WO2021196275A1 - Application of emetine in preparation of drug for treating or preventing novel coronaviruses sars-cov-2 - Google Patents

Application of emetine in preparation of drug for treating or preventing novel coronaviruses sars-cov-2 Download PDF

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WO2021196275A1
WO2021196275A1 PCT/CN2020/084674 CN2020084674W WO2021196275A1 WO 2021196275 A1 WO2021196275 A1 WO 2021196275A1 CN 2020084674 W CN2020084674 W CN 2020084674W WO 2021196275 A1 WO2021196275 A1 WO 2021196275A1
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cov
concentration
sars
pharmaceutically acceptable
drug
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Chinese (zh)
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刘青松
王傲莉
孙永
王文超
胡晨
吴宏
何军
苏斌
王黎
王蓓蕾
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中国科学院合肥物质科学研究院
安徽省疾病预防控制中心
安徽中科拓苒药物科学研究有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

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  • the present invention belongs to the technical field of biomedicine, and specifically relates to a new application of imitrine in the preparation of medicines for the treatment or prevention of SARS-CoV-2 infection by the novel coronavirus.
  • coronaviruses belong to the genus Coronavirus of the order Nidovirales, Coronaviridae (Coronaviridae).
  • Viruses of the genus Coronavirus are RNA viruses with an envelope and a linear single-stranded positive-stranded genome. They are a large group of viruses that are widespread in nature and are known to cause colds and Middle East respiratory syndrome (MERS-CoV). And severe acute respiratory syndrome (caused by SARS-CoV) and other more serious diseases.
  • the new coronavirus SARS-CoV-2 is the seventh species discovered in 2019 (the remaining six species are HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV) that can infect human coronaviruses. Named by the World Health Organization on January 12, 2020, it can cause a new type of coronavirus pneumonia, COVID-19, and pose a serious threat to human health.
  • Monoclonal antibodies, peptides, and small molecule compounds are usually hotspots in antiviral drug research.
  • Emetine also known as emetine, is an isoquinoline alkaloid extracted from the Rubiaceae plant Emetine. Its molecular formula is C 29 H 40 N 2 O 4 and its structural formula is shown below.
  • the antiviral studies on evapotrin and its derivatives have shown that it is effective against dengue fever virus, human immunodeficiency virus, newcastle disease virus, petit pestis virus, varicella virus, herpes virus, and MERS-CoV, HCoV- OC43, MHV-A59, HCoV-NL63 and many other DNA viruses and RNA viruses have inhibitory activity.
  • the patent document CN108721293A selected non-toxic concentration of etorrhine for broad-spectrum anti-coronavirus research, and found that it can effectively inhibit ⁇ group coronavirus HCoV-OC43, MERS-CoV, MHV-A59 and ⁇ group coronavirus HCoV- in vitro. NL63 replicates and shows a dose-effect correlation.
  • the present invention aims to provide the application of imitrine or its pharmaceutically acceptable salt in the preparation of drugs for the treatment or prevention of coronavirus infection and a method for the treatment and prevention of SARS-CoV-2 infection by the novel coronavirus.
  • the coronavirus is a novel coronavirus SARS-CoV-2.
  • the medicament is any one of the following pharmaceutically acceptable dosage forms prepared by using tuberine or a pharmaceutically acceptable salt thereof as the active ingredient of the medicament: tablets, capsules, granules, oral liquids , Sustained release formulations, controlled release formulations, nano formulations or injections.
  • the effective concentration of the etotrorrhizine or a pharmaceutically acceptable salt thereof is 0.005 ⁇ M or more, preferably 0.010 ⁇ M or more.
  • the concentration of the totranine or its pharmaceutically acceptable salt in the blood is 2 ⁇ M or less, preferably 1 ⁇ M or less.
  • a method for treating SARS-CoV-2 infection with a new type of coronavirus which comprises administering to patients infected with the new type of coronavirus SARS-CoV-2 a therapeutically effective amount of imitorine or its pharmacological agent. Accepted salt drugs.
  • the therapeutically effective amount is such that the effective concentration of the etopyrine or a pharmaceutically acceptable salt thereof is 0.005 ⁇ M or more and 2 ⁇ M or less.
  • the therapeutically effective amount is such that the effective concentration of the etofibrine or a pharmaceutically acceptable salt thereof is 0.010 ⁇ M or more and 1 ⁇ M or less.
  • a method for preventing SARS-CoV-2 infection by a new type of coronavirus comprises administering to a subject a preventively effective amount of a drug containing etopyrine or a pharmaceutically acceptable salt thereof.
  • the prophylactically effective amount is such that the concentration of the etorrhine or its pharmaceutically acceptable salt is 0.010 ⁇ M or more and 2 ⁇ M or less.
  • the prophylactically effective amount is such that the concentration of the action concentration of the etopyrine or a pharmaceutically acceptable salt thereof is 0.010 ⁇ M or more and 1 ⁇ M or less.
  • the subject is a human.
  • the present invention uses the new coronavirus SARS-CoV-2 as a model virus, and through in vitro antiviral effects studies, it provides the preparation of imitrine or its pharmaceutically acceptable salt for the treatment or prevention of the new coronavirus SARS-CoV -2
  • the new application in drugs for infection reveals that torazine or its pharmaceutically acceptable salt can effectively inhibit the replication of the new coronavirus SARS-CoV-2, and show a dose-effect correlation, and it is resistant to the new coronavirus SARS-CoV -2 activity, and has the effect of preventing the new coronavirus SARS-CoV-2 infection, and accordingly provide a method for the treatment or prevention of the new coronavirus SARS-CoV-2 infection, for the new coronavirus SARS-CoV-2 infection Prevention, control and treatment are of great significance.
  • Figure 1 is a graph showing the inhibitory effect of different concentrations of sugarcine on the new coronavirus SARS-CoV-2;
  • panel A represents the virus infection rate curve of the new coronavirus SARS-CoV-2 under different concentrations of sugarcine and The cytotoxicity curve on Vero cells;
  • B panel represents the index data of A panel;
  • Panel C represents the Western blot test results of different concentrations of imitorine in the treatment of new coronavirus SARS-CoV-2 infection in vitro;
  • Figure 2 is a graph showing the preventive effect of different concentrations of sugarcine on the new coronavirus SARS-CoV-2;
  • Panel A represents the virus infection rate curve of the new coronavirus SARS-CoV-2 under different concentrations of sugarcine;
  • Panel B represents the index data of Panel A;
  • Panel C represents the Western blot test results of different concentrations of urotrorrhizine to prevent new coronavirus SARS-CoV-2 infection in vitro.
  • the present invention aims to provide a new application of imitrine in the preparation of drugs for the treatment or prevention of new coronavirus SARS-CoV-2 infection, which uses the new coronavirus SARS-CoV-2 as a model virus, and through in vitro antiviral effect research, It has been found that imitrine can significantly inhibit the replication of the new coronavirus SARS-CoV-2 in vitro, has biological activity against the new coronavirus SARS-CoV-2, and can be used to treat or prevent the new coronavirus SARS-CoV-2 infection Method.
  • the etofibrine of the present invention can be in the free base form as shown above, or it can be made into a pharmaceutically acceptable salt.
  • the types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of a compound with a pharmaceutically acceptable inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, Nitric acid, phosphoric acid, metaphosphoric acid, etc.; or formed by reaction with organic acids such as acetic acid, propionic acid, caproic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, lemon Acid, succinic acid, maleic acid, tartaric acid, fumaric acid, trifluoroacetic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic
  • virus culture and detection are performed in a third-level biosafety laboratory, and are operated under the biosafety regulations of microbiology and biomedical laboratories.
  • the primers used were synthesized by General Biotech Co., Ltd.; the probes used were synthesized by General Biogenes Co., Ltd.
  • Example 1 Isolation and cultivation of new coronavirus SARS-COV-2
  • This embodiment is to separate and cultivate the new coronavirus SARS-COV-2 to obtain the virus liquid of the new coronavirus SARS-COV-2, which specifically includes the following steps:
  • step 1.3 When Vero cells grow to more than 75% of the bottom area of the T75 flask, add the supernatant obtained from the 24-well plate or 48-well plate in step 1.2 to the Vero cells in the T75 culture flask at 37°C, 5% Incubate in a CO 2 incubator for 1 hour; then add 13 mL of DMEM medium containing 5% FBS, and continue to incubate for 48 hours; use an inverted microscope to observe whether Vero cells reach approximately 75% CPE.
  • This experiment measures the virulence of the viral liquid of the new coronavirus SARS-COV-2 obtained in step 1.4 above, specifically: the Vero cells in the logarithmic growth phase are digested and counted and then inoculated on a transparent 96-well cell culture plate (BeaverBio, On China), the cell density is 2 ⁇ 10 4 cells/well, cultured in a 37°C, 5% CO 2 incubator, and the culture medium is DMEM medium containing 10% FBS and 1% double antibody.
  • the cells grow into a monolayer, discard the culture medium, and add the SARS-CoV-2 virus solution serially diluted with the culture medium 10 times, the dilutions of which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 12 replicate wells for each dilution concentration, and control of normal cells and undiluted virus solution at the same time, then place them in a 37°C, 5% CO 2 incubator, and observe daily Cell lesions and CPE conditions were recorded, and observed until CPE appeared, when the lesions no longer developed, the results were recorded, and the 50% tissue cell infection amount of the virus (TCID 50 ) was recorded.
