serum (FBS) comes from American Thermo Fisher Technology; glucose comes from Beijing Soleibao Technology Co., Ltd.; analytical pure NaCl comes from BBI Life Science Co., Ltd.; analytical pure KCl comes from BBI Life Science Co., Ltd.; NaHCO 3 comes from BBI Life Science Co., Ltd.; HEPES comes from Beijing Soleibao Technology Co., Ltd.; CaCl 2 comes from BBI Life Science Co., Ltd.; analytical pure MgCl 2 comes from BBI Life Science Co., Ltd.; KH 2 PO 4 comes from BBI Life Science Co., Ltd.; MgSO 4 Comes from BBI Life Science Co., Ltd.; 100× penicillin streptomycin mixture comes from Beijing Soleibao Technology Co., Ltd.
主要实验仪器Main experimental equipment
豪华型超净工作台来自于上海智诚分析仪器制造公司;恒温振荡器来自于常州国华电器有限公司;全自动雪花制冰机来自于常熟市雪科电器有限公司;压力蒸汽灭菌锅来自于太原兴育科教仪器设备制造有限公司;细胞培养箱来自于德国Eppendorf公司;倒置显微镜来自于日本Olympus IX51;PP-380垂直电极拉制仪来自于日本Narishige公司;MF-200微电极抛光仪来自于日本Narishige公司;医用冷藏冷冻箱来自于澳柯玛股份有限公司;数字转速型蠕动泵来自于保定创锐泵业有限公司;酸度计来自于奥豪斯仪器有限公司;VORTEX涡旋混合器来自于海门市其林贝尔仪器有限公司The luxurious ultra-clean workbench comes from Shanghai Zhicheng Analytical Instrument Manufacturing Company; the constant temperature oscillator comes from Changzhou Guohua Electric Co., Ltd.; the automatic snowflake ice maker comes from Changshu Xueke Electric Co., Ltd.; the pressure steam sterilizer comes from Taiyuan Xingyu Science and Education Equipment Manufacturing Co., Ltd.; cell incubator from Eppendorf, Germany; inverted microscope from Olympus IX51, Japan; PP-380 vertical electrode drawing instrument from Narishige, Japan; MF-200 microelectrode polishing instrument from Japan It is from Narishige Company in Japan; the medical refrigerator is from Aucma Co., Ltd.; the digital speed type peristaltic pump is from Baoding Chuangrui Pump Co., Ltd.; the acidity meter is from Ohaus Instrument Co., Ltd.; the VORTEX vortex mixer is from Haimen City Qilin Bell Instrument Co., Ltd.
效果例1 S6促进胰岛素分泌呈葡萄糖浓度依赖性Effect Example 1 S6 promotes insulin secretion in a glucose concentration-dependent manner
实验前准备:1)实验分为3组,Ep(Eppendorf)管编号(每组7个Ep管);2)配制KRBH溶液,置培养箱中孵育30min,用NaOH调制pH至7.4;3)配制不同浓度的待测样品:2.8mmol/L葡萄糖溶液(2.8G)、8.3mmol/L葡萄糖溶液(8.3G)16.7mmol/L葡萄糖溶液(16.7G)、2.8G+10μM S6、8.3G+10μM S6、16.7G+10μM S6。Preparation before experiment: 1) Experiment is divided into 3 groups, Ep (Eppendorf) tube number (7 Ep tubes per group); 2) Prepare KRBH solution, incubate in an incubator for 30 minutes, and adjust pH to 7.4 with NaOH; 3) Prepare Samples to be tested with different concentrations: 2.8mmol/L glucose solution (2.8G), 8.3mmol/L glucose solution (8.3G), 16.7mmol/L glucose solution (16.7G), 2.8G+10μM S6, 8.3G+10μM S6 , 16.7G+10μM S6.
实验步骤:1)向每个Ep管中加入500μL 2.8G,在体式显微镜下挑取5个胰岛(大小均一,边缘光滑)至Ep管中,放至培养箱内孵育30min;2)用移液枪将上清液吸出弃掉,然后每组依次各加入500μL 2.8G、8.3G、16.7G,置培养箱中孵育30min;3)取出Ep管,用移液枪将上清液吸出至提前标记好的Ep管中,混匀,封口,4℃保存,并按照上一轮的加药顺序依次加入500μL 2.8G+10μM S6、8.3G+10μM S6、16.7G+10μM S6,置培养箱中孵育30min;4)取出Ep管,将上清液吸出至已编号的Ep管中,封口,4℃保存。检测各组胰岛素的含量。Experimental steps: 1) Add 500μL 2.8G to each Ep tube, pick 5 islets (uniform in size, smooth edges) into the Ep tube under a stereomicroscope, and place them in the incubator to incubate for 30 minutes; 2) Use pipetting Aspirate and discard the supernatant with a gun, and then add 500μL 2.8G, 8.3G, 16.7G to each group in turn, and incubate in an incubator for 30 minutes; 3) Take out the Ep tube and aspirate the supernatant with a pipette to the mark in advance In a good Ep tube, mix, seal, store at 4°C, and add 500μL 2.8G+10μM S6, 8.3G+10μM S6, 16.7G+10μM S6, and incubate in the incubator according to the order of the previous round of dosing. 30min; 4) Take out the Ep tube, aspirate the supernatant into the numbered Ep tube, seal, and store at 4°C. Detect the content of insulin in each group.
实验结果如图1所示,图1表示S6对不同葡萄糖条件下大鼠胰岛胰岛素分泌的影响,从图中可以看出,在2.8mM葡萄糖(2.8G),8.3mM葡萄糖(8.3G)和16.7mM葡萄糖(16.7G)的条件下用10μM S6处理胰岛,n=7,**p<0.01;***p<0.001,从图中可以看出,在基础葡萄糖浓度(2.8G)下,S6未发挥促胰岛素分泌的作用,而在中等葡萄糖浓度(8.3G)及高葡萄糖浓度(16.7G)下的促胰岛素分泌作用显著,且后者作用明显强于前者,上述结果表明S6以葡萄糖浓度依赖性的方式促进胰岛素分泌。The experimental results are shown in Figure 1. Figure 1 shows the effect of S6 on rat pancreatic islet insulin secretion under different glucose conditions. It can be seen from the figure that at 2.8mM glucose (2.8G), 8.3mM glucose (8.3G) and 16.7 The islets were treated with 10μM S6 under the condition of mM glucose (16.7G), n=7, **p<0.01; ***p<0.001, as can be seen from the figure, under the basal glucose concentration (2.8G), S6 The effect of promoting insulin secretion is not exerted, but the effect of promoting insulin secretion under medium glucose concentration (8.3G) and high glucose concentration (16.7G) is significant, and the latter has a significantly stronger effect than the former. The above results indicate that S6 is dependent on glucose concentration. Promote insulin secretion in a sexual way.
上述结果表明S6在高葡萄糖浓度(8.3G,16.7G)下发挥促胰岛素分泌的作用,而且此作用具有葡萄糖浓度依赖性,表明S6可以在GLP-1R表达的胰岛组织中发挥促进胰岛素分泌的作用,并且极大的减少了诱发低血糖的风险。基于以上结果,我们可以初步断定S6在治疗T2DM中的可能性。The above results indicate that S6 can promote insulin secretion under high glucose concentrations (8.3G, 16.7G), and this effect is glucose concentration-dependent, indicating that S6 can promote insulin secretion in GLP-1R-expressing pancreatic islets. , And greatly reduce the risk of inducing hypoglycemia. Based on the above results, we can preliminarily determine the possibility of S6 in the treatment of T2DM.
接下来,我们进一步探究S6诱导胰岛素分泌的电生理机制,在大鼠原代胰岛β细胞上观察S6对细胞内钙离子浓度变化的影响。Next, we further explore the electrophysiological mechanism of S6 inducing insulin secretion, and observe the effect of S6 on the changes of intracellular calcium ion concentration on rat primary pancreatic islet β cells.
效果例2 S6升高β细胞内的钙离子浓度Effect Example 2 S6 increases the calcium ion concentration in β cells
实验前准备:1)分离胰岛β细胞,在细胞培养箱内培养3~4h;2)配制实验所需的KRBH溶液(调pH至7.35~7.45)以及待测药物溶液:2.8G、8.3G、8.3G+10μM S6。Preparations before the experiment: 1) Isolate the islet β cells and culture them in a cell incubator for 3 to 4 hours; 2) Prepare the KRBH solution required for the experiment (adjust the pH to 7.35 to 7.45) and the drug solution to be tested: 2.8G, 8.3G, 8.3G+10μM S6.
