WO2022088047A1 - Itf2357在制备预防和治疗冠状病毒的药物中的应用 - Google Patents

Itf2357在制备预防和治疗冠状病毒的药物中的应用 Download PDF

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WO2022088047A1
WO2022088047A1 PCT/CN2020/125280 CN2020125280W WO2022088047A1 WO 2022088047 A1 WO2022088047 A1 WO 2022088047A1 CN 2020125280 W CN2020125280 W CN 2020125280W WO 2022088047 A1 WO2022088047 A1 WO 2022088047A1
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hydrochloride
itf2357
stereoisomers
cov
sars
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PCT/CN2020/125280
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French (fr)
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袁曙光
李红昌
邹荣峰
崔文强
刘科
李红春
陈显翀
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中国科学院深圳先进技术研究院
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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • A61K31/325—Carbamic acids; Thiocarbamic acids; Anhydrides or salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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 invention belongs to the field of medicine, and specifically relates to the application of ITF2357 in the preparation of medicines for preventing and treating coronavirus.
  • the new coronavirus pneumonia (Corona Virus Disease 2019) is an infectious disease caused by the infection of the human body by the new coronavirus (SARS-Cov-2). Its symptoms mainly include fever, fatigue, dry cough, dyspnea and renal failure [The Lancet , 2020, 395(10223): 507-513; The Lancet, 2020, 395(10223): 497-506].
  • the coronavirus belongs to the genus Coronavirus in the family Coronaviridae.
  • the virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an envelope, with a diameter of about 80-120 nm.
  • RNA viruses Its genetic material is the largest among all RNA viruses, and generally only infects humans, mice, pigs, cats, and dogs. , avian vertebrates.
  • the coronavirus was first isolated from chickens in 1937. The shape of coronavirus particles is irregular, with a diameter of about 60-220 nm.
  • the virus has an envelope structure with three proteins on it: spike glycoprotein (S, Spike Protein), small envelope glycoprotein (E, Envelope Protein) and membrane glycoprotein (M, Membrane Protein), and a few species also have hemagglutination.
  • S spike glycoprotein
  • E small envelope glycoprotein
  • M membrane glycoprotein
  • Glucoprotein HE protein, Haemaglutinin-esterase
  • SARS-Cov-2 virus particles The diameter of SARS-Cov-2 virus particles is between 60 and 140 nm, and there are spikes of 9 to 12 nm outside the envelope, which are shaped like a corolla.
  • Genome sequencing revealed that SARS-Cov-2 is a single-stranded RNA coronavirus. By comparing the gene sequences of other virus samples, it was found that SARS-Cov-2 was similar to SARS-CoV (79.5%) [Nature, 2020] and bat coronavirus (96%) [bioRxiv, 2020, 2020.01.22.914952], and speculated that The virus may have originated from bats [bioRxiv, 2020, 2020.01.24.915157; Nature, 2020].
  • the 2019-nCoV virus belongs to ⁇ CoV and is the seventh member of the HCoV family that is different from SARS-CoV and MERS-CoV [New England Journal of Medicine, 2020], and the remaining six members include HCoV 229E, NL63, OC43, HKU1, SARS -CoV and MERS-CoV.
  • the novel coronavirus pneumonia is caused by a new type of coronavirus. It is the same coronavirus as the well-known SARS-CoV that causes atypical pneumonia, but the type is different. Although its fatality rate is lower than that of SARS-CoV, it is far more infectious. to SARS-CoV.
  • the purpose of the present invention is to provide the application of ITF2357 in the preparation of medicines for preventing or treating the new type of coronavirus.
  • the present invention provides ITF2357 or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of preventing and/or treating coronavirus The use of medicines for diseases.
  • Another aspect of the present invention provides ITF2357 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof in the preparation of a method for preventing coronavirus from entering cells application in medicine.
  • the coronaviruses are novel coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.
  • the disease caused by the coronavirus is pneumonia or its complications caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV.
  • ITF2357 is shown in structural formula (1)
  • ITF2357 or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs is used as the only active ingredient in the preparation of preventive and/or use in medicines for the treatment of diseases caused by coronavirus.
  • ITF2357 or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs and other antiviral drugs are prepared Application of the composition as an active ingredient in the preparation of a medicament for preventing and/or treating diseases caused by coronavirus.