  • TCID 50 50% tissue cell infection amount of the virus
  • L the logarithm of the highest dilution
  • D the difference between the logarithms of the dilution
  • S is the sum of the ratios of positive wells.
  • the determination of virus infectivity is one of the commonly used methods to assess its virulence. Usually, half of the tissue (cell) culture infectious dose (50% tissue culture infectious dose, TCID 50 ) of cell culture is measured to evaluate the infectivity (virulence) of the virus. ), the TCID 50 in this experiment is 10 -4.25 .
  • the culture plate was placed in a 37°C, 5% CO 2 incubator for 3 days, and the CCK-8 method was used to determine the toxicity of different concentrations of etorrhine to Vero cells to evaluate the toxic effect of etorrhine on the cells.
  • CC 50 refers to the concentration of the drug that causes 50% of the cells to become pathological.
  • the primer sequence used was: SA-F: 5'-CAATGGTTTAACAGGCACAGG-3' (SEQ ID NO: 1); SA-R: 5'-CTCAAGTGTCTGTGGATCACG-3' (SEQ ID NO: 2); Probe sequence: SA-probe: 5'-FAM-GGCAGAGACATTGCTGACACTACTGATGC-BHQ-3' (SEQ ID NO: 3, where FAM is a fluorescent reporter group and BHQ is a fluorescent quenching group)
  • the PCR system is: real-time fluorescence quantitative one-step PCR reaction solution 2 ⁇ One Step RT-PCR Buffer III (TAKARA) 10 ⁇ L, PrimeScript RT Enzyme Mix II (TAKARA) 0.4 ⁇ L, 5 U/ ⁇ L TaKaRa Ex Taq HS (TAKARA) 0.4 ⁇ L , SA-F 0.4 ⁇ L (final concentration 0.1 ⁇ 1.0 ⁇ M), SA-R 0.4 ⁇ L (final concentration 0.1 ⁇ 1.0 ⁇ M), SA-probe 0.8 ⁇ L
  • the amount of template RNA (10pg-100ng) used is 2 ⁇ L.
  • the LightCycler Real Time PCR amplification instrument is used for the amplification reaction.
  • the amplification procedure is: reverse transcription reaction: 42°C 5min; 95°C 10sec; 1 cycle; PCR reaction procedure: 95°C 5sec; 60°C 20sec; 40 Cycles.
  • the experimental settings are: normal cell control group (the culture medium is DMEM medium containing 10% FBS and 1% double antibody plus 0.1% DMSO), the normal virus control group (the culture medium contains 10% FBS and 1% double antibody The DMEM medium plus 0.1% DMSO), imitrine (8 concentrations) + virus group (3 parallel).
  • the left side shows the inhibitory effect of different concentrations of saponine on the new coronavirus SARS-CoV-2. It can be seen that with the increase of the concentration, the inhibitory effect of the new type of coronavirus SARS-CoV-2 is more obvious (that is, the infection rate of the virus is significantly reduced), which proves that it can strongly inhibit the SARS-CoV-2 virus. Copy. Web shown in FIG.
  • emetine was obtained by calculation of an IC novel coronavirus is SARS-CoV-2 50 (IC 50 refers to a drug concentration effective to inhibit viral infection of cells by 50%) was 0.007 ⁇ 0.002 ⁇ M
  • concentration of etorrhizine does not have any toxic side effects on Vero cells (the CC 50 of etorrhizine to Vero cells is 1.96 ⁇ 0.37 ⁇ M).
  • the western blot method was used to detect the nucleocapsid expression of SARS-CoV-2 virus in each group of test samples (the antibody used was purchased from Sino biological, Cat: 40588-T62, China), and GAPDH was used as an internal reference.
  • test results are shown in panel C in Figure 1.
  • the four groups of concentrations (0.01 ⁇ M, 0.03 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, as the treatment group) of stigmatine can all show resistance to the new coronavirus
  • the nucleocapsid expression of SARS-CoV-2 is significantly inhibited, and the estimated EC 50 of the new coronavirus SARS-CoV-2 is about 0.01 ⁇ M; especially when the concentration of the effect of it exceeds 0.1 ⁇ M.
  • the effective concentration of etorrhine (that is, the concentration of etofibrine in the blood of the patient after the drug is administered to a virus-infected patient) can be 0.005 ⁇ M or more, more preferably 0.01 ⁇ M or more, which can be effectively treated Infection with the new coronavirus SARS-COV-2.
  • the concentration of the drug in the blood is preferably 2 ⁇ M or less, more preferably 1 ⁇ M or less.
  • the dosage form of the drug can be any one of the following pharmaceutically acceptable dosage forms made with evapotranine as the active ingredient of the drug: tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, nanometers Preparation or injection.
  • Virus infection select a virus solution with a TCID 50 of 20-30 to infect cells, and the volume of each well is 20 ⁇ L; infection for 1-2 hours (note: during the virus infection period, the drug is still there); infection During the process, gently shake left and right to ensure full contact between the virus and the cells;
  • the test results are shown in Panel A in Figure 2, which shows the preventive effect of different concentrations of spitrootine on the new coronavirus SARS-CoV-2. It can be seen that with the increase of the concentration, the preventive effect of spitrootine on the new coronavirus SARS-CoV-2 infection is more obvious (that is, the infection rate of the virus is significantly reduced), as shown in panel B in Figure 2, after calculating spitrootine
  • the IC 50 for the prevention of SARS-CoV-2 infection with the new coronavirus is 0.019 ⁇ 0.009 ⁇ M, and the concentration of imitine at this time is not toxic to Vero cells (the CC 50 of imitine to Vero cells is 1.96 ⁇ 0.37 ⁇ M). Side effects, which proves that a safe concentration of turbinine can effectively prevent the infection of the new coronavirus SARS-CoV-2 in vitro.
  • Virus infection select the virus solution with a TCID 50 of 20-30 to infect the cells, with a volume of 0.5 mL per well; infection for 1-2 hours; gently shake left and right during the infection process to ensure full contact between the virus and the cells;
  • test results are shown in panel C in Figure 2. It can be seen that, compared with the negative control, the four groups of concentrations (0.01 ⁇ M, 0.03 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, as the prevention group) of ephedrine can all show resistance to the new type.
  • the nucleocapsid expression of the coronavirus SARS-CoV-2 is significantly inhibited, and the estimated EC 50 of the new coronavirus SARS-CoV-2 is about 0.01 ⁇ M; especially when the concentration of the effect exceeds 0.01 ⁇ M.
  • Example 3 From the results of Example 3 above, it can be seen that in the presence of a safe concentration of serotonin, it can effectively prevent the infection of the new coronavirus SARS-CoV-2, so it can be used to prepare and prevent the new coronavirus SARS-COV- 2 Infectious drugs, and preferably, after the drug is administered, the concentration of etorrhine can be 0.01 ⁇ M or more, preferably 0.02 ⁇ M or more and 2 ⁇ M or less, which can effectively prevent the infection of the new coronavirus SARS-COV-2.
  • the dosage form of the drug can be any one of the following pharmaceutically acceptable dosage forms made with etotrorrhizine or its derivatives or medicinal salts as the active ingredient of the drug: tablets, capsules, granules, oral liquids, suspensions Release formulations, controlled release formulations, nano formulations or injections.
  • the present invention provides the application of imitrine or its pharmaceutically acceptable salt in the preparation of drugs for treating or preventing coronavirus infection, which can realize the prevention and control of SARS-CoV-2 infection of the new coronavirus and is suitable for industrial application.

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Abstract

An application of emetine in preparation of a drug for treating or preventing novel coronaviruses SARS-CoV-2. Anti-novel coronavirus SARS-CoV-2 research is conducted by selecting emetine having a safe concentration, proving that emetine can effectively inhibit replication of novel coronaviruses SARS-CoV-2, and prevent infection of the novel coronaviruses SARS-CoV-2, and is used for further developing an anti-novel coronavirus SARS-CoV-2 drug.

Description

吐根碱在制备治疗或预防新型冠状病毒SARS-CoV-2感染的药物中的应用Application of imitrine in the preparation of medicines for the treatment or prevention of new coronavirus SARS-CoV-2 infection 技术领域Technical field
本发明属于生物医药技术领域，具体涉及吐根碱在制备治疗或预防新型冠状病毒SARS-CoV-2感染的药物中的新应用。The present invention belongs to the technical field of biomedicine, and specifically relates to a new application of imitrine in the preparation of medicines for the treatment or prevention of SARS-CoV-2 infection by the novel coronavirus.
背景技术Background technique
冠状病毒在系统分类上属套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus)。冠状病毒属的病毒是具囊膜(envelope)、基因组为线性单股正链的RNA病毒，是自然界广泛存在的一大类病毒，已知可引起感冒以及中东呼吸综合征(MERS-CoV引起)和严重急性呼吸综合征(SARS-CoV引起)等较严重疾病。In the systematic classification, coronaviruses belong to the genus Coronavirus of the order Nidovirales, Coronaviridae (Coronaviridae). Viruses of the genus Coronavirus are RNA viruses with an envelope and a linear single-stranded positive-stranded genome. They are a large group of viruses that are widespread in nature and are known to cause colds and Middle East respiratory syndrome (MERS-CoV). And severe acute respiratory syndrome (caused by SARS-CoV) and other more serious diseases.