实验步骤:1)取细胞玻片至35mm的培养皿内,用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,加入Fura2-AM工作液,用锡箔纸包裹后于培养箱内孵育30min。用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,以充分去除残留的Fura2-AM工作液;2)在检测系统的荧光显微镜浴槽内加入适量的含2.8mM葡萄糖的KRBH溶液,放置细胞于浴槽内。分别进行不同待测药物的干预,在激发波长380nm和340nm,发射波长510nm下,观察细胞的荧光强度变化。Experimental steps: 1) Take the cell slide into a 35mm petri dish, wash the cells twice with KRBH solution containing 2.8mM glucose, add Fura2-AM working solution, wrap it with foil and incubate in an incubator for 30min. Wash the cells twice with KRBH solution containing 2.8 mM glucose to fully remove the remaining Fura2-AM working solution; 2) Add an appropriate amount of KRBH solution containing 2.8 mM glucose to the fluorescent microscope bath of the detection system, and place the cells in the bath . Intervene with different drugs to be tested, and observe the changes in the fluorescence intensity of the cells at excitation wavelengths of 380nm and 340nm and emission wavelengths of 510nm.
实验结果如图2-A和图2-B所示,图A表示在β细胞中存在8.3mM葡萄糖的情况下,S6对[Ca 2+]i的影响,[Ca 2+]i的变化以F340/F380的比例绘制;B表示响应于S6的F-F 0的平均值,F表示不同处理在峰前和峰后15s的平均值,F 0表示对于2.8mM葡萄糖浓度最小值前后15s的平均值,n=13,***p<0.001。从图中可以看出,在8.3G葡萄糖浓度下,细胞内Ca 2+水平明显升高;在此基础上继续添加S6,细胞内Ca 2+浓度仍能进一步增加。因此,可以推断S6的促胰岛素分泌作用是通过升高细胞内Ca 2+浓度来实现,并且在8.3G的葡萄糖浓度显著促进细胞内钙离子浓度升高。 The experimental results are shown in Figure 2-A and Figure 2-B. Figure A shows the effect of S6 on [Ca 2+ ]i in the presence of 8.3mM glucose in β cells. The change in [Ca 2+ ]i is The ratio of F340/F380 is drawn; B represents the average value of FF 0 in response to S6, F represents the average value of 15s before and after the peak of different treatments, and F 0 represents the average value of 15s before and after the minimum value of 2.8mM glucose concentration. n=13, ***p<0.001. It can be seen from the figure that at a glucose concentration of 8.3G, the intracellular Ca 2+ level is significantly increased; on this basis, by continuing to add S6, the intracellular Ca 2+ concentration can still increase further. Therefore, it can be inferred that the insulin secretion effect of S6 is achieved by increasing the intracellular Ca 2+ concentration, and the glucose concentration of 8.3G significantly promotes the increase of intracellular calcium ion concentration.
效果例3 S6未影响β细胞电压依赖性钙(VDCC)通道Effect Example 3 S6 did not affect β cell voltage-dependent calcium (VDCC) channels
实验步骤:1)在显微镜浴槽内加入适量检测VDCC的电极外液,然后将细胞玻片放入浴槽边缘处,并在显微镜视野内找到细胞;2)充灌电极内液;3)电极入液,高阻封接。将细胞钳制在-70mV,进行快电容补偿;全细胞模式下破膜后,给予慢电容补偿。给予-50mV~+30mV的电压刺激且以10mV为一个步阶,记录50ms内电流的变化。在记录电压依赖性钙通道时,细胞外液用Ba 2+代替Ca 2+,其主要目的是延缓钙通道的衰减,而且Ba 2+电流可将信号适当放大以便于分析。 Experimental steps: 1) Add a proper amount of electrode external liquid to detect VDCC in the microscope bath, then put the cell slide into the edge of the bath, and find the cells in the microscope field of view; 2) Fill the electrode inner liquid; 3) Fill the electrode with liquid , High resistance sealing. Clamp the cell at -70mV for fast capacitance compensation; after the membrane is broken in the whole cell mode, give slow capacitance compensation. Give a voltage stimulation of -50mV~+30mV with a step of 10mV, and record the change of current within 50ms. When recording voltage-dependent calcium channels, Ba 2+ is used to replace Ca 2+ in extracellular fluid. The main purpose is to delay the attenuation of calcium channels, and Ba 2+ current can appropriately amplify the signal for easy analysis.
实验结果如图3所示,A表示在有或没有S6的情况下记录电流曲线;B表示存在或不存在S6时,电压门控的Ca 2+通道的电流-电压关系曲线;C表示在0mV处记录的平均电流密度,n=7。从图3可以看出,在测试电压为0mV下,与对照组相比(电流密度为-5.5022±1.4896pA/pF),S6处理组电流密度为(-6.9781±1.0858pA/pF),两组间没有显著性差异。因此,S6未影响β细胞电压依赖性钙通道,说明S6对VDCC通道没有促进作用。 The experimental results are shown in Figure 3. A represents the current curve recorded with or without S6; B represents the current-voltage curve of the voltage-gated Ca 2+ channel in the presence or absence of S6; C represents 0mV The average current density recorded at n=7. It can be seen from Figure 3 that when the test voltage is 0mV, compared with the control group (current density is -5.5022±1.4896pA/pF), the current density of the S6 treatment group is (-6.9781±1.0858pA/pF). There is no significant difference between. Therefore, S6 did not affect β-cell voltage-dependent calcium channels, indicating that S6 did not promote VDCC channels.
效果例4 S6抑制了β细胞电压依赖性钾(Kv)通道,延长β细胞动作电位时程Effect Example 4 S6 inhibits the voltage-dependent potassium (Kv) channel of β cells and prolongs the action potential duration of β cells
实验步骤:1)在显微镜浴槽内加入适量检测电极外液,将含有β细胞的玻片放入浴槽内,并在镜下找到细胞;2)充灌适量电极内液,轻敲电极管壁除去起泡;3)电极入液,入液过程中可给予正压,既能吹走电极尖端周围的脏物,又利于高阻封接,入液后将电极放至细胞的正上方,慢慢下移电极;当电极接触到细胞膜表面时,继续下压电极,待电阻增加0.5MΩ左右时给予负压直至高阻封接。电压钳模式下将细胞钳制在-70mV,进行快电容补偿;全细胞模式破膜后,进行慢电容补偿,将细胞膜电容调至≥7MΩ。给予-70mV~+80mV的电压刺激并以10mV为一个步阶,记录400ms内电流的变化。破膜后,在电流钳模式下给予150pA、4ms的电流刺激。计算细胞去极化开始到复极化至距离静息电位以上10mV所需的时间。Experimental steps: 1) Add an appropriate amount of detection electrode outer liquid into the microscope bath, put the glass slide containing β cells in the bath, and find the cells under the microscope; 2) Fill an appropriate amount of electrode inner liquid, tap the electrode tube wall to remove Foaming; 3) When the electrode enters the liquid, a positive pressure can be applied during the liquid entering process, which can blow away the dirt around the electrode tip and facilitate high-resistance sealing. After entering the liquid, place the electrode directly above the cell and slowly Move the electrode down; when the electrode touches the surface of the cell membrane, continue to press the electrode down, and when the resistance increases by about 0.5 MΩ, give a negative pressure until the high-resistance seal is reached. In the voltage clamp mode, the cell is clamped at -70mV for fast capacitance compensation; after the membrane is broken in the whole cell mode, slow capacitance compensation is performed, and the cell membrane capacitance is adjusted to ≥7MΩ. Give voltage stimulation of -70mV~+80mV and take 10mV as a step, and record the change of current within 400ms. After rupture of the membrane, a current stimulation of 150pA and 4ms was given in the current clamp mode. Calculate the time from the start of cell depolarization to the time it takes for the cell to repolarize to 10 mV above the resting potential.