  • antiviral drugs are selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemannan, acyclovir Sodium, Adefovir, Alovudine, Avesuto, Tricyclodecamine Hydrochloride, Alanodine, Aridone, Ateviridine Mesylate, Afrilidine, Cidofovir, Silicate panthefylline, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desilovir, didanosine, dioxalisine, eduridine, emivirine, eltra Sitapine, Enviraden, Enviroxime, Heptin, Famciclovir, Chlorphenhydramine Hydrochloride, Noncitabine, Nonauridine, Phosphorate, Foscarnet Sodium, Phos
  • Another aspect of the present invention provides a pharmaceutical composition for treating or preventing diseases caused by coronaviruses of the family Coronaviridae, comprising ITF2357 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, Variant, ester, amide or prodrug.
  • the pharmaceutical composition further includes pharmaceutically acceptable excipients.
  • the dosage form of the pharmaceutical composition is an oral preparation, a pulmonary inhalation preparation, a mucosal administration preparation, an ophthalmic preparation or an injection.
  • the oral preparation is selected from granules, powders, pills, tablets, capsules or oral liquids.
  • Another aspect of the present invention provides the use of ITF2357 as a disinfectant against Coronaviridae.
  • Another aspect of the present invention provides a method for treating diseases caused by coronaviruses, comprising adding a therapeutically effective amount of ITF2357 or a pharmaceutically acceptable salt, isotope, stereoisomer, stereoisomer thereof A mixture, tautomer, ester or prodrug is administered to a subject.
  • Another aspect of the present invention provides a method for preventing a subject from being infected with a Coronaviridae virus, comprising adding a therapeutically effective amount of ITF2357 or a pharmaceutically acceptable salt, isotope, stereoisomer, stereoisomer thereof Mixtures of isomers, tautomers, esters or prodrugs are administered to subjects prior to infection.
  • the invention proves for the first time that ITF2357 has an inhibitory effect on the novel coronavirus of new coronary pneumonia.
  • ITF2357 is used as an effective drug in the treatment of novel coronavirus infection.
  • Figure 1 shows the production and verification of SARS-2-S pseudotype particles.
  • SARS-2-S protein can be successfully expressed in mammalian cells.
  • SARS-2-S pseudovirion has S protein modification.
  • C The SARS-2-S pseudovirion can successfully infect host cells and integrate the reporter gene.
  • D SARS-2-S pseudovirions enter susceptible cells by recognizing ACE2 receptors.
  • E SARS-2-S pseudovirions were able to infect human 293T cells expressing ACE2-GFP, but not into 293T cells without ACE2 expression.
  • ACE2-GFP green; Flag tag, red, showing the S protein;
  • F SARS-2-S pseudovirion capable of binding to the ACE2 receptor with co-localized signals at different intracellular locations (membrane and cytoplasm).
  • G Time-dependent entry of SARS-2-S pseudovirions into ACE2-GFP/293T cells.
  • Figure 2 shows that ITF2357 effectively inhibits pseudovirion infection.
  • Immunofluorescence staining was used to determine the inhibitory effect of the screened clinical drugs on the infection of host cells by SARS-2-S pseudovirus particles. After the pseudovirus particles infect ACE2-GFP expressing 293T cells, the intracellular localization of pseudovirus particles was observed by fixed staining. Blue, DAPI; red, Flag antibody labeled S protein; green, ACE2-GFP signal.
  • treatment refers to reversing, alleviating, inhibiting the progression of, or preventing the diseases or conditions to which the term applies, or one or more symptoms of these diseases or conditions, or the One or more symptoms of a disorder.
  • terapéuticaally effective amount is the amount of Compound 1, or a pharmaceutically acceptable salt thereof, present in the compositions described herein that, when such compositions are administered by the chosen route of administration, at The desired level of drug is provided in the secretions and tissues of the airways and lungs, or alternatively in the bloodstream of the subject to be treated, to produce the desired physiological response or desired biological effect.
  • the precise amount will depend on many factors, such as the specific activity of the composition, the delivery device used, the physical properties of the composition, its intended use, and animal considerations such as the severity of the disease state, etc., and can be determined by one of skill in the art based on The information provided herein makes it easy to determine the exact amount.
  • the active ingredients of the present invention are administered by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient.
  • An advantage of the compounds of the present invention is that they are orally bioavailable and can be administered orally.