新型冠状病毒SARS-CoV-2是2019年发现的第7种(其余六种分别是HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV)可以感染人的冠状病毒，于2020年1月12日被世界卫生组织命名，其可以引发新型冠状病毒肺炎COVID-19，对人类的身体健康产生严重的威胁。The new coronavirus SARS-CoV-2 is the seventh species discovered in 2019 (the remaining six species are HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV) that can infect human coronaviruses. Named by the World Health Organization on January 12, 2020, it can cause a new type of coronavirus pneumonia, COVID-19, and pose a serious threat to human health.
单克隆抗体、多肽和小分子化合物通常是抗病毒药物研究的热点，通过对FDA已批准的药物库进行高通量筛选，探讨已有药物的新用途，已成为药物研发的一种重要途径。由于候选药物已具有关于药理药效测试、功能靶点以及临床安全性等资料，有利于进一步的毒理学评价、药代动力学评价和制剂研发等，可以大大减少研发风险、缩短研发时间和研发成本，有着广阔的应用前景。Monoclonal antibodies, peptides, and small molecule compounds are usually hotspots in antiviral drug research. Through high-throughput screening of FDA-approved drug libraries and exploring new uses of existing drugs, it has become an important way for drug development. Since the candidate drug already has information on pharmacological efficacy testing, functional targets, and clinical safety, it is conducive to further toxicological evaluation, pharmacokinetic evaluation, and formulation development, etc., which can greatly reduce R&D risks, shorten R&D time and R&D Cost has broad application prospects.
吐根碱(emetine)也称依米丁，是从茜草科植物吐根中提取的一种异喹啉生物碱，分子式为C 29H 40N 2O 4，其结构式如下文所示。对吐根碱及衍生物的抗病毒研究表明，吐根碱及衍生物对登革热病毒、人免疫缺陷病毒、新城疫病毒、小反刍兽疫病毒、水牛痘病毒、疱疹病毒以及MERS-CoV、HCoV-OC43、MHV-A59、HCoV-NL63等多种DNA病毒和RNA病毒均有抑制活性。其中MERS-CoV抗病毒药物高通量筛选研究发现，吐根碱具有一定的抗冠状病毒的活性，对0.0001MOI剂量的MERS-CoV感染的药物半数有效浓度(Concentration for 50％of maximal effect，EC 50)为14.7μM，半数细胞毒性浓度(Concentration cytotoxicity 50％，CC 50)为17μM，药物安全指数为1.5，提示吐根碱具有一定的抗冠状病毒的活性(Chan JF，Chan KH，Kao RY，等.Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.J Infect，2013，67，606-616)。专利文献CN108721293A选用无毒性浓度的吐根碱进行广谱抗冠状病毒研究，发现吐根碱能在体外有效抑制β群冠状病毒HCoV-OC43、MERS-CoV、MHV-A59以及α 群冠状病毒HCoV-NL63的复制，并呈现剂量效应相关性，针对MOI＝0.01剂量的HCoV-OC43、HCoV-NL63、MERS-CoV和MHV-A59四种冠状病毒的药物EC 50分别为0.30μM、1.75μM、0.35μM和0.12μM，显示出吐根碱具有广谱抗冠状病毒活性。 Emetine, also known as emetine, is an isoquinoline alkaloid extracted from the Rubiaceae plant Emetine. Its molecular formula is C 29 H 40 N 2 O 4 and its structural formula is shown below. The antiviral studies on evapotrin and its derivatives have shown that it is effective against dengue fever virus, human immunodeficiency virus, newcastle disease virus, petit pestis virus, varicella virus, herpes virus, and MERS-CoV, HCoV- OC43, MHV-A59, HCoV-NL63 and many other DNA viruses and RNA viruses have inhibitory activity. Among them, the high-throughput screening study of MERS-CoV antiviral drugs has found that stigmatine has a certain anti-coronavirus activity, and the half effective concentration of the drug for MERS-CoV infection at 0.0001MOI dose (Concentration for 50% of maximal effect, EC 50 ) is 14.7 μM, the half cytotoxicity concentration (Concentration cytotoxicity 50%, CC 50 ) is 17 μM, and the drug safety index is 1.5, suggesting that it has certain anti-coronavirus activity (Chan JF, Chan KH, Kao RY, Etc. Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus. J Infect, 2013, 67, 606-616). The patent document CN108721293A selected non-toxic concentration of etorrhine for broad-spectrum anti-coronavirus research, and found that it can effectively inhibit β group coronavirus HCoV-OC43, MERS-CoV, MHV-A59 and α group coronavirus HCoV- in vitro. NL63 replicates and shows a dose-effect correlation. The EC 50 for the four coronaviruses of HCoV-OC43, HCoV-NL63, MERS-CoV and MHV-A59 at MOI=0.01 doses are 0.30μM, 1.75μM, 0.35μM, respectively And 0.12μM, showing that ephemidine has broad-spectrum anti-coronavirus activity.
然而，目前尚未见吐根碱在抗新型冠状病毒SARS-CoV-2中的应用报道。However, there has been no report on the application of oxiranine in the anti-new coronavirus SARS-CoV-2.
发明内容Summary of the invention
本发明旨在提供吐根碱或其药学可接受的盐在制备治疗或预防冠状病毒感染的药物中的应用及治疗和预防新型冠状病毒SARS-CoV-2感染的方法。The present invention aims to provide the application of imitrine or its pharmaceutically acceptable salt in the preparation of drugs for the treatment or prevention of coronavirus infection and a method for the treatment and prevention of SARS-CoV-2 infection by the novel coronavirus.
在本发明的第一个方面，提供了吐根碱或其药学可接受的盐在制备治疗或预防冠状病毒感染的药物中的应用，所述冠状病毒为新型冠状病毒SARS-CoV-2。In the first aspect of the present invention, there is provided the use of imitrine or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment or prevention of coronavirus infections, and the coronavirus is a novel coronavirus SARS-CoV-2.
在一个实施方案中，所述药物是以吐根碱或其药学可接受的盐作为药物活性成分制成的以下任何一种药学上可接受的剂型：片剂、胶囊剂、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。In one embodiment, the medicament is any one of the following pharmaceutically acceptable dosage forms prepared by using tuberine or a pharmaceutically acceptable salt thereof as the active ingredient of the medicament: tablets, capsules, granules, oral liquids , Sustained release formulations, controlled release formulations, nano formulations or injections.
在一个实施方案中，当所述药物在施用后，所述吐根碱或其药学可接受的盐的作用浓度为0.005μM以上，优选为0.010μM以上。In one embodiment, when the drug is administered, the effective concentration of the etotrorrhizine or a pharmaceutically acceptable salt thereof is 0.005 μM or more, preferably 0.010 μM or more.
为了避免所述药物对于正常细胞产生不可接受的毒性，所述吐根碱或其药学可接受的盐在血液中的作用浓度为2μM以下，优选为1μM以下。In order to avoid the unacceptable toxicity of the drug to normal cells, the concentration of the totranine or its pharmaceutically acceptable salt in the blood is 2 μM or less, preferably 1 μM or less.
在本发明的第二方面，提供一种治疗新型冠状病毒SARS-CoV-2感染的方法，其包括向新型冠状病毒SARS-CoV-2感染患者施用治疗有效量的含有吐根碱或其药学可接受的盐的药物。In the second aspect of the present invention, there is provided a method for treating SARS-CoV-2 infection with a new type of coronavirus, which comprises administering to patients infected with the new type of coronavirus SARS-CoV-2 a therapeutically effective amount of imitorine or its pharmacological agent. Accepted salt drugs.
在一个实施方案中，所述治疗有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.005μM以上且为2μM以下。In one embodiment, the therapeutically effective amount is such that the effective concentration of the etopyrine or a pharmaceutically acceptable salt thereof is 0.005 μM or more and 2 μM or less.
在一个实施方案中，所述治疗有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为1μM以下。In one embodiment, the therapeutically effective amount is such that the effective concentration of the etofibrine or a pharmaceutically acceptable salt thereof is 0.010 μM or more and 1 μM or less.
在本发明的第三方面，提供一种预防新型冠状病毒SARS-CoV-2感染的方法，其包括向受试者施用预防有效量的含有吐根碱或其药学可接受的盐的药物。In the third aspect of the present invention, a method for preventing SARS-CoV-2 infection by a new type of coronavirus is provided, which comprises administering to a subject a preventively effective amount of a drug containing etopyrine or a pharmaceutically acceptable salt thereof.
在一个实施方案中，所述预防有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为2μM以下。In one embodiment, the prophylactically effective amount is such that the concentration of the etorrhine or its pharmaceutically acceptable salt is 0.010 μM or more and 2 μM or less.
在一个实施方案中，所述预防有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为1μM以下。In one embodiment, the prophylactically effective amount is such that the concentration of the action concentration of the etopyrine or a pharmaceutically acceptable salt thereof is 0.010 μM or more and 1 μM or less.
在一个实施方案中，所述受试者为人。In one embodiment, the subject is a human.