实验结果:通过全细胞膜片钳实验对Kv电流和动作电位进行检测,结果如图4所示,A表示使用或不使用S6记录的当前迹线,B表示Kv通道的电流-电压曲线,C表示以80mV记录的Kv通道的平均电流密度,n=7,**P<0.01。如图4的A、B、C数据显示,在测试电压为80mV下,与对照组相比,S6处理组显著性抑制了Kv电流。本实验动作电位持续时间(APD)由4ms,150pA电流注入引起,图D和E表示代表性动作电位波形,F表示平均APD,n=7;***P<0.001。如图4的D、E、F所示,与对照组相比,S6明显延长了动作电位时程。因此,S6诱导的胰岛素分泌作用是通过抑制Kv通道,延长动作电位时程来完成的。Experimental results: Kv current and action potential were detected by whole-cell patch clamp experiment. The results are shown in Figure 4. A represents the current trace recorded with or without S6, B represents the current-voltage curve of the Kv channel, and C represents The average current density of the Kv channel recorded at 80mV, n=7, **P<0.01. As shown in the data of A, B, and C in Fig. 4, when the test voltage is 80mV, compared with the control group, the S6 treatment group significantly suppressed the Kv current. The action potential duration (APD) in this experiment was caused by 4ms and 150pA current injection. Figures D and E represent representative action potential waveforms, and F represents the average APD, n=7; ***P<0.001. As shown in D, E, and F of Figure 4, compared with the control group, S6 significantly prolonged the action potential duration. Therefore, the insulin secretion induced by S6 is accomplished by inhibiting Kv channels and prolonging the duration of action potentials.
效果例5 Kv通道部分参与S6介导的胰岛素分泌作用Effect Example 5 Kv channel is partly involved in S6-mediated insulin secretion
实验1 Experiment 1
实验前准备:1)分离胰岛β细胞,在细胞培养箱内培养3~4h;2)配制实验所需的KRBH溶液(调pH至7.35~7.45)以及待测药物溶液:2.8G、8.3G、8.3G+10μMS6、8.3G+20mM TEA、8.3G+20mM TEA+S6;3)配置Fura-2AM工作液:取1.5mL的Ep管,加入2μL 1mM的Fura2-AM工作液和1mL含2.8mM葡萄糖的KRBH溶液,配制成终浓度为2μM的荧光染料,充分混匀后避光保存,备用。Preparations before the experiment: 1) Isolate the islet β cells and culture them in a cell incubator for 3 to 4 hours; 2) Prepare the KRBH solution required for the experiment (adjust the pH to 7.35 to 7.45) and the drug solution to be tested: 2.8G, 8.3G, 8.3G+10μMS6, 8.3G+20mM TEA, 8.3G+20mM TEA+S6; 3) Configure Fura-2AM working solution: Take 1.5mL Ep tube, add 2μL 1mM Fura2-AM working solution and 1mL containing 2.8mM glucose The KRBH solution is prepared into a fluorescent dye with a final concentration of 2μM, thoroughly mixed and stored in the dark for later use.
实验步骤:1)取细胞玻片至35mm的培养皿内,用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,加入Fura2-AM工作液,用锡箔纸包裹后于培养箱内孵育30min。用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,以充分去除残留的Fura2-AM工作液;2)在检测系统的荧光显微镜浴槽内加入适量的含2.8mM葡萄糖的KRBH溶液,放置细胞于浴槽内。分别进行不同待测药物的干预,在激发波长380nm和340nm,发射波长510nm下,观察细胞的荧光强度变化。Experimental steps: 1) Take the cell slide into a 35mm petri dish, wash the cells twice with KRBH solution containing 2.8mM glucose, add Fura2-AM working solution, wrap it with foil and incubate in an incubator for 30min. Wash the cells twice with KRBH solution containing 2.8 mM glucose to fully remove the remaining Fura2-AM working solution; 2) Add an appropriate amount of KRBH solution containing 2.8 mM glucose to the fluorescent microscope bath of the detection system, and place the cells in the bath . Intervene with different drugs to be tested, and observe the changes in the fluorescence intensity of the cells at excitation wavelengths of 380nm and 340nm and emission wavelengths of 510nm.
实验结果:为了探究S6诱导的胰岛素分泌作用是否仅与Kv通道有关,我们使用Kv通道阻断剂TEA进行观察,结果如图5的A和B所示,A表示在存在8.3mM葡萄糖的情况下,20mM氯化四乙铵(TEA)对S6诱导的[Ca 2+]i升高的影响,[Ca 2+]i的变化以在不同条件下的F340/F380的比例绘制;B表示在存在或不存在8.3mM的TEA时,响应S6的F-F 0平均值,n=18,***p<0.001。如图5的A和B所示,钙离子成像实验显示,TEA在8.3mM葡萄糖条件下显着增加了细胞内Ca 2+水平,紧接着我们同时给予Kv通道阻断剂TEA和S6,发现[Ca 2+]i仍能够进一步增加。 Experimental results: In order to explore whether the insulin secretion induced by S6 is only related to the Kv channel, we used the Kv channel blocker TEA to observe. The results are shown in Figure 5 A and B. A indicates that in the presence of 8.3mM glucose , The effect of 20mM tetraethylammonium chloride (TEA) on the increase of [Ca 2+ ]i induced by S6, the change of [Ca 2+ ]i is plotted with the ratio of F340/F380 under different conditions; B indicates the presence of Or in the absence of 8.3mM TEA, the average value of FF 0 in response to S6, n=18, ***p<0.001. As shown in Figure 5 A and B, the calcium ion imaging experiment showed that TEA significantly increased the intracellular Ca 2+ level under the condition of 8.3mM glucose. Then we gave the Kv channel blockers TEA and S6 at the same time, and found [ Ca 2+ ]i can still be increased further.
实验2 Experiment 2
实验前准备:1)实验分为3组,Ep管编号;2)配制待测药物,2.8G、8.3G、8.3G+20mM TEA、8.3G+10μM S6、8.3G+20mM TEA+10μM S6。Preparation before the experiment: 1) The experiment is divided into 3 groups, Ep tube number; 2) The drug to be tested is prepared, 2.8G, 8.3G, 8.3G+20mM TEA, 8.3G+10μM S6, 8.3G+20mM TEA+10μM S6.
实验步骤:1)预孵育;2)取出Ep管,弃上清,每组依次各加入500μL 2.8G、8.3G、8.3G+20mM TEA,置培养箱中孵育30min;3)取出Ep管,将上清液吸出至提前标记好的Ep管中,混匀,封口,4℃保存,按照上一轮的加药顺序依次加入500μL 8.3G+10μM S6、8.3G+20mM TEA+S6,置培养箱中孵育30min;4)取出Ep管,用移液枪将上清液吸出至已编号的Ep管中,封口,4℃保存,检测各组胰岛素的含量。Experimental steps: 1) Pre-incubation; 2) Take out the Ep tube, discard the supernatant, add 500 μL 2.8G, 8.3G, 8.3G+20mM TEA to each group in turn, and incubate in the incubator for 30 minutes; 3) Take out the Ep tube and add Aspirate the supernatant into the pre-labeled Ep tube, mix, seal, store at 4°C, add 500μL 8.3G+10μM S6, 8.3G+20mM TEA+S6 in the order of the previous round of dosing, and place in the incubator Incubate for 30min in medium; 4) Take out the Ep tube, aspirate the supernatant into the numbered Ep tube with a pipette, seal, store at 4°C, and detect the insulin content of each group.
实验结果如图5的C所示,C表示在不存在或存在8.3mM葡萄糖的TEA下,S6对胰岛素分泌的作用,n=6,*p<0.05,**p<0.01,***p<0.001。我们发现S6和TEA分别在8.3mM葡萄糖条件下显着增强了胰岛素的分泌,同时给予TEA和S6时,胰岛素分泌的能力进一步增强,这些结果共同表明,S6的促胰岛素分泌作用除了Kv通道抑制作用,还涉及其他因素。The experimental results are shown in C of Figure 5. C represents the effect of S6 on insulin secretion in the absence or presence of 8.3mM glucose TEA, n=6, *p<0.05, **p<0.01, ***p <0.001. We found that S6 and TEA significantly enhanced the secretion of insulin under the condition of 8.3mM glucose, respectively. When TEA and S6 were given at the same time, the ability of insulin secretion was further enhanced. These results together indicate that the insulin secretion effect of S6 is in addition to the Kv channel inhibitory effect. , Other factors are also involved.