  • the effective dose of the active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, delivery method and pharmaceutical formulation, and will be determined by the clinician using routine dose escalation studies.
  • ITF2357 or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs and other antiviral drugs are prepared
  • the application of the composition as an active ingredient in the preparation of a medicament for preventing and/or treating diseases caused by coronavirus, for the above-mentioned two or more active ingredients in a unit dosage form combination is administered to a patient simultaneously or sequentially.
  • Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.
  • the construction method adopts the article Xiuyuan Ou et al. (published on March 27, 2020). Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature communications, 11(1), 1-12. Published methods.
  • the DNA sequence of SARS-2-S protein was modified without changing its amino acid sequence by codon optimization method, which facilitated the abundant expression of SARS-2-S in 293T cells (Fig. 1A).
  • the three-plasmid expression system was transfected in 293T cells, and SARS-2-S was modified on the outer membrane of the pseudovirion to form SARS-2 -S pseudovirion. It can be judged by immunoblotting that this pseudoviral particle removed the original VSV-G envelope glycoprotein and replaced it with SARS-2-S, the envelope glycoprotein of the new coronavirus ( Figure 1B).
  • the pseudovirion carrying the Luciferase reporter gene was used to infect host cells 293T or ACE2/293T cells.
  • the Luciferase cell activity assay showed that the SARS-2-S pseudovirus particles had high infectivity to ACE2/293T cells, and the infection efficiency was about 100 times higher than that of 293T cells ( Figure 1C).
  • pseudovirus particles were also used to infect monkey kidney epithelial Vero-E6 cells with endogenous ACE2 expression, and immunofluorescence staining showed that SARS-2-S pseudovirus particles could well enter Vero-E6 cells; Knockdown of the endogenous ACE2 receptor greatly reduced the infection efficiency of pseudovirions (Fig.
  • SARS-2-S pseudovirus particles were not only localized on the cell membrane, but also could enter cells with ACE2 receptors and transported to the perinuclear cells of cells perinuclear area (Fig. 1F).
  • the entry of SARS-2-S pseudovirions into host cells was also time-dependent, reaching a saturation phase after approximately 2 h (Fig. 1G).
  • Example 1 Using the SARS-2-S pseudovirus particle in vitro infection model (Luciferase reporter system and immunofluorescence staining localization system) constructed in Example 1, the in vitro cell biological verification of ITF2357 was carried out.
  • ACE2-GFP stably transfected 293T cells were inoculated in a 96-well plate, and the cells were pretreated with different concentrations of ITF2357 for 2 hours, then SARS-2-S pseudovirus particles were added to infect for 3 hours, and then the supernatant was removed and replaced with fresh complete medium. After 40 hours of infection, the cells were lysed by the Luciferase Assay System (Promega) and the reaction substrate was added, and the luminescence intensity of luciferase was measured by Glomax 96. The bioluminescence intensity was proportional to the infection efficiency of virus particles.
  • the results of Luciferase activity assay showed that ITF2357 had an obvious concentration-dependent effect and could effectively inhibit the infection efficiency of SARS-2-S pseudovirus particles.
  • the EC50 concentration of ITF2357 is approximately 6.8 ⁇ 1.0 ⁇ M.
  • the amount of pseudovirus particles used for infection is much higher than that of Luciferase reporter assay, which is closer to the amount of virus infecting human cells under physiological conditions. . Therefore, the Luciferase reporter system can better reflect the antiviral effect of clinical drugs under physiological conditions. This shows that ITF2357 has a great potential for anti-coronavirus infection.
  • the action pathway and target of this clinical drug are clear steps in inhibiting the infection of host cells by the new coronavirus pneumonia virus, which has great research value and drug screening potential.