基于以上技术方案，本发明以新型冠状病毒SARS-CoV-2作为模式病毒，通过体外抗病毒效果研究，提供了吐根碱或其药学可接受的盐在制备治疗或预防新型冠状病毒 SARS-CoV-2感染的药物中的新应用，揭示吐根碱或其药学可接受的盐能够有效抑制新型冠状病毒SARS-CoV-2的复制，并呈现剂量效应相关性，具有抗新型冠状病毒SARS-CoV-2活性，并具有预防新型冠状病毒SARS-CoV-2感染的效果，据此提供一种治疗或预防新型冠状病毒SARS-CoV-2感染的方法，对于新型冠状病毒SARS-CoV-2感染的防控和治疗具有重要意义。Based on the above technical solutions, the present invention uses the new coronavirus SARS-CoV-2 as a model virus, and through in vitro antiviral effects studies, it provides the preparation of imitrine or its pharmaceutically acceptable salt for the treatment or prevention of the new coronavirus SARS-CoV -2 The new application in drugs for infection reveals that torazine or its pharmaceutically acceptable salt can effectively inhibit the replication of the new coronavirus SARS-CoV-2, and show a dose-effect correlation, and it is resistant to the new coronavirus SARS-CoV -2 activity, and has the effect of preventing the new coronavirus SARS-CoV-2 infection, and accordingly provide a method for the treatment or prevention of the new coronavirus SARS-CoV-2 infection, for the new coronavirus SARS-CoV-2 infection Prevention, control and treatment are of great significance.
附图说明Description of the drawings
图1为示出不同浓度吐根碱对新型冠状病毒SARS-CoV-2的抑制效果的图；其中A幅表示不同浓度吐根碱条件下新型冠状病毒SARS-CoV-2的病毒感染率曲线和对Vero细胞的细胞毒性曲线；B幅表示A幅的指标数据；C幅表示不同浓度吐根碱在体外治疗新型冠状病毒SARS-CoV-2感染的Western blot检测结果；Figure 1 is a graph showing the inhibitory effect of different concentrations of sugarcine on the new coronavirus SARS-CoV-2; panel A represents the virus infection rate curve of the new coronavirus SARS-CoV-2 under different concentrations of sugarcine and The cytotoxicity curve on Vero cells; B panel represents the index data of A panel; Panel C represents the Western blot test results of different concentrations of imitorine in the treatment of new coronavirus SARS-CoV-2 infection in vitro;
图2为示出不同浓度吐根碱对新型冠状病毒SARS-CoV-2的预防效果的图；其中A幅表示不同浓度吐根碱条件下新型冠状病毒SARS-CoV-2的病毒感染率曲线；B幅表示A幅的指标数据；C幅表示不同浓度吐根碱在体外预防新型冠状病毒SARS-CoV-2感染的Western blot检测结果。Figure 2 is a graph showing the preventive effect of different concentrations of sugarcine on the new coronavirus SARS-CoV-2; Panel A represents the virus infection rate curve of the new coronavirus SARS-CoV-2 under different concentrations of sugarcine; Panel B represents the index data of Panel A; Panel C represents the Western blot test results of different concentrations of urotrorrhizine to prevent new coronavirus SARS-CoV-2 infection in vitro.
具体实施方式Detailed ways
本发明旨在提供吐根碱在制备治疗或预防新型冠状病毒SARS-CoV-2感染的药物中的新应用，其以新型冠状病毒SARS-CoV-2作为模式病毒，通过体外抗病毒效果研究，发现吐根碱能在体外显著抑制新型冠状病毒SARS-CoV-2的复制，具有抗新型冠状病毒SARS-CoV-2的生物活性，可以用于治疗或预防新型冠状病毒SARS-CoV-2感染的方法中。The present invention aims to provide a new application of imitrine in the preparation of drugs for the treatment or prevention of new coronavirus SARS-CoV-2 infection, which uses the new coronavirus SARS-CoV-2 as a model virus, and through in vitro antiviral effect research, It has been found that imitrine can significantly inhibit the replication of the new coronavirus SARS-CoV-2 in vitro, has biological activity against the new coronavirus SARS-CoV-2, and can be used to treat or prevent the new coronavirus SARS-CoV-2 infection Method.
吐根碱的游离碱形式的化学结构如下。The chemical structure of the free base form of imitrine is as follows.
Figure PCTCN2020084674-appb-000001
Figure PCTCN2020084674-appb-000001
本发明的吐根碱可以是如上所示的游离碱形式，也可以被制成药学可接受的盐。药学可接受的盐的类型包括但不限于：(1)酸加成盐、通过将化合物的游离碱形式与药学可接受的无机酸反应形成，所述无机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸、偏磷酸等；或与有机酸反应形成，所述有机酸如乙酸、丙酸、己酸、环戊烷丙酸、羟基乙酸、丙酮酸、乳酸、丙二酸、苹果酸、柠檬酸、琥珀酸、马来酸、酒石酸、反丁烯 二酸、三氟乙酸、苯甲酸、3-(4-羟基苯甲酰基)苯甲酸、肉桂酸、扁桃酸、甲烷磺酸、乙烷磺酸、1，2-乙二磺酸、2-羟基乙磺酸、苯磺酸、甲苯磺酸、4-甲基双环-[2.2.2]辛-2-烯-1-甲酸、2-萘磺酸、叔丁基乙酸、葡庚糖酸、4,4'-亚甲基双-(3-羟基-2-烯-1-甲酸)、3-苯基丙酸、三甲基乙酸、十二烷基硫酸、葡糖酸、谷氨酸、水杨酸、羟基萘酸、硬脂酸、粘康酸等。本发明优选的吐根碱药学可接受的盐是如下结构所示的盐酸盐。The etofibrine of the present invention can be in the free base form as shown above, or it can be made into a pharmaceutically acceptable salt. The types of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of a compound with a pharmaceutically acceptable inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, Nitric acid, phosphoric acid, metaphosphoric acid, etc.; or formed by reaction with organic acids such as acetic acid, propionic acid, caproic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, lemon Acid, succinic acid, maleic acid, tartaric acid, fumaric acid, trifluoroacetic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid Acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, 2-naphthalene Sulfonic acid, tert-butyl acetic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, ten Dialkyl sulfuric acid, gluconic acid, glutamic acid, salicylic acid, hydroxynaphthoic acid, stearic acid, muconic acid, etc. The preferred pharmaceutically acceptable salt of etopyrine in the present invention is the hydrochloride shown in the following structure.
Figure PCTCN2020084674-appb-000002
Figure PCTCN2020084674-appb-000002
以下结合具体实施例，对本发明进一步阐述。应当理解的是，具体实施例仅用于进一步说明本发明，而不是用于限制本发明的内容。The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments are only used to further illustrate the present invention, rather than to limit the content of the present invention.
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。The methods used in the following examples are conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。The ways to obtain various biological materials described in the examples are only to provide an experimental way to achieve specific disclosure purposes, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the prompts in the examples.
以下实施例中所有病毒培养和检测均在生物安全三级实验室进行，在微生物和生物医学实验室的生物安全规定下操作。In the following examples, all virus culture and detection are performed in a third-level biosafety laboratory, and are operated under the biosafety regulations of microbiology and biomedical laboratories.
所用引物由通用生物科技有限公司合成；所用探针由通用生物基因公司合成。The primers used were synthesized by General Biotech Co., Ltd.; the probes used were synthesized by General Biogenes Co., Ltd.
实施例1：新型冠状病毒SARS-COV-2的分离培养Example 1: Isolation and cultivation of new coronavirus SARS-COV-2
该实施例为对新型冠状病毒SARS-COV-2进行分离培养，以获得新型冠状病毒SARS-COV-2的病毒液，具体包括以下步骤：This embodiment is to separate and cultivate the new coronavirus SARS-COV-2 to obtain the virus liquid of the new coronavirus SARS-COV-2, which specifically includes the following steps:
1.1、首先在T75培养瓶中培养Vero细胞(非洲绿猴肾细胞，ATCC，美国)，培养基为DMEM培养基(Corning，美国)、10％血清(FBS)(ExCellBio，新西兰)和1％双抗(Corning，美国)，培养条件为37℃，5％CO 2的培养箱，备用。 1.1. First, culture Vero cells (African green monkey kidney cells, ATCC, USA) in a T75 culture flask with DMEM medium (Corning, USA), 10% serum (FBS) (ExCellBio, New Zealand) and 1% double Anti-(Corning, USA), culture condition is 37°C, 5% CO 2 incubator, spare.
1.2、从确诊感染新型冠状病毒SARS-COV-2的患者的咽拭子分离(由中国疾病预防控制中心(CDC)完成)获取新型冠状病毒SARS-COV-2，随后将该病毒感染在24 孔板或48孔板中培养至单层的Vero细胞，感染后待Vero细胞出现75％细胞病变(CPE)后取上清继续感染步骤1.1中备用的T75培养瓶中的Vero细胞进行扩增。1.2. Obtain the new coronavirus SARS-COV-2 from the throat swabs of patients confirmed to be infected with the new coronavirus SARS-COV-2 (completed by the Chinese Center for Disease Control and Prevention (CDC)), and then infect the virus in 24 wells Vero cells cultured to a single layer in a plate or 48-well plate, after infection, after the Vero cells appear 75% cytopathic (CPE), the supernatant is taken to continue to infect the Vero cells in the spare T75 culture flask in step 1.1 for expansion.