效果例6 证明S6介导的细胞内Ca 2+浓度升高的另一个原因是细胞内钙库释放 Effect Example 6 It is proved that another reason for the increase of intracellular Ca 2+ concentration mediated by S6 is the release of intracellular calcium pool
实验1 Experiment 1
实验前准备:1)分离胰岛β细胞,在细胞培养箱内培养3~4h;2)配制实验所需的KRBH溶液(调pH至7.35~7.45)以及待测药物溶液:2.8G、8.3G、8.3G+10μMS6、8.3G+0.1μM Azelnidipine、8.3G+0.1μM Azelnidipine+S6;3)配置Fura-2AM工作液:取1.5mL的Ep管,加入2μL 1mM的Fura2-AM工作液和1mL含2.8mM葡萄糖的KRBH溶液,配制成终浓度为2μM的荧光染料,充分混匀后避光保存,备用。Preparations before the experiment: 1) Isolate the islet β cells and culture them in a cell incubator for 3 to 4 hours; 2) Prepare the KRBH solution required for the experiment (adjust the pH to 7.35 to 7.45) and the drug solution to be tested: 2.8G, 8.3G, 8.3G+10μMS6, 8.3G+0.1μM Azelnidipine, 8.3G+0.1μM Azelnidipine+S6; 3) Configure Fura-2AM working solution: Take 1.5mL Ep tube, add 2μL 1mM Fura2-AM working solution and 1mL containing 2.8 The KRBH solution of mM glucose is prepared as a fluorescent dye with a final concentration of 2μM, and it is well mixed and stored in the dark for later use.
实验步骤:1)取细胞玻片至35mm的培养皿内,用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,加入Fura2-AM工作液,用锡箔纸包裹后于培养箱内孵育30min。用含2.8mM葡萄糖的KRBH溶液清洗细胞2次,以充分去除残留的Fura2-AM工作液;2)在检测系统的荧光显微镜浴槽内加入适量的含2.8mM葡萄糖的KRBH溶液,放置细胞于浴槽内。分别进行不同待测药物的干预,在激发波长380nm和340nm,发射波长510nm下,观察细胞的荧光强度变化。Experimental steps: 1) Take the cell slide into a 35mm petri dish, wash the cells twice with KRBH solution containing 2.8mM glucose, add Fura2-AM working solution, wrap it with foil and incubate in an incubator for 30min. Wash the cells twice with KRBH solution containing 2.8 mM glucose to fully remove the remaining Fura2-AM working solution; 2) Add an appropriate amount of KRBH solution containing 2.8 mM glucose to the fluorescent microscope bath of the detection system, and place the cells in the bath . Intervene with different drugs to be tested, and observe the changes in the fluorescence intensity of the cells at excitation wavelengths of 380nm and 340nm and emission wavelengths of 510nm.
实验结果:为了探究[Ca 2+]i的增加与细胞内钙库释放的关系,我们使用L型Ca 2+通道拮抗剂Azelnidipine分析S6在促进细胞内Ca 2+浓度升高的作用,结果如图6的A和B图所示,A表示在8.3mM葡萄糖存在下,L型Ca 2+通道拮抗剂Azelnidipine对S6诱导的[Ca 2+]i升高的影响,[Ca 2+]i的变化以在不同条件下F340/F380的比例绘制;B表示在存在或不存在Azelnidipine时,响应S6的F-F 0平均值为8.3mM,n=14,***p<0.001。如图6的A和B图钙离子成像实验显示,在8.3G葡萄糖条件下,细胞内钙水平显著增加;随后我们发现同时给予S6和Azelnidipine时,[Ca 2+]i仍明显增加,这些现象提示细胞内Ca 2+的增加可能源自细胞内钙动员。 Experimental results: In order to explore the relationship between the increase of [Ca 2+ ]i and the release of intracellular calcium stores, we used the L-type Ca 2+ channel antagonist Azelnidipine to analyze the effect of S6 in promoting the increase of intracellular Ca 2+ concentration. The results are as follows Panels a and B shown in FIG. 6, a indicates the presence of 8.3mM glucose, L-type Ca 2+ channel antagonist Azelnidipine on S6 induced [Ca 2+] I elevated, [Ca 2+] i in The changes are plotted with the ratio of F340/F380 under different conditions; B indicates that in the presence or absence of Azelnidipine, the average value of FF 0 in response to S6 is 8.3mM, n=14, ***p<0.001. The calcium ion imaging experiments in Figure 6 A and B show that under the condition of 8.3G glucose, the intracellular calcium level increased significantly; then we found that when S6 and Azelnidipine were administered at the same time, [Ca 2+ ]i was still significantly increased. These phenomena It suggests that the increase of intracellular Ca 2+ may originate from intracellular calcium mobilization.
实验2 Experiment 2
实验前准备:配制实验所需的无Ca 2+-KRBH溶液(调节pH至7.35~7.45)以及待测药物溶液:2.8G、8.3G、8.3G+10μM S6。 Preparation before experiment: prepare the Ca 2+ -KRBH solution (adjust the pH to 7.35~7.45) needed for the experiment and the drug solution to be tested: 2.8G, 8.3G, 8.3G+10μM S6.
实验步骤:1)取出细胞,用含2.8mM葡萄糖的KRBH溶液清洗2次,加入Fura2-AM工作液孵育30min,孵育完成后用含2.8mM葡萄糖的KRBH溶液清洗细胞2次;2)分别进行不同待测药物的干预,在激发波长380nm和340nm,发射波长510nm下,观察细胞的荧光强度变化。Experimental steps: 1) Take out the cells, wash them twice with KRBH solution containing 2.8mM glucose, add Fura2-AM working solution and incubate for 30min, after incubation, wash the cells twice with KRBH solution containing 2.8mM glucose; 2) perform different With the intervention of the drug to be tested, the fluorescence intensity changes of the cells were observed at excitation wavelengths of 380nm and 340nm and emission wavelengths of 510nm.
实验结果:我们在去除细胞外Ca 2+情况下,测量细胞内钙浓度的变化,结果如图6的C图和D图所示,C表示在没有细胞外钙的情况下,S6诱导[Ca 2+]i升高,[Ca 2+]i的变化以在不同条件下的F340/F380的比例绘制;D表示在不存在细胞外钙的情况下,响应S6的F-F 0平均值为8.3mM,n=9,***p<0.001。如图6的C和D图数据显示,在没有细 胞外Ca 2+的存在下,S6仍明显增加了细胞内钙的浓度,以上结果共同表明S6通过细胞内Ca 2+库释放增强了[Ca 2+]i。 Experimental results: We measured the changes in intracellular calcium concentration under the condition of removing extracellular Ca 2+ . The results are shown in Figure 6 C and D. C indicates that in the absence of extracellular calcium, S6 induces [Ca 2+ ]i increases, the change of [Ca 2+ ]i is plotted with the ratio of F340/F380 under different conditions; D means that in the absence of extracellular calcium, the average value of FF 0 in response to S6 is 8.3mM , N=9, ***p<0.001. The data in Figure 6 C and D shows that in the absence of extracellular Ca 2+ , S6 still significantly increases the intracellular calcium concentration. The above results together indicate that S6 enhances the release of intracellular Ca 2+ pools [Ca 2+ ]i.
二、验证S6通过激活GLP-1R促进大鼠胰岛素分泌2. Verify that S6 promotes insulin secretion in rats by activating GLP-1R
为了证明S6是否是有效的GLP-1R激动剂,我们首先引入了一种新兴的名为细胞热转变分析(Cellular Thermal Shift Assay,CETSA)的实验来监测和量化药物到达并结合蛋白靶标的程度。其原理是每种蛋白质都有自身的热熔解曲线,当用药物处理细胞时,未与药物结合的蛋白质将在加热过程中变性失活并产生沉淀,而能与药物结合的蛋白质能稳定存在。然后通过western blot检测不同温度下药物处理组和对照组胞浆内残留的可溶性蛋白来估算热稳定性的变化。另外该方法适用范围广泛,可以在活细胞、细胞裂解液和组织内检测药物和靶标蛋白的相互作用。In order to prove whether S6 is an effective GLP-1R agonist, we first introduced a new experiment called Cellular Thermal Shift Assay (CETSA) to monitor and quantify the extent to which the drug reaches and binds to the protein target. The principle is that each protein has its own thermal melting curve. When the cells are treated with drugs, the protein that is not bound to the drug will be denatured and inactivated during the heating process and precipitated, while the protein that can bind to the drug can exist stably. Then, the residual soluble protein in the cytoplasm of the drug treatment group and the control group was detected by western blot to estimate the change of thermal stability. In addition, the method has a wide range of applications, and can detect the interaction of drugs and target proteins in living cells, cell lysates and tissues.