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Abstract

ITF2357在制备预防和治疗冠状病毒的药物中的应用。ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药在制备预防和治疗冠状病毒所致疾病的药物中的应用。所述的冠状病毒为新型冠状病毒SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1和MERS-CoV。冠状病毒所致疾病为SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1或MERS-CoV任一病毒引起的肺炎或其并发症。首次证实了ITF2357对新冠肺炎新型冠状病毒抑制作用，半数有效浓度低，具有极大潜力。

Description

ITF2357在制备预防和治疗冠状病毒的药物中的应用 技术领域
本发明属于药物领域，具体涉及ITF2357在制备预防和治疗冠状病毒的药物中的应用。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019)是由新型冠状病毒(SARS-Cov-2)感染人体而引起的传染性疾病，其症状主要包括发热、乏力、干咳、呼吸困难和肾衰竭等[The Lancet,2020,395(10223):507-513；The Lancet,2020,395(10223):497-506]。冠状病毒在系统分类上属冠状病毒科(Coronaviridae)冠状病毒属(Corona virus)。冠状病毒属的病毒是具外套膜(envelope)的正链单股RNA病毒，直径约80～120nm，其遗传物质是所有RNA病毒中最大的，一般只会感染人、鼠、猪、猫、犬、禽类脊椎动物。冠状病毒于1937年被首次从鸡身上分离出来。冠状病毒粒子形状并不规则，直径约60～220nm。病毒具有包膜结构，上面有三种蛋白：刺突糖蛋白(S，Spike Protein)、小包膜糖蛋白(E，Envelope Protein)和膜糖蛋白(M，Membrane Protein)，少数种类还有血凝素糖蛋白(HE蛋白，Haemaglutinin－esterase)[Nederlands Tijdschrift Voor Geneeskunde,2014,158(158):A8119-A8119]。
SARS-Cov-2病毒颗粒直径在60～140nm之间，包膜外有9～12nm的尖刺，形似花冠。基因组测序表明，SARS-Cov-2是一种单链RNA冠状病毒。通过与其他病毒样品基因序列的比较，发现SARS-Cov-2与SARS-CoV(79.5％)[Nature,2020]和蝙蝠冠状病毒(96％)相似[bioRxiv,2020,2020.01.22.914952]，并推测该病毒可能起源于蝙蝠[bioRxiv,2020,2020.01.24.915157；Nature,2020]。2019-nCoV病毒属于βCoV，是区别于SARS-CoV和MERS-CoV的HCoV家族的第7个成员[New England Journal of Medicine,2020]，其余6个成员包括HCoV 229E、NL63、OC43、HKU1、SARS-CoV和MERS-CoV。
引起新型冠状病毒肺炎的是一种新型冠状病毒，它与人们熟知的引起非典型性肺炎的SARS-CoV同属冠状病毒，但类型不同，其致死率虽低于SARS-CoV但传染性远远高于SARS-CoV。
到目前为止依旧没有任何特效药物能够治愈新型冠状病毒肺炎。目前的治疗方法大多是对症治疗，特别是对一些重症患者疗效较差。因此，开发有效的冠状病毒肺炎治疗特效药物成为了当下一个迫在眉睫需要解决的课题。
发明内容
本发明的目的在于提供ITF2357在制备预防或治疗抗新冠肺炎新型冠状病毒的药物中的应用。
具体而言，为解决的本发明的技术问题，采用如下技术方案：
本发明提供了ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药在制备预防和/或治疗冠状病毒所致疾病的药物中的应用。
本发明另一个方面提供了ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药在制备阻止冠状病毒进入细胞的药物中的应用。
在本发明的技术方案中，所述的冠状病毒为新型冠状病毒SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1和MERS-CoV。
在本发明的技术方案中，冠状病毒所致疾病为SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1或MERS-CoV任一病毒引起的肺炎或其并发症。