1.3、当Vero细胞长到超过T75瓶底面积75％时，将步骤1.2中从24孔板或48孔板中获取的上清添加至T75培养瓶中的Vero细胞中，在37℃，5％CO 2的培养箱中孵育1小时；随后添加13mL的含5％FBS的DMEM培养基，继续孵育48小时；用倒置显微镜观察Vero细胞是否达到大约75％CPE。 1.3. When Vero cells grow to more than 75% of the bottom area of the T75 flask, add the supernatant obtained from the 24-well plate or 48-well plate in step 1.2 to the Vero cells in the T75 culture flask at 37°C, 5% Incubate in a CO 2 incubator for 1 hour; then add 13 mL of DMEM medium containing 5% FBS, and continue to incubate for 48 hours; use an inverted microscope to observe whether Vero cells reach approximately 75% CPE.
1.4、收集T75瓶中细胞和上清，反复冻融3次裂解细胞，2000rpm条件下离心10分钟除去细胞碎片，上清作为新型冠状病毒SARS-COV-2的病毒液分装冻存，-80℃保存备用。1.4. Collect the cells and supernatant in the T75 flask, freeze and thaw 3 times to lyse the cells, centrifuge at 2000 rpm for 10 minutes to remove cell debris, and store the supernatant as the virus solution of the new coronavirus SARS-COV-2. Store at ℃ for later use.
1.5、病毒毒力测定1.5, virus virulence determination
该实验对上述步骤1.4获得的新型冠状病毒SARS-COV-2的病毒液的毒力进行测定，具体为：将对数生长期的Vero细胞消化计数后接种在透明96孔细胞培养板(BeaverBio，中国)上，细胞密度为2×10 4个细胞/每孔，37℃、5％CO 2培养箱中培养，培养液为含10％FBS、1％双抗的DMEM培养基。待细胞长成单层后，弃培养液，加入用培养液10倍系列稀释的SARS-CoV-2病毒液，其稀释度分别为10 -1、10 -2、10 -3、10 -4、10 -5、10 -6、10 -7，每个稀释浓度设置12个复孔，同时设立正常细胞和未稀释病毒液对照，然后置37℃、5％CO 2培养箱中培养，每日观察细胞病变并记录CPE情况，观察直至CPE出现为止，当病变不再发展时记录结果，并记录病毒的50％组织细胞感染量(TCID 50)。TCID 50的计算公式为：Log(TCID 50)＝L-D(S-0.5)；其中L为最高稀释度的对数，D为稀释度对数之间的差，S为阳性孔比率总和。病毒感染能力测定是评估其毒力的常用方法之一，通常测定细胞培养物的半数组织(细胞)培养物感染量(50％tissue culture infectious dose，TCID 50)来评估病毒的感染能力(毒力)，本实验中TCID 50为10 -4.25。 This experiment measures the virulence of the viral liquid of the new coronavirus SARS-COV-2 obtained in step 1.4 above, specifically: the Vero cells in the logarithmic growth phase are digested and counted and then inoculated on a transparent 96-well cell culture plate (BeaverBio, On China), the cell density is 2×10 4 cells/well, cultured in a 37°C, 5% CO 2 incubator, and the culture medium is DMEM medium containing 10% FBS and 1% double antibody. After the cells grow into a monolayer, discard the culture medium, and add the SARS-CoV-2 virus solution serially diluted with the culture medium 10 times, the dilutions of which are 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 12 replicate wells for each dilution concentration, and control of normal cells and undiluted virus solution at the same time, then place them in a 37°C, 5% CO 2 incubator, and observe daily Cell lesions and CPE conditions were recorded, and observed until CPE appeared, when the lesions no longer developed, the results were recorded, and the 50% tissue cell infection amount of the virus (TCID 50 ) was recorded. The calculation formula of TCID 50 is: Log(TCID 50 )=LD(S-0.5); where L is the logarithm of the highest dilution, D is the difference between the logarithms of the dilution, and S is the sum of the ratios of positive wells. The determination of virus infectivity is one of the commonly used methods to assess its virulence. Usually, half of the tissue (cell) culture infectious dose (50% tissue culture infectious dose, TCID 50 ) of cell culture is measured to evaluate the infectivity (virulence) of the virus. ), the TCID 50 in this experiment is 10 -4.25 .
实施例2：吐根碱的体外抑制病毒效果检测Example 2: Detection of the in vitro antiviral effect of imitrine
2.1、吐根碱的细胞毒性测定2.1. Cytotoxicity determination of imitrine
1)将吐根碱盐酸盐(购自MCE，中国)采用二甲基亚砜(DMSO)进行梯度稀释，初始浓度为20mM，3倍比稀释，共7个浓度，吸取每个浓度的吐根碱溶液1μL至1mL含2％FBS、1％双抗的DMEM维持液中，作为药物溶液，共7组，用于后续实验。1) Dilute spitrootine hydrochloride (purchased from MCE, China) with dimethyl sulfoxide (DMSO). The initial concentration is 20 mM, and it is diluted by 3 times. There are 7 concentrations in total. 1 μL to 1 mL of the rootine solution in DMEM maintenance solution containing 2% FBS and 1% bi-antibody as a drug solution, a total of 7 groups, used for subsequent experiments.
2)将对数生长期的Vero细胞消化计数后接种在透明96孔细胞培养板上，细胞密度为2×10 5个细胞/每孔，37℃、5％CO 2培养箱中培养，待细胞长成单层后，弃细胞培养液，分别加入上述7组含不同浓度吐根碱的药物溶液0.1mL，每个浓度设置3个复孔，同时设立正常细胞和空白对照。将培养板置37℃、5％CO 2培养箱中培养3天，采用CCK-8方法测定不同浓度吐根碱对Vero细胞的毒性，以评价吐根碱对细胞 的毒性作用。 2) Digest and count Vero cells in the logarithmic growth phase and inoculate them on a transparent 96-well cell culture plate at a cell density of 2×10 5 cells/well, culture in a 37°C, 5% CO 2 incubator, and wait for the cells to After growing into a monolayer, discard the cell culture medium, and add 0.1 mL of the above 7 groups of drug solutions containing different concentrations of ephedrine, each with 3 multiple wells for each concentration, and set up normal cells and a blank control at the same time. The culture plate was placed in a 37°C, 5% CO 2 incubator for 3 days, and the CCK-8 method was used to determine the toxicity of different concentrations of etorrhine to Vero cells to evaluate the toxic effect of etorrhine on the cells.
如图1中A幅所示，右侧表示不同浓度吐根碱对Vero细胞的细胞毒性检测结果，可见随着浓度升高，吐根碱对Vero细胞的细胞毒性增强，图1中B幅示出了吐根碱对Vero细胞的CC 50为1.96±0.37μM。CC 50是指使得50％的细胞发生病变时的药物浓度。 As shown in panel A in Figure 1, the right side shows the cytotoxicity test results of different concentrations of etorrhine on Vero cells. It can be seen that as the concentration increases, the cytotoxicity of etorrhine on Vero cells increases. Panel B in Figure 1 shows The CC 50 of emmetrorrhizine to Vero cells was 1.96±0.37μM. CC 50 refers to the concentration of the drug that causes 50% of the cells to become pathological.