主要药品与试剂Main drugs and reagents
Tris来自于北京索莱宝科技有限公司;GLP-1R Antibody Rabbit Polyclonal来自于武汉三鹰生物技术有限公司;HRP-conjugated Affinipure Rabbit&Anti-Goat IgG(H+L)来自于武汉三鹰生物技术有限公司;SDS-PAGE凝胶制备试剂盒来自于北京索莱宝科技有限公司;Glycine来自于北京索莱宝科技有限公司;SDS来自于北京索莱宝科技有限公司;Tween-20来自于北京索莱宝科技有限公司;脱脂奶粉来自于博士德生物工程有限公司;5X loading buffer来自于博士德生物工程有限公司;M5Prestained Plus Protein Ladder来自于北京聚合美生物科技有限公司;硝酸纤维素膜NC膜来自于美国Millipore;Biodlight TM ECL Chemiluminescent HRP Substrate(High Sensitivity)来自于美国bioworld;BamHI、XhoL、EcoRI、NotI来自于美国Fermentas;BglII、NheI、NsiI、HindIII来自于美国Fermentas;Smal、EcoRV来自于美国Fermentas;DNA连接酶、DNA marker来自于美国Fermentas;
Figure PCTCN2021096380-appb-000004
Entry One Step Cloning Kit来自于南京诺唯赞生物科技有限公司;Pfu DNA聚合酶来自于上海生工生物有限公司;琼脂酶、DNA凝胶回收试剂盒来自于天根生化科技(北京)有限公司;高纯度质粒小提试剂盒来自于天根生化科技(北京)有限公司;中量抽提试剂盒来自于杭州爱思进生物科技有限公司;酵母提取物、胰蛋白胨来自于美国Theromo Fisher科技;Kanamycin、Ampicillin来自于Sigma-Aldrich(上海)贸易有限公司;透析式E.coli无细胞重组蛋白表达试剂盒来自于杭州谨澳生物科技公司;透析式TOB无细胞重组蛋白表达试剂盒来自于杭州谨澳生物科技公司;透析式Yeast无细胞重组蛋白表达试剂盒来自于杭州谨澳生物科技公司。
Tris is from Beijing Soleibao Technology Co., Ltd.; GLP-1R Antibody Rabbit Polyclonal is from Wuhan Sanying Biotechnology Co., Ltd.; HRP-conjugated Affinipure Rabbit&Anti-Goat IgG (H+L) is from Wuhan Sanying Biotechnology Co., Ltd.; SDS-PAGE gel preparation kit comes from Beijing Soleibao Technology Co., Ltd.; Glycine comes from Beijing Soleibao Technology Co., Ltd.; SDS comes from Beijing Soleibao Technology Co., Ltd.; Tween-20 comes from Beijing Soleibao Technology Co., Ltd. Co., Ltd.; skimmed milk powder from Boster Bioengineering Co., Ltd.; 5X loading buffer from Boster Bioengineering Co., Ltd.; M5Prestained Plus Protein Ladder from Beijing Polymei Biotechnology Co., Ltd.; nitrocellulose membrane NC membrane from Millipore, USA ; Biodlight TM ECL Chemiluminescent HRP Substrate (High Sensitivity) comes from bioworld in the United States; BamHI, XhoL, EcoRI, NotI are from Fermentas in the United States; BglII, NheI, NsiI, HindIII are from Fermentas in the United States; Smal and EcoRV are from Fermentas in the United States; DNA connection Enzyme and DNA marker are from Fermentas, USA;
Figure PCTCN2021096380-appb-000004
Entry One Step Cloning Kit is from Nanjing Novazan Biotechnology Co., Ltd.; Pfu DNA polymerase is from Shanghai Shenggong Biology Co., Ltd.; Agarase and DNA gel recovery kits are from Tiangen Biochemical Technology (Beijing) Co., Ltd.; The high-purity plasmid small extraction kit was from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the medium-volume extraction kit was from Hangzhou Aisijin Biotechnology Co., Ltd.; the yeast extract and tryptone were from the United States Thermo Fisher Technology; Kanamycin , Ampicillin is from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Dialysis E.coli cell-free recombinant protein expression kit is from Hangzhou Jinao Biotechnology Company; Dialysis TOB cell-free recombinant protein expression kit is from Hangzhou Jinao Biotechnology company; the dialysis Yeast cell-free recombinant protein expression kit comes from Hangzhou Jinao Biotechnology Company.
主要实验仪器Main experimental equipment
低温高速离心机来自于德国Eppendorf公司;多功能酶标仪来自于美国Bio Tek公司;Western blot垂直电泳系统来自于美国Bio Tek公司;Western blot半干转膜系统来自于美国Bio Tek公司;凝胶成像系统来自于美国Bio Tek公司;钙成像LAMBDA10-B系统来自于北京MDE公司;PCR仪来自于美国Bio-rad公司;恒温振荡培养箱来自于太仓华美生化仪器有限公司;恒温磁力搅拌器来自于上海司乐仪器有限公司;电热恒温培养箱来自于上海福玛实验设备有限公司;微型漩涡混合仪来自于上海沪西分析仪 器厂有限公司;数显恒温水浴锅来自于国华电器有限公司。The low-temperature high-speed centrifuge comes from Eppendorf, Germany; the multi-function microplate reader comes from BioTek, USA; the Western blot vertical electrophoresis system comes from BioTek, USA; The Western blot semi-dry transfer membrane system comes from BioTek, USA; Gel The imaging system is from BioTek, USA; the calcium imaging LAMBDA10-B system is from Beijing MDE; the PCR machine is from Bio-rad, the USA; the constant temperature oscillating incubator is from Taicang Huamei Biochemical Instrument Co., Ltd.; the constant temperature magnetic stirrer is from Taicang Huamei Biochemical Instrument Co., Ltd. Shanghai Sile Instrument Co., Ltd.; the electrothermal constant temperature incubator is from Shanghai Fuma Experimental Equipment Co., Ltd.; the miniature vortex mixer is from Shanghai Huxi Analytical Instrument Factory Co., Ltd.; the digital thermostat water bath is from Guohua Electric Co., Ltd.
效果例7 CETSA验证GLP-1R与S6的靶向作用Effect Example 7 CETSA verifies the targeting effect of GLP-1R and S6
无细胞GLP-1R蛋白的表达:1)构建大鼠GLP-1R载体:用PCR扩增技术获得大鼠GLP-1R的序列片段,琼脂糖凝胶中回收GLP-1R rat基因片段,将目的基因GLP-1R克隆到载体pEX-3中,用EcoRI和BamHI双酶切pEX-3,电泳,回收载体pEX-3;利用
Figure PCTCN2021096380-appb-000005
Entry One Step Cloning Kit,把扩增片段重组克隆到pEX-3载体中;将重组连接产物转化为感受态细胞,挑取克隆菌落,少量抽提得到质粒,用EcoRI和BamHI双酶切鉴定,电泳,挑取阳性克隆;最后对重组质粒测序验证后进行大量抽提,取适量阳性克隆对应的菌液测序,剩余的菌液用甘油保存;将测序结果与目的基因序列进行比对,核对无误后,用甘油菌液接菌LB培养基,大量质粒抽提,得到足够量的重组质粒。2)无细胞GLP-1R蛋白的表达:采用杭州谨澳生物科技Z1高保真DNA聚合酶G-POL-001或Z2超保真DNA聚合酶G-POL-002对目的基因进行扩增,为了提高蛋白表达的成功率,我们分别采用E.coli透析式无细胞蛋白表达体系,TOB透析式无细胞蛋白表达体系,Yeast透析式无细胞蛋白表达体系进行表达,最后用SDS-PAGE观测表达结果。
Expression of cell-free GLP-1R protein: 1) Construction of rat GLP-1R vector: The sequence fragment of rat GLP-1R was obtained by PCR amplification technology, and the GLP-1R rat gene fragment was recovered from agarose gel, and the target gene GLP-1R was cloned into the vector pEX-3, pEX-3 was digested with EcoRI and BamHI, and the vector pEX-3 was recovered by electrophoresis; use
Figure PCTCN2021096380-appb-000005
Entry One Step Cloning Kit, recombinantly clone the amplified fragments into pEX-3 vector; transform the recombination ligation product into competent cells, pick the cloned colonies, extract a small amount of plasmids, use EcoRI and BamHI double enzyme digestion to identify, electrophoresis , Pick the positive clones; finally, after the recombinant plasmid is sequenced and verified, a large amount of extraction is carried out, and an appropriate amount of the bacterial solution corresponding to the positive clone is sequenced, and the remaining bacterial solution is stored in glycerol; the sequencing result is compared with the target gene sequence, and the result is verified. , Inoculate LB medium with glycerol bacteria liquid, extract a large amount of plasmids, and obtain a sufficient amount of recombinant plasmids. 2) Expression of cell-free GLP-1R protein: Hangzhou Jinao Biotechnology Z1 high-fidelity DNA polymerase G-POL-001 or Z2 ultra-fidelity DNA polymerase G-POL-002 is used to amplify the target gene, in order to improve For the success rate of protein expression, we used E.coli dialysis cell-free protein expression system, TOB dialysis cell-free protein expression system, Yeast dialysis cell-free protein expression system for expression, and finally observed the expression results with SDS-PAGE.
实验步骤:1)从-80℃冰箱取出用TOB表达系统表达的含有GLP-1R的总蛋白溶液,置于冰上,取出实验所需总蛋白的体积,实验分为2组,实验干预组加入终浓度为10μM S6,对照组加入同体积的DMSO,置于37℃培养箱内孵育45min。孵育结束后,将实验干预组和对照组分别等分为5组,然后用RT-PCR仪在不同温度下(47~67℃)加热3min,室温冷却3min,4℃,20000g 20min离心,将上清吸出至200μL的Ep管中,加入5×Loading buffer,95℃加热5min后,于-80℃冰箱内保存。2)蛋白印迹(Western-Blot)分析:a配制10%的分离胶和5%的浓缩胶,蛋白上样体积为5μL;b电泳:先以80V恒压分离30min,待蛋白样品进入分离胶后换成120V继续分离1h,停止电泳:c转膜:恒压半干湿法转膜,20V,25min;d封闭:将NC膜放入5%脱脂奶粉中,37℃孵育1h;e孵育一抗,用TBST将一抗稀释至1:3000,室温孵育2h,最后用TBST洗涤三次,每次10min;f孵育二抗,用TBST将二抗稀释至1:10000,室温下孵育1h后用TBST洗涤三次,每次10min,g显影:配制ECL发光液,并使用ChemiDicTM XRS+成像系统对蛋白条带进行鉴定。Experimental steps: 1) Take out the total protein solution containing GLP-1R expressed by TOB expression system from the -80℃ refrigerator, place it on ice, and take out the volume of total protein required for the experiment. The experiment is divided into 2 groups, and the experimental intervention group is added The final concentration was 10μM S6. The control group was added with the same volume of DMSO and incubated in a 37°C incubator for 45 minutes. After the incubation, the experimental intervention group and the control group were divided into 5 groups, and then heated by RT-PCR instrument at different temperatures (47~67℃) for 3min, cooled at room temperature for 3min, 4℃, 20,000g 20min centrifugation, and the Aspirate it to a 200μL Ep tube, add 5×Loading buffer, heat at 95°C for 5 minutes, and store at -80°C in a refrigerator. 2) Western-Blot analysis: a prepare 10% separating gel and 5% concentrated gel, and the protein loading volume is 5μL; b electrophoresis: first separate at 80V constant pressure for 30 minutes, and wait until the protein sample enters the separating gel Change to 120V and continue to separate for 1 hour, stop electrophoresis: c transfer: constant pressure semi-dry and wet transfer, 20V, 25min; d block: put the NC membrane into 5% skimmed milk powder and incubate at 37°C for 1 hour; e incubate the primary antibody , Dilute the primary antibody to 1:3000 with TBST, incubate at room temperature for 2 hours, and finally wash with TBST three times for 10 minutes each; fIncubate the secondary antibody, dilute the secondary antibody to 1:10000 with TBST, incubate at room temperature for 1 hour and then wash with TBST Three times, 10min each time, g development: prepare ECL luminescent solution, and use ChemiDicTM XRS+ imaging system to identify protein bands.
实验结果:Experimental results:
1,pEX3-GLP-1R表达载体的鉴定1. Identification of pEX3-GLP-1R expression vector
对质粒进行如下检测:1)采用序列比对的方法进行基因序列检测:测序结果与目的序列一致;2)采用序列比对的方法进行向量序列检测:侧翼20bp序列与载体一致;3)采用琼脂糖电泳的方法对片段大小进行检测:质粒无污染带,片段大小正确;4)采用紫外分光光度法对浓度进行检测:OD260/280为1.8-2.0。The plasmids were tested as follows: 1) Gene sequence detection using sequence alignment: the sequencing result was consistent with the target sequence; 2) Vector sequence detection using sequence alignment: the flanking 20bp sequence was consistent with the vector; 3) using agar Carbohydrate electrophoresis method to detect the fragment size: the plasmid has no contamination band, the fragment size is correct; 4) The concentration is detected by ultraviolet spectrophotometry: OD260/280 is 1.8-2.0.
2,TOB透析式蛋白表达体系的GLP-1R蛋白的鉴定2. Identification of GLP-1R protein in TOB dialysis protein expression system
分别取5μL GLP-lR E.coli、GLP-lR TOB、GLP-lR Yeast的上清和总蛋白进行SDS-PAGE检测,如图7的A图所示,GLP-lR目的蛋白在E.coli表达体系和Yeast表达体系未观测到目的蛋白过量表达;目的蛋白(*)在TOB表达体系疑似表达,为可溶蛋白。 随后我们对GLP-lR TOB样品进行了western blot检测,结果如图7的B图所示,证实GLP-lR目的蛋白在TOB表达体系表达。Take 5μL of GLP-lR E.coli, GLP-lR TOB, GLP-lR Yeast supernatant and total protein for SDS-PAGE detection, as shown in Figure 7 A, the GLP-lR target protein is expressed in E. coli expression system No overexpression of the target protein was observed in the Yeast expression system; the target protein (*) is suspected to be expressed in the TOB expression system and is a soluble protein. Subsequently, we performed western blot detection on GLP-1R TOB samples, and the results are shown in Figure 7B, confirming that the GLP-1R target protein is expressed in the TOB expression system.
3,GLP-1R是S6的特异性靶标3. GLP-1R is a specific target of S6
在前一部分中,已经验证S6能够发挥促进大鼠胰岛素分泌的作用,本发明还验证了S6为有效的小分子GLP-1R激动剂。首先使用CETSA技术来观察GLP-1R和S6的关系,通过Western-Blot检测蛋白质样品,观察GLP-1R表达的变化。如图8所示,图A表示使用DMSO和S6(10μM)处理的细胞裂解物的CETSA使用Western印迹检测S6和GLP-1R之间的相互作用,定量数据如B中显示,n=3,独立实验,*p<0.05。随着温度的升高,对照组(DMSO)GLP-1R的表达越来越不稳定;而S6处理组的GLP-1R表达虽然也随着温度升高而减弱,但相对于DMSO处理组而言是稳定的,尤其是在62℃时两组间的差距最大,这说明与GLP-1R结合后增强了GLP-1R的热稳定性,进而说明GLP-1R是S6的靶蛋白。In the previous part, it has been verified that S6 can play a role in promoting insulin secretion in rats. The present invention also verified that S6 is an effective small molecule GLP-1R agonist. First, use CETSA technology to observe the relationship between GLP-1R and S6, detect protein samples through Western-Blot, and observe the changes in GLP-1R expression. As shown in Figure 8, Figure A shows the CETSA of cell lysates treated with DMSO and S6 (10 μM). Western blotting was used to detect the interaction between S6 and GLP-1R. The quantitative data is shown in B, n=3, independent Experiment, *p<0.05. With the increase of temperature, the expression of GLP-1R in the control group (DMSO) became more and more unstable; while the expression of GLP-1R in the S6 treatment group also decreased with the increase in temperature, but compared with the DMSO treatment group It is stable, especially at 62°C. The gap between the two groups is the largest. This indicates that the combination with GLP-1R enhances the thermal stability of GLP-1R, and further indicates that GLP-1R is the target protein of S6.