2.2、吐根碱对SARS-CoV-2病毒的体外抑制作用检测2.2. Detection of the in vitro inhibitory effect of imitrine on SARS-CoV-2 virus
1)将吐根碱采用DMSO进行梯度稀释，初始浓度为1mM，3倍比稀释，共8个浓度，吸取每个浓度的吐根碱溶液1μL至1mL含2％FBS、1％双抗的DMEM维持培养基中，作为细胞维持液，共8组，用于后续实验；1) Use DMSO to carry out a gradient dilution of stigmine, the initial concentration is 1mM, 3 times the dilution ratio, a total of 8 concentrations, draw each concentration of stigmine solution 1μL to 1mL DMEM containing 2% FBS, 1% double antibody In the maintenance medium, as the cell maintenance solution, there are a total of 8 groups for subsequent experiments;
2)取一瓶单层致密Vero细胞，首先将细胞消化后加入新鲜DMEM培养基(含10％FBS和1％双抗)，分散均匀后将细胞密度稀释至2.0×10 5个细胞/mL； 2) Take a bottle of monolayer dense Vero cells, first digest the cells and add fresh DMEM medium (containing 10% FBS and 1% bi-antibody), and dilute the cell density to 2.0×10 5 cells/mL after evenly dispersing;
3)在96孔板中每孔加入100μL细胞悬液(2.0×10 4个/孔)，置于细胞培养箱，37℃、5％CO 2条件下培养24h后观察细胞生长情况； 3) Add 100 μL of cell suspension (2.0×10 4 cells/well) to each well of a 96-well plate, place it in a cell incubator, and observe the cell growth after 24 hours at 37°C and 5% CO 2;
4)待细胞贴壁达到底面积95％左右时，吸出细胞培养板孔内的培养液，每孔以100μL维持培养基(含2％FBS、1％双抗的DMEM维持培养基)洗涤2遍；选择TCID 50为20～30的病毒液(根据实施例1中1.5的方法计算获得，下同)感染细胞，每孔染毒体积为20μL；感染1.5h；感染过程中轻轻左右摇匀，保证病毒与细胞充分接触； 4) When the cell adhesion reaches about 95% of the bottom area, aspirate the culture solution in the cell culture plate well, and wash each well with 100μL maintenance medium (DMEM maintenance medium containing 2% FBS and 1% double antibody) twice ; Select a virus solution with a TCID 50 of 20-30 (calculated according to the method 1.5 in Example 1, the same below) to infect cells, with a volume of 20 μL per well; infection for 1.5 hours; gently shake left and right during infection, Ensure that the virus is in full contact with the cell;
5)吸去上清，然后使用维持培养基洗去未吸附的病毒(2次)，每孔分别加入上述8组含有不同浓度吐根碱的细胞维持液100μL，每个浓度设置3个平行，48小时后冻融一次收集病毒细胞，进行PCR检测，使用的引物序列为：SA-F：5’-CAATGGTTTAACAGGCACAGG-3’(SEQ ID NO:1)；SA-R：5’-CTCAAGTGTCTGTGGATCACG-3’(SEQ ID NO:2)；探针序列：SA-probe：5’-FAM-GGCAGAGACATTGCTGACACTACTGATGC-BHQ-3’(SEQ ID NO:3，其中FAM为荧光报告基团，BHQ为荧光淬灭基团)；PCR体系为：实时荧光定量一步法PCR反应液2×One Step RT-PCR Buffer III(TAKARA)10μL、PrimeScript RT Enzyme Mix Ⅱ(TAKARA)0.4μL、5U/μL TaKaRa Ex Taq HS(TAKARA)0.4μL、SA-F 0.4μL(终浓度0.1～1.0μM)、SA-R 0.4μL(终浓度0.1～1.0μM)、SA-probe 0.8μL、RNase Free ddH2O 5.6μL。在使用时，模板RNA(10pg～100ng)的使用量为2μL。PCR检测使用LightCycler Real Time PCR扩增仪进行扩增反应，扩增程序为：反转录反应：42℃ 5min；95℃ 10sec；1个循环；PCR反应程序：95℃ 5sec；60℃ 20sec；40个循环。5) Aspirate the supernatant, and then use the maintenance medium to wash away the unadsorbed virus (2 times), add 100μL of the above 8 groups of cell maintenance solutions containing different concentrations of ephemidine to each well, and set 3 parallel for each concentration. After 48 hours, the virus cells were collected by freezing and thawing for PCR detection. The primer sequence used was: SA-F: 5'-CAATGGTTTAACAGGCACAGG-3' (SEQ ID NO: 1); SA-R: 5'-CTCAAGTGTCTGTGGATCACG-3' (SEQ ID NO: 2); Probe sequence: SA-probe: 5'-FAM-GGCAGAGACATTGCTGACACTACTGATGC-BHQ-3' (SEQ ID NO: 3, where FAM is a fluorescent reporter group and BHQ is a fluorescent quenching group) The PCR system is: real-time fluorescence quantitative one-step PCR reaction solution 2×One Step RT-PCR Buffer III (TAKARA) 10 μL, PrimeScript RT Enzyme Mix II (TAKARA) 0.4 μL, 5 U/μL TaKaRa Ex Taq HS (TAKARA) 0.4 μL , SA-F 0.4μL (final concentration 0.1～1.0μM), SA-R 0.4μL (final concentration 0.1～1.0μM), SA-probe 0.8μL, RNase Free ddH2O 5.6μL. When in use, the amount of template RNA (10pg-100ng) used is 2μL. For PCR detection, the LightCycler Real Time PCR amplification instrument is used for the amplification reaction. The amplification procedure is: reverse transcription reaction: 42°C 5min; 95°C 10sec; 1 cycle; PCR reaction procedure: 95°C 5sec; 60°C 20sec; 40 Cycles.
6)实验设置为：正常细胞对照组(培养液为含10％FBS和1％双抗的DMEM培养基加0.1％DMSO)、正常病毒对照组(培养液为含10％FBS和1％双抗的DMEM培养基加0.1％DMSO)、吐根碱(8个浓度)+病毒组(3个平行)。6) The experimental settings are: normal cell control group (the culture medium is DMEM medium containing 10% FBS and 1% double antibody plus 0.1% DMSO), the normal virus control group (the culture medium contains 10% FBS and 1% double antibody The DMEM medium plus 0.1% DMSO), imitrine (8 concentrations) + virus group (3 parallel).
如图1中A幅所示，左侧表示不同浓度吐根碱对新型冠状病毒SARS-CoV-2的抑制 效果。可见，随着浓度的增加，吐根碱对新型冠状病毒SARS-CoV-2的抑制效果越明显(即病毒的感染率明显下降)，证明吐根碱能够很强地抑制SARS-CoV-2病毒的复制。如图1中B幅所示，经计算获得的吐根碱对新型冠状病毒SARS-CoV-2的IC 50(IC 50是指能够有效抑制50％细胞感染病毒的药物浓度)为0.007±0.002μM，并且此时的吐根碱浓度对Vero细胞(吐根碱对Vero细胞的CC 50为1.96±0.37μM)没有任何毒副作用，选择指数(Selectivity index，SI，SI＝CC 50/EC 50，SI＞1为有效)为280，这证明安全浓度的吐根碱在体外可以有效抑制新型冠状病毒SARS-CoV-2的活性。 As shown in Panel A in Figure 1, the left side shows the inhibitory effect of different concentrations of saponine on the new coronavirus SARS-CoV-2. It can be seen that with the increase of the concentration, the inhibitory effect of the new type of coronavirus SARS-CoV-2 is more obvious (that is, the infection rate of the virus is significantly reduced), which proves that it can strongly inhibit the SARS-CoV-2 virus. Copy. Web shown in FIG. 1 B, emetine was obtained by calculation of an IC novel coronavirus is SARS-CoV-2 50 (IC 50 refers to a drug concentration effective to inhibit viral infection of cells by 50%) was 0.007 ± 0.002μM At this time, the concentration of etorrhizine does not have any toxic side effects on Vero cells (the CC 50 of etorrhizine to Vero cells is 1.96±0.37 μM). The selection index (Selectivity index, SI, SI=CC 50 /EC 50 , SI >1 is effective) is 280, which proves that a safe concentration of torazine can effectively inhibit the activity of the new coronavirus SARS-CoV-2 in vitro.
2.3、Western blot检测吐根碱对SARS-CoV-2病毒的体外抑制效果2.3 Western blot to detect the in vitro inhibitory effect of spitrootine on SARS-CoV-2 virus
1)取一瓶单层致密Vero细胞，首先将细胞消化后加入新鲜DMEM培养基(含10％FBS和1％双抗)，分散均匀后将细胞密度稀释至5.0×10 5个细胞/mL； 1) Take a bottle of single-layer dense Vero cells, first digest the cells and add fresh DMEM medium (containing 10% FBS and 1% bi-antibody), and dilute the cell density to 5.0×10 5 cells/mL after evenly dispersing;
2)在6孔板中每孔加入1mL细胞悬液(5.0×10 5个/孔)，置于细胞培养箱，37℃、5％CO 2条件下培养过夜后观察细胞生长情况； 2) Add 1 mL of cell suspension (5.0×10 5 cells/well) to each well of a 6-well plate, place it in a cell incubator, incubate overnight at 37°C and 5% CO 2 and observe the cell growth;
3)待细胞贴壁达到底面积80％左右，吸出细胞培养板孔内的培养液，每孔以1mL维持培养基洗涤2遍；选择TCID 50为20～30的病毒液感染细胞，每孔染毒体积为0.5mL；感染1-2h；感染过程中轻轻左右摇匀，保证病毒与细胞充分接触； 3) After the cells have adhered to about 80% of the bottom area, aspirate the culture medium in the cell culture plate wells, and wash each well with 1 mL of maintenance medium twice; select a virus with a TCID 50 of 20-30 to infect the cells, and stain each well. The volume of the virus is 0.5mL; the infection is 1-2h; gently shake left and right during the infection process to ensure full contact between the virus and the cells;
4)倾去上清，然后使用维持培养基洗去未吸附的病毒(2次)。将吐根碱采用DMSO进行稀释，初始浓度分别为：0.01mM、0.03mM、0.1mM、0.3mM，同时设立阴性对照(即不含有吐根碱的DMSO)和阳性药物(5mM的瑞德西韦，购自MCE，中国)对照，吸取每个浓度的吐根碱溶液、阴性对照和阳性对照1μL至1mL含2％FBS、1％双抗的DMEM维持液中，作为细胞维持液(共6组)，分别加入6孔板的细胞中，置于培养箱内培养24小时；24小时后，取出培养板，去掉上清，PBS洗涤1遍；4) Pour off the supernatant, and then use the maintenance medium to wash away the unadsorbed virus (2 times). Dilute it with DMSO, the initial concentrations are: 0.01mM, 0.03mM, 0.1mM, 0.3mM, and set up a negative control (ie DMSO without it) and a positive drug (5mM remdesivir) , Purchased from MCE, China) control, pipette 1μL of each concentration of stigmatine solution, negative control and positive control into 1mL DMEM maintenance solution containing 2% FBS and 1% double antibody as cell maintenance solution (6 groups in total) ), were added to the cells in the 6-well plate and placed in the incubator for 24 hours; after 24 hours, take out the culture plate, remove the supernatant, and wash once with PBS;
5)每孔中加入200μL的裂解液(含有蛋白酶/磷酸酶抑制剂，1:100稀释)，轻轻晃动，使其充分与细胞接触，然后置于4℃冰箱45min，每隔15min轻轻晃动6孔板并轻拍6孔板的边缘；45min后，取出培养板，将裂解液吸入至1.5mL EP管中，再加入25μl的loading buffer；经56℃ 30min灭活后，100℃加热15min，12000rpm离心2min后获得检测样品，放置于-20℃保存。5) Add 200μL of lysate (containing protease/phosphatase inhibitor, 1:100 dilution) to each well, shake gently to make it fully contact with the cells, and then place in a refrigerator at 4°C for 45 minutes, shaking gently every 15 minutes 6-well plate and lightly pat the edge of the 6-well plate; 45 minutes later, take out the culture plate, suck the lysate into a 1.5mL EP tube, and add 25μl of loading buffer; after inactivation at 56°C for 30 minutes, heat at 100°C for 15 minutes, After centrifugation at 12000 rpm for 2 min, the test sample was obtained and stored at -20°C.