效果例8 S6通过GLP-1R促进胰岛素分泌Effect Example 8 S6 promotes insulin secretion through GLP-1R
实验前准备:1)实验分3组,Ep管编号;2)配制待测药物:2.8G、8.3G、8.3G+100nM Exendin(9-39)、8.3G+S6+Exendin(9-39)、8.3G+S6。Preparation before the experiment: 1) The experiment is divided into 3 groups, Ep tube number; 2) Preparation of drugs to be tested: 2.8G, 8.3G, 8.3G+100nM Exendin(9-39), 8.3G+S6+Exendin(9-39) , 8.3G+S6.
实验步骤:1)预孵育;2)取出Ep管弃上清,每组各自加入500μL 2.8G、8.3G、8.3G+Exendin(9-39),孵育30min;3)将上清液吸出至提前标记好的Ep管中,混匀,封口,4℃保存;4)按照前一次的给药顺序,依次各自给予500μL8.3G+S6+Exendin(9-39)、8.3G+S6,孵育30min后,上清液吸出至已准备好的Ep管中,混匀,封口。检测各组上清液中胰岛素的含量。Experimental steps: 1) Pre-incubation; 2) Take out the Ep tube and discard the supernatant, add 500 μL 2.8G, 8.3G, 8.3G+Exendin (9-39) to each group, and incubate for 30 minutes; 3) Aspirate the supernatant to advance In the labeled Ep tube, mix well, seal, and store at 4°C; 4) In accordance with the order of the previous administration, administer 500μL 8.3G+S6+Exendin(9-39), 8.3G+S6, respectively, and incubate for 30min. , Aspirate the supernatant into the prepared Ep tube, mix well, and seal. Detect the content of insulin in the supernatant of each group.
实验结果:通过使用GLP-1R拮抗剂Exendin(9-39),检查S6增强胰岛素分泌是否依赖于大鼠胰岛中的GLP-1R活化,如图9所示,表示S6中存在或不存在Exendin(9-39)的情况下,S6通过GLP-1R诱导葡萄糖依赖性胰岛素分泌,n=6;*P<0.05,**P<0.01。结果显示Exendin(9-39)阻断了S6促进的胰岛素分泌作用,表明S6对胰岛素分泌的作用与GLP-1R激活有关,这与先前实验结果相同。Experimental results: By using the GLP-1R antagonist Exendin(9-39), it was checked whether the enhancement of insulin secretion by S6 is dependent on the activation of GLP-1R in rat pancreatic islets. As shown in Figure 9, it indicates the presence or absence of Exendin( 9-39), S6 induces glucose-dependent insulin secretion through GLP-1R, n=6; *P<0.05, **P<0.01. The results showed that Exendin (9-39) blocked the insulin secretion promoted by S6, indicating that the effect of S6 on insulin secretion is related to the activation of GLP-1R, which is the same as the previous experimental results.
效果例9 S6通过GLP-1R增加细胞内Ca 2+浓度 Effect Example 9 S6 increases intracellular Ca 2+ concentration through GLP-1R
实验前准备:1)分离胰岛β细胞,在细胞培养箱内培养3~4h;2)配制实验所需的KRBH溶液(调pH至7.35~7.45)并配制待测溶液:2.8G、8.3G、8.3G+Exendin(9-39)、8.3G+S6+Exendin(9-39)、8.3G+S6;3)配置Fura-2AM工作液。Preparations before the experiment: 1) Isolate pancreatic islet β cells and culture them in a cell incubator for 3 to 4 hours; 2) Prepare the KRBH solution required for the experiment (adjust the pH to 7.35 to 7.45) and prepare the test solution: 2.8G, 8.3G, 8.3G+Exendin(9-39), 8.3G+S6+Exendin(9-39), 8.3G+S6; 3) Configure Fura-2AM working fluid.
实验步骤:在2.8G、8.3G、8.3G+Exendin(9-39)、8.3G+S6+Exendin(9-39)、和8.3G+S6干预下,观察细胞的荧光强度变化。Experimental procedure: Under the intervention of 2.8G, 8.3G, 8.3G+Exendin(9-39), 8.3G+S6+Exendin(9-39), and 8.3G+S6, observe the changes in the fluorescence intensity of the cells.
实验结果:为进一步验证S6升高细胞内Ca 2+浓度与GLP-1R的关系,我们使用Exendin(9-39)对细胞内Ca 2+浓度变化进行检测。如图10所示,图A表示Exendin(9-39)对S6诱导的影响[Ca 2+]i在8.3mM葡萄糖存在下,[Ca 2+]i的变化以F340/F380的比例绘制,图B表示在存在或不存在Exendin(9-39)时对S6响应的F-F0平均值,n=9,***P<0.001。实验结果显示Exendin(9-39)减弱了S6诱导的细胞内Ca 2+增强的作用,而单独使用时没有影响细胞内Ca 2+浓度变化。这些发现表明,S6通过GLP-1R引起[Ca 2+]i 增加,从而导致胰岛素囊泡的胞吐作用增强。 Experimental results: In order to further verify the relationship between S6's increased intracellular Ca 2+ concentration and GLP-1R, we used Exendin (9-39) to detect changes in intracellular Ca 2+ concentration. As shown in Figure 10, Figure A shows the effect of Exendin(9-39) on S6 induction [Ca 2+ ]i in the presence of 8.3mM glucose, the change of [Ca 2+ ]i is plotted at the ratio of F340/F380. B represents the average F-F0 response to S6 in the presence or absence of Exendin (9-39), n=9, ***P<0.001. The experimental results showed that Exendin (9-39) attenuated the effect of S6-induced intracellular Ca 2+ enhancement, but did not affect the intracellular Ca 2+ concentration change when used alone. These findings indicate that S6 causes an increase in [Ca 2+ ]i through GLP-1R, leading to enhanced exocytosis of insulin vesicles.
效果例10 S6通过GLP-1R抑制Kv通道电流Effect example 10 S6 suppresses Kv channel current through GLP-1R
实验前准备:1)分离好胰岛β细胞,在细胞培养箱内培养24h,待用;2)电极的制备;3)配制实验所需的药物浓度:11.1G、11.1G+S6、11.1G+Exendin(9-39)、11.1G+S6+Exendin(9-39)。Preparations before the experiment: 1) Isolate the pancreatic islet β cells, culture them in a cell incubator for 24 hours, ready to use; 2) Preparation of electrodes; 3) Preparation of the drug concentration required for the experiment: 11.1G, 11.1G+S6, 11.1G+ Exendin(9-39), 11.1G+S6+Exendin(9-39).
实验步骤:记录不同药物干预下β细胞上Kv通道电流。Experimental procedure: Record the Kv channel current on β cells under the intervention of different drugs.
实验结果如图11所示,图A表示存在或不存在Exendin(9-39)的情况下记录的细胞电流曲线;图B表示Kv通道的电流-电压曲线;图C表示80mV下Kv通道的平均电流密度,n=7;*P<0.05。实验结果证明S6明显减弱了Kv通道电流,而用Exendin(9-39)后,S6抑制Kv通道电流的作用被逆转了。The experimental results are shown in Figure 11. Figure A shows the cell current curve recorded in the presence or absence of Exendin (9-39); Figure B shows the current-voltage curve of the Kv channel; Figure C shows the average of the Kv channel at 80mV Current density, n=7; *P<0.05. The experimental results proved that S6 significantly reduced the Kv channel current, and after using Exendin (9-39), the effect of S6 on the Kv channel current was reversed.
GLP-1R属于G蛋白偶联受体B族亚类,当GLP-1及其类似物与GLP-1R结合后,可以触发一系列的信号反应,最终调节胰岛素的分泌活动。在胰岛素分泌实验中,我们观察在GLP-1R阻断剂Exendin(9-39)存在下,S6减弱了对大鼠胰岛素分泌的作用,表明S6通过GLP-1R发挥促胰岛素分泌的作用,再次印证了S6作用于GLP-1R。紧接着在钙离子成像技术实验中,我们观察S6诱导细胞内Ca 2+浓度升高的作用是否也与GLP-1R有关,结果发现在Exendin(9-39)存在下,S6升高细胞内钙离子浓度的能力被消除,这表明S6升高细胞内钙离子浓度是通过激活GLP-1R完成的。而且在膜片钳实验中我们发现S6通过激活GLP-1R抑制Kv通道,进而延长动作电位时程,导致电压依赖性钙通道开放时间延长,最终导致细胞内Ca 2+浓度增加。 GLP-1R belongs to the B subclass of G protein-coupled receptors. When GLP-1 and its analogues bind to GLP-1R, they can trigger a series of signal responses and ultimately regulate the secretion of insulin. In the insulin secretion experiment, we observed that in the presence of the GLP-1R blocker Exendin (9-39), S6 attenuated the effect of insulin secretion in rats, indicating that S6 can promote insulin secretion through GLP-1R, again confirming S6 acts on GLP-1R. Immediately in the calcium ion imaging technology experiment, we observed whether the effect of S6 in inducing the increase of intracellular Ca 2+ concentration is also related to GLP-1R, and found that in the presence of Exendin (9-39), S6 increased intracellular calcium The ability of ion concentration is eliminated, which indicates that S6 increases the intracellular calcium ion concentration by activating GLP-1R. Moreover, in the patch clamp experiment, we found that S6 inhibits Kv channels by activating GLP-1R, thereby prolonging the duration of the action potential, leading to a prolonged opening time of voltage-dependent calcium channels, and ultimately leading to an increase in intracellular Ca 2+ concentration.
效果例11 化合物S6的急性毒性试验Effect Example 11 Acute toxicity test of compound S6
将雄性小鼠随机分为5组,每组10只,将化合物S6溶于生理盐水中进行灌喂给药,第1-6组的给药剂量分别为1.5g/kg、3.0g/kg、5.0g/kg、7.5g/kg、15.0g/kg。给药后将老鼠按常规标准进行饲养,10天后记录各组死亡情况,用SPSS13.0软件计算药物半数致死量(LD50)。药物急性毒性试验结果如下表所示,结果显示本发明提供的化合物LD50值为6.2g/kg,95%可信限为4.3-7.9g/kg,毒性较低。Male mice were randomly divided into 5 groups with 10 mice in each group. Compound S6 was dissolved in physiological saline for oral administration. The dosages of groups 1-6 were 1.5g/kg, 3.0g/kg, 5.0g/kg, 7.5g/kg, 15.0g/kg. After the administration, the mice were reared according to conventional standards. After 10 days, the death of each group was recorded, and SPSS13.0 software was used to calculate the median lethal dose (LD50) of the drug. The results of the acute toxicity test of the drug are shown in the following table. The results show that the LD50 value of the compound provided by the present invention is 6.2 g/kg, the 95% confidence limit is 4.3-7.9 g/kg, and the toxicity is low.
表1 药物急性毒性试验结果Table 1 Results of drug acute toxicity test
组别Group 给药剂量g/kgDosage g/kg 10天小鼠死亡数Number of mouse deaths in 10 days 成活率Survival rate
11 1.51.5 00 100%100%
22 3.03.0 11 90%90%
33 5.05.0 22 80%80%
44 7.57.5 66 40%40%
55 15.015.0 1010 00
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. Scope.

Claims (10)

  1. 一种小分子GLP-1R激动剂,其为通式(I)的化合物,或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物,A small molecule GLP-1R agonist, which is a compound of general formula (I), or pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds,
    Figure PCTCN2021096380-appb-100001
    Figure PCTCN2021096380-appb-100001
    其中,A和B独立的选自C、N或O,B与相邻碳原子之间的
    Figure PCTCN2021096380-appb-100002
    表示单键或双键;
    Among them, A and B are independently selected from C, N or O, between B and adjacent carbon atoms
    Figure PCTCN2021096380-appb-100002
    Represents a single bond or a double bond;
    R 1、R 2独立的选自:-H、-OH、-CF 3、卤素、C 1-10直链烷基、C 3-10支链烷基、C 3-8环烷基,或R 1、R 2与R 1和R 2之间的碳原子形成C 3-8环烷基、芳香五元环基团或芳香六元环基团; R 1 and R 2 are independently selected from: -H, -OH, -CF 3 , halogen, C 1-10 straight chain alkyl, C 3-10 branched chain alkyl, C 3-8 cycloalkyl, or R 1. The carbon atoms between R 2 and R 1 and R 2 form a C 3-8 cycloalkyl group, an aromatic five-membered ring group or an aromatic six-membered ring group;
    R 3选自-H、C 1-5直链烷基、C 3-8环烷基; R 3 is selected from -H, C 1-5 straight chain alkyl, C 3-8 cycloalkyl;
    R 4、R 5独立的选自-H、-OH、卤素、-CN、C 1-5直链/支链烷基、-N(C 0-10烷基)(C 0-10烷基)、-OC 0-10烷基、C 3-6环烷基。 R 4 and R 5 are independently selected from -H, -OH, halogen, -CN, C 1-5 straight chain/branched chain alkyl, -N (C 0-10 alkyl) (C 0-10 alkyl) , -OC 0-10 alkyl, C 3-6 cycloalkyl.
  2. 根据权利要求1所述的小分子GLP-1R激动剂,其特征在于,所述A为N,B为C、N或O,且当B为O时,B与相邻的碳原子之间是碳氧单键;所述R 1、R 2独立的选自:-OH、-CH 3、-Cl、-C 2H 5,或R 1、R 2与R 1和R 2之间的碳原子形成芳香六元环基团;R 3选自-CH 3、-C 2H 5;R 4、R 5独立的选自-H、-OH、卤素、-CH 3The small molecule GLP-1R agonist according to claim 1, wherein the A is N, B is C, N or O, and when B is O, there is a gap between B and adjacent carbon atoms. Carbon-oxygen single bond; said R 1 , R 2 are independently selected from: -OH, -CH 3 , -Cl, -C 2 H 5 , or R 1 , R 2 and the carbon atom between R 1 and R 2 An aromatic six-membered ring group is formed; R 3 is selected from -CH 3 , -C 2 H 5 ; R 4 and R 5 are independently selected from -H, -OH, halogen, and -CH 3 .
  3. 根据权利要求2所述的小分子GLP-1R激动剂,其特征在于,所述式(Ⅰ)化合物具有如下立体构型:The small molecule GLP-1R agonist according to claim 2, wherein the compound of formula (I) has the following three-dimensional configuration:
    Figure PCTCN2021096380-appb-100003
    Figure PCTCN2021096380-appb-100003
    Figure PCTCN2021096380-appb-100004
    Figure PCTCN2021096380-appb-100004
  4. 一种药物组合物,所述药物组合物包含权利要求1-3任一所述的通式(I)的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物和氘代化合物,还包括药剂学上可接受的辅料,所述辅料选自:载体、稀释剂、粘合剂、润滑剂和润湿剂中的至少一种。A pharmaceutical composition comprising the compound of general formula (I) according to any one of claims 1-3 or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, or solvate thereof And deuterated compounds, and also include pharmaceutically acceptable excipients, which are selected from at least one of a carrier, a diluent, a binder, a lubricant, and a wetting agent.
  5. 根据权利要求4所述的组合物,其特征在于,所述药物组合物包含治疗有效量的通式(I)的化合物,所述药物组合物适于口服给药。The composition according to claim 4, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of general formula (I), and the pharmaceutical composition is suitable for oral administration.
  6. 权利要求1-3任一项所述的小分子GLP-1R激动剂在制备治疗GLP-1R受体介导的疾病的药物用途。The use of the small molecule GLP-1R agonist of any one of claims 1 to 3 in the preparation of drugs for the treatment of GLP-1R receptor-mediated diseases.
  7. 根据权利要求6所述的用途,其特征在于,所述GLP-1R受体介导的疾病包括:糖尿病、癫痫、脑缺血性疾病或神经退行性疾病。The use according to claim 6, wherein the GLP-1R receptor-mediated diseases include diabetes, epilepsy, cerebral ischemic diseases or neurodegenerative diseases.
  8. 根据权利要求7所述的用途权,所述糖尿病为二型糖尿病。According to the right to use according to claim 7, the diabetes is type 2 diabetes.
  9. 权利要求1-3任一项所述的小分子GLP-1R激动剂在制备Kv通道抑制剂中的用途。Use of the small molecule GLP-1R agonist of any one of claims 1 to 3 in the preparation of Kv channel inhibitors.
  10. 权利要求1-3任一项所述的小分子GLP-1R激动剂在制备治疗与Kv通道相关疾病的药物中的用途,所述与Kv通道相关的疾病包括:糖尿病、癫痫、脑缺血性疾病或神经退行性疾病。Use of the small molecule GLP-1R agonist according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of Kv channel related diseases, the Kv channel related diseases including: diabetes, epilepsy, cerebral ischemia Disease or neurodegenerative disease.
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