6)采用western blot方法检测各组检测样品中SARS-CoV-2病毒的核壳体表达量(其中使用的抗体购自Sino biological，Cat:40588-T62，中国)，用GAPDH作为内参。6) The western blot method was used to detect the nucleocapsid expression of SARS-CoV-2 virus in each group of test samples (the antibody used was purchased from Sino biological, Cat: 40588-T62, China), and GAPDH was used as an internal reference.
检测结果如图1中C幅所示，相对于阴性对照，使用的四组浓度(0.01μM、0.03μM、0.1μM、0.3μM，作为治疗组)的吐根碱均能表现出对新型冠状病毒SARS-CoV-2的核壳体表达的显著抑制，经估算的吐根碱对新型冠状病毒SARS-CoV-2的EC 50为约0.01μM；尤其是当吐根碱的作用浓度超过0.1μM时，甚至未检测到SARS-CoV-2的核壳体表达；而采用浓度为5μM的瑞德西韦阳性药物组仍然能够检测到SARS-CoV-2的核壳 体表达，表明吐根碱对SARS-CoV-2的抑制作用更强。 The test results are shown in panel C in Figure 1. Compared with the negative control, the four groups of concentrations (0.01μM, 0.03μM, 0.1μM, 0.3μM, as the treatment group) of stigmatine can all show resistance to the new coronavirus The nucleocapsid expression of SARS-CoV-2 is significantly inhibited, and the estimated EC 50 of the new coronavirus SARS-CoV-2 is about 0.01 μM; especially when the concentration of the effect of it exceeds 0.1 μM. , Even the nucleocapsid expression of SARS-CoV-2 was not detected; however, the nucleocapsid expression of SARS-CoV-2 was still detected in the redsivir-positive drug group at a concentration of 5μM, indicating that the effect of spitrootine on SARS -The inhibitory effect of CoV-2 is stronger.
由上述实施例2的结果可知，安全浓度的吐根碱能够很强地抑制SARS-CoV-2病毒的复制，因此吐根碱可以用于制备治疗新型冠状病毒SARS-COV-2感染的药物，并且优选当施用该药物后，吐根碱的作用浓度(即向病毒感染患者施用药物后，患者血液中的吐根碱的浓度)可以为0.005μM以上，更优选为0.01μM以上，可以有效治疗新型冠状病毒SARS-COV-2感染。另外，为了避免所述药物对于正常细胞产生不可接受的毒性，所述吐根碱在血液中的作用浓度优选为2μM以下，更优选为1μM以下。From the results of Example 2 above, it can be seen that a safe concentration of etorrhine can strongly inhibit the replication of SARS-CoV-2 virus, so it can be used to prepare drugs for the treatment of new coronavirus SARS-COV-2 infection. And it is preferable that after administration of the drug, the effective concentration of etorrhine (that is, the concentration of etofibrine in the blood of the patient after the drug is administered to a virus-infected patient) can be 0.005 μM or more, more preferably 0.01 μM or more, which can be effectively treated Infection with the new coronavirus SARS-COV-2. In addition, in order to avoid the unacceptable toxicity of the drug to normal cells, the concentration of the drug in the blood is preferably 2 μM or less, more preferably 1 μM or less.
该药物的剂型可以为以吐根碱作为药物活性成分，制成的以下任何一种药学上可接受的剂型：片剂、胶囊剂、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。The dosage form of the drug can be any one of the following pharmaceutically acceptable dosage forms made with evapotranine as the active ingredient of the drug: tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, nanometers Preparation or injection.
实施例3：吐根碱的体外病毒预防效果检测Example 3: In Vitro Virus Preventive Effect Test of Epicine
3.1、吐根碱对SARS-CoV-2病毒的预防作用检测3.1. Detection of the preventive effect of spitrootine on SARS-CoV-2 virus
1)按照实施例2中2.2的方法获得8个浓度的细胞维持液，用于后续实验；1) Obtain 8 concentrations of cell maintenance solution according to the method in 2.2 in Example 2 for use in subsequent experiments;
2)取一瓶单层致密Vero细胞，首先将细胞消化后加入新鲜DMEM培养基(含10％FBS和1％双抗)，分散均匀后将细胞密度稀释至2.0×10 5个细胞/mL； 2) Take a bottle of monolayer dense Vero cells, first digest the cells and add fresh DMEM medium (containing 10% FBS and 1% bi-antibody), and dilute the cell density to 2.0×10 5 cells/mL after evenly dispersing;
3)在96孔板中每孔加入100μL细胞悬液(2.0×10 4个/孔)，置于细胞培养箱，37℃、5％CO 2条件下培养24h后观察细胞生长情况； 3) Add 100 μL of cell suspension (2.0×10 4 cells/well) to each well of a 96-well plate, place it in a cell incubator, and observe the cell growth after 24 hours at 37°C and 5% CO 2;
4)待细胞贴壁达到底面积80％左右时，吸出细胞培养板孔内的培养液，每孔中分别加入上述8个浓度的细胞维持液100μL，每个浓度设置3个平行，于37℃、5％CO 2条件下培养3-5h； 4) When the cell adhesion reaches about 80% of the bottom area, aspirate the culture medium in the cell culture plate wells, add 100μL of the cell maintenance solution of the above 8 concentrations to each well, set 3 parallel for each concentration, at 37℃ , Cultivate 3-5h under the condition of 5% CO 2;
5)感染病毒：选择TCID 50为20～30的病毒液感染细胞，每孔染毒体积为20μL；感染1-2小时(注意：在感染病毒期间，作为药物的吐根碱依然存在)；感染过程中轻轻左右摇匀，保证病毒与细胞充分接触； 5) Virus infection: select a virus solution with a TCID 50 of 20-30 to infect cells, and the volume of each well is 20 μL; infection for 1-2 hours (note: during the virus infection period, the drug is still there); infection During the process, gently shake left and right to ensure full contact between the virus and the cells;
6)倾去上清，然后使用维持培养基洗去未吸附的病毒(2次)，并加入维持培养基，每孔100μL，48小时后收集细胞及上清，随后按照上述实施例2中2.2的方法进行PCR检测。6) Pour off the supernatant, then wash off the unadsorbed virus with the maintenance medium (2 times), and add the maintenance medium, 100 μL per well, collect the cells and supernatant after 48 hours, and then follow 2.2 in Example 2 above. Method for PCR detection.
检测结果如图2中A幅所示，表示不同浓度吐根碱对新型冠状病毒SARS-CoV-2的预防效果。可见，随着浓度的增加，吐根碱对新型冠状病毒SARS-CoV-2感染的预防效果越明显(即病毒的感染率明显下降)，如图2中B幅所示，经计算吐根碱对新型冠状病毒SARS-CoV-2感染预防的IC 50为0.019±0.009μM，并且此时的吐根碱浓度对Vero细胞(吐根碱对Vero细胞的CC 50为1.96±0.37μM)没有任何毒副作用，这证明安全浓度的吐根碱在体外可以有效预防新型冠状病毒SARS-CoV-2的感染。 The test results are shown in Panel A in Figure 2, which shows the preventive effect of different concentrations of spitrootine on the new coronavirus SARS-CoV-2. It can be seen that with the increase of the concentration, the preventive effect of spitrootine on the new coronavirus SARS-CoV-2 infection is more obvious (that is, the infection rate of the virus is significantly reduced), as shown in panel B in Figure 2, after calculating spitrootine The IC 50 for the prevention of SARS-CoV-2 infection with the new coronavirus is 0.019±0.009μM, and the concentration of imitine at this time is not toxic to Vero cells (the CC 50 of imitine to Vero cells is 1.96±0.37μM). Side effects, which proves that a safe concentration of turbinine can effectively prevent the infection of the new coronavirus SARS-CoV-2 in vitro.
3.2、Western blot检测吐根碱对SARS-CoV-2病毒的体外预防效果3.2. Western blot detection of the in vitro preventive effect of spitrootine on SARS-CoV-2 virus
1)取一瓶单层致密Vero细胞，首先将细胞消化后加入新鲜DMEM培养基(含10％FBS和1％双抗)，分散均匀后将细胞密度稀释至4.0×10 5个细胞/mL； 1) Take a bottle of single-layer dense Vero cells, first digest the cells and add fresh DMEM medium (containing 10% FBS and 1% bi-antibody), and dilute the cell density to 4.0×10 5 cells/mL after evenly dispersing;
2)在6孔板中每孔加入1mL细胞悬液(4.0×10 5个/孔)，置于细胞培养箱，37℃、5％CO 2条件下培养过夜后观察细胞生长情况； 2) Add 1 mL of cell suspension (4.0×10 5 cells/well) to each well of a 6-well plate, place it in a cell incubator, incubate overnight at 37°C and 5% CO 2 and observe the cell growth;
3)待细胞贴壁达到底面积80％左右时，吸出细胞培养板孔内的培养液，每孔中分别加入上述实施例2的2.3中步骤3)的6组细胞维持液3mL，每组设置3个平行，于37℃、5％CO 2条件下培养3.5h； 3) When the cell adhesion reaches about 80% of the bottom area, aspirate the culture solution from the cell culture plate wells, and add 3 mL of the 6 groups of cell maintenance solution in step 2.3 of Example 2 above to each well, and set each group 3 in parallel, cultured at 37℃ and 5% CO 2 for 3.5h;
4)感染病毒：选择TCID 50为20～30的病毒液感染细胞，每孔染毒体积为0.5mL；感染1-2h；感染过程中轻轻左右摇匀，保证病毒与细胞充分接触； 4) Virus infection: select the virus solution with a TCID 50 of 20-30 to infect the cells, with a volume of 0.5 mL per well; infection for 1-2 hours; gently shake left and right during the infection process to ensure full contact between the virus and the cells;
5)倾去上清，然后使用维持培养基洗去未吸附的病毒(2次)，并加入维持培养基，每孔100μL，24小时后，取出培养板，去掉上清，PBS洗涤1遍，加入200μL的裂解液，随后按照上述实施例2中2.3的方法进行Western blot检测。5) Pour off the supernatant, then use the maintenance medium to wash away the unadsorbed virus (2 times), and add the maintenance medium, 100 μL per well. After 24 hours, take out the culture plate, remove the supernatant, and wash once with PBS. Add 200 μL of lysate, and then perform Western blot detection according to the method 2.3 in Example 2 above.
检测结果如图2中C幅所示，可见，相对于阴性对照，使用的四组浓度(0.01μM、0.03μM、0.1μM、0.3μM，作为预防组)的吐根碱均能表现出对新型冠状病毒SARS-CoV-2的核壳体表达的显著抑制，估算的吐根碱对新型冠状病毒SARS-CoV-2的EC 50为约0.01μM；尤其是当吐根碱的作用浓度超过0.01μM时，甚至未检测到SARS-CoV-2的核壳体表达，证明吐根碱在体外可有效预防SARS-CoV-2的感染；而采用浓度为5μM的瑞德西韦阳性药物组未表现出对SARS-CoV-2的核壳体表达抑制，因此不能预防SARS-CoV-2病毒的感染。 The test results are shown in panel C in Figure 2. It can be seen that, compared with the negative control, the four groups of concentrations (0.01μM, 0.03μM, 0.1μM, 0.3μM, as the prevention group) of ephedrine can all show resistance to the new type. The nucleocapsid expression of the coronavirus SARS-CoV-2 is significantly inhibited, and the estimated EC 50 of the new coronavirus SARS-CoV-2 is about 0.01 μM; especially when the concentration of the effect exceeds 0.01 μM. When the SARS-CoV-2 nucleocapsid expression was not even detected at the time, it proved that imitrine can effectively prevent SARS-CoV-2 infection in vitro; while the group of positive drugs with a concentration of 5μM remdesivir did not show It inhibits the expression of SARS-CoV-2 nucleocapsid, so it cannot prevent SARS-CoV-2 virus infection.
由上述实施例3结果可知，在安全浓度的吐根碱存在的情况下，可以有效预防新型冠状病毒SARS-CoV-2的感染，因此吐根碱可以用于制备预防新型冠状病毒SARS-COV-2感染的药物，并且优选当施用该药物后，吐根碱的作用浓度可以为0.01μM以上，优选0.02μM以上且2μM以下，可以有效预防新型冠状病毒SARS-COV-2的感染。From the results of Example 3 above, it can be seen that in the presence of a safe concentration of serotonin, it can effectively prevent the infection of the new coronavirus SARS-CoV-2, so it can be used to prepare and prevent the new coronavirus SARS-COV- 2 Infectious drugs, and preferably, after the drug is administered, the concentration of etorrhine can be 0.01 μM or more, preferably 0.02 μM or more and 2 μM or less, which can effectively prevent the infection of the new coronavirus SARS-COV-2.
该药物的剂型可以为以吐根碱或其衍生物或药用盐作为药物活性成分，制成的以下任何一种药学上可接受的剂型：片剂、胶囊剂、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。The dosage form of the drug can be any one of the following pharmaceutically acceptable dosage forms made with etotrorrhizine or its derivatives or medicinal salts as the active ingredient of the drug: tablets, capsules, granules, oral liquids, suspensions Release formulations, controlled release formulations, nano formulations or injections.
此处描述的实施例只用于说明(作为例证)，技术人员所做的各种修改或变更也应包括在专利申请的实质范围内。The embodiments described here are only for illustration (as an illustration), and various modifications or changes made by technical personnel should also be included in the essential scope of the patent application.
工业应用性Industrial applicability
本发明提供了吐根碱或其药学可接受的盐在制备治疗或预防冠状病毒感染的药物中的应用，可以实现对新型冠状病毒SARS-CoV-2感染的防控，适于工业应用。The present invention provides the application of imitrine or its pharmaceutically acceptable salt in the preparation of drugs for treating or preventing coronavirus infection, which can realize the prevention and control of SARS-CoV-2 infection of the new coronavirus and is suitable for industrial application.

Claims (11)

  1. 吐根碱或其药学可接受的盐在制备治疗或预防冠状病毒感染的药物中的应用，其中所述冠状病毒为新型冠状病毒SARS-CoV-2。The application of imitrine or a pharmaceutically acceptable salt thereof in the preparation of a medicine for the treatment or prevention of coronavirus infection, wherein the coronavirus is a novel coronavirus SARS-CoV-2.
  2. 根据权利要求1所述的应用，其中所述药物是以吐根碱或其药学可接受的盐作为药物活性成分制成的选自以下的剂型：片剂、胶囊剂、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。The application according to claim 1, wherein the drug is made of tuberine or a pharmaceutically acceptable salt thereof as the active ingredient of the drug in a dosage form selected from the following: tablets, capsules, granules, oral liquids, Sustained release formulations, controlled release formulations, nano formulations or injections.
  3. 根据权利要求1或2所述的应用，其中当所述药物在施用后，所述吐根碱或其药学可接受的盐的作用浓度为0.005μM以上且为2μM以下。The use according to claim 1 or 2, wherein after the drug is administered, the effective concentration of the etorrhizine or its pharmaceutically acceptable salt is 0.005 μM or more and 2 μM or less.
  4. 根据权利要求1或2所述的应用，其中当所述药物在施用后，所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为1μM以下。The application according to claim 1 or 2, wherein after the drug is administered, the concentration of the etorrhizine or its pharmaceutically acceptable salt is 0.010 μM or more and 1 μM or less.
  5. 一种治疗新型冠状病毒SARS-CoV-2感染的方法，其包括向新型冠状病毒SARS-CoV-2感染患者施用治疗有效量的含有吐根碱或其药学可接受的盐的药物。A method for treating a novel coronavirus SARS-CoV-2 infection, which comprises administering a therapeutically effective amount of a medicine containing imitrine or a pharmaceutically acceptable salt thereof to patients infected with the novel coronavirus SARS-CoV-2.
  6. 根据权利要求5所述的方法，其中所述治疗有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.005μM以上且为2μM以下。The method according to claim 5, wherein the therapeutically effective amount is such that the effective concentration of the etopyrine or a pharmaceutically acceptable salt thereof is 0.005 μM or more and 2 μM or less.
  7. 根据权利要求5所述的方法，其中所述治疗有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为1μM以下。The method according to claim 5, wherein the therapeutically effective amount is such that the concentration of the effect of the etopyrine or a pharmaceutically acceptable salt thereof is 0.010 μM or more and 1 μM or less.
  8. 一种预防新型冠状病毒SARS-CoV-2感染的方法，其包括向受试者施用预防有效量的含有吐根碱或其药学可接受的盐的药物。A method for preventing a novel coronavirus SARS-CoV-2 infection, which comprises administering to a subject a preventively effective amount of a drug containing etopyrine or a pharmaceutically acceptable salt thereof.
  9. 根据权利要求8所述的方法，其中所述预防有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为2μM以下。8. The method according to claim 8, wherein the prophylactically effective amount is such that the concentration of the action concentration of torazine or a pharmaceutically acceptable salt thereof is 0.010 μM or more and 2 μM or less.
  10. 根据权利要求8所述的方法，其中所述预防有效量为使得所述吐根碱或其药学可接受的盐的作用浓度为0.010μM以上且为1μM以下。The method according to claim 8, wherein the prophylactically effective amount is such that the concentration of the action concentration of the torazine or a pharmaceutically acceptable salt thereof is 0.010 μM or more and 1 μM or less.
  11. 根据权利要求8所述的方法，其中所述受试者为人。The method of claim 8, wherein the subject is a human.
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