在本发明的技术方案中，ITF2357如结构式(1)所示的
Figure PCTCN2020125280-appb-000001
在本发明的技术方案中，ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药作为唯一活性成分在制备预防和/或治疗冠状病毒所致疾病的药物中的应用。
在本发明的技术方案中，ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药与其他抗病毒药物制备的组合物作为活性成分在制备预防和/或治疗冠状病毒所致疾病的药物中的应用。
在本发明的技术方案中，其他抗病毒药物选自更昔洛韦、阿昔洛韦、金刚烷胺、金刚乙胺、奥司他韦、阿巴卡韦、醋孟南、阿昔洛韦钠、阿德福韦、阿洛夫定、阿韦舒托、盐酸三环癸胺、阿拉诺丁、阿立酮、阿替韦啶甲磺酸酯、阿夫立定、西多福韦、西潘茶碱、恩曲他滨、盐酸阿糖胞苷、甲磺酸地拉韦啶、地昔洛韦、去羟肌苷、二噁沙利、依度尿苷、乙米韦林、依曲西他平、恩韦拉登、恩韦肟、贺普丁、泛昔洛韦、盐酸氯苯氢异喹、非西他滨、非阿尿苷、磷利酯、膦甲酸钠、膦乙酸钠、甘西洛维钠、碘苷、茚地那韦、乙氧丁酮醛、拉米 夫定、洛布卡韦、洛德腺苷、洛匹那韦、盐酸美莫汀、甲红硫脲、那非那韦、奈韦拉平、喷昔洛韦、吡罗达韦、利巴韦林、甲磺酸沙奎那韦、利托那韦、盐酸索金刚胺、索立夫定、匍枝青霉菌素、司他夫定、替诺福韦、盐酸梯络龙、曲氟尿苷、盐酸伐昔洛韦、阿糖腺苷、磷酸阿糖腺苷、阿糖腺苷磷酸钠、替拉那韦、韦罗肟、扎西他滨、齐多夫定、净韦肟。
本发明另一个方面提供了一种治疗或预防冠状病毒科病毒所致疾病的药物组合物，其包括ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药。
在本发明的技术方案中，药物组合物的还包括药学上可接受的辅料。
在本发明的技术方案中，药物组合物的剂型为口服制剂、肺部吸入制剂、粘膜给药制剂、眼用制剂或注射剂。
在本发明的技术方案中，口服制剂选自颗粒剂、粉磨剂、丸剂、片剂、胶囊或口服液。
本发明另一个方面提供了ITF2357作为抗冠状病毒科病毒的消毒剂的用途。
本发明另一个方面提供了一种用于治疗冠状病毒科病毒所致疾病的方法，包括将治疗有效量的ITF2357或其药物学上可接受的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯或前药给药于受试者。
本发明另一个方面提供了一种用于预防受试者感染冠状病毒科病毒的方法，包括将治疗有效量的ITF2357或其药物学上可接受的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯或前药给药在感染前给予受试者。
有益效果
本发明首次证实了ITF2357对新冠肺炎新型冠状病毒抑制作用，其治疗指数高，半数有效浓度低；而且在染毒前施用ITF2357可有效增加防病毒的效果。ITF2357用作治疗抗新冠肺炎新型冠状病毒感染方面的有效药物。
附图说明
图1为新冠假病毒颗粒(SARS-2-S pseudotype particle)的生产与验证。
其中，(A)SARS-2-S蛋白可以成功在哺乳动物细胞中表达。(B)SARS-2-S假病毒颗粒具有S蛋白修饰。(C)SARS-2-S假病毒颗粒可成功侵染宿主细胞并整合报告基因。(D)SARS-2-S假病毒颗粒通过识别ACE2受体进入易感染细胞。(E)SARS-2-S假病毒颗粒能够侵染ACE2-GFP表达的人293T细胞，而不进入无ACE2表达的293T细胞。ACE2-GFP，绿色；Flag标签，红色，显示S蛋白；(F)SARS-2-S假病毒颗粒能够与ACE2受体结合，在细胞内 的不同位置(细胞膜和细胞质)具有共定位信号。(G)SARS-2-S假病毒颗粒进入ACE2-GFP/293T细胞具有时间依赖性。
图2为ITF2357有效抑制假病毒颗粒侵染。免疫荧光染色法确定筛选出的临床药物对SARS-2-S假病毒颗粒侵染宿主细胞的抑制作用。假病毒颗粒侵染ACE2-GFP表达的293T细胞后，固定染色观察假病毒颗粒的细胞内定位。蓝色，DAPI；红色，Flag抗体标识S蛋白；绿色，ACE2-GFP信号。
具体实施方式
在本发明中，本文所用的术语治疗是指逆转、减轻、抑制该术语所适用的疾病或病症或者这些疾病或病症的一种或多种症状的进展，或预防这些疾病或病症或者这些疾病或病症的一种或多种症状。
如本文所用的术语“治疗有效量”是存在于本文所述组合物中的化合物1或其药学上可接受的盐的量，其是当通过所选择的施用途径施用这样的组合物时，在气道和肺的分泌物和组织中或者可选地在待治疗的受试者的血流中提供所需水平的药物，以产生预期的生理反应或所需的生物学效应。精确的量将取决于许多因素，例如组合物的比活性，所用的递送装置，组合物的物理特性，其预期用途，以及动物考虑因素例如疾病状态的严重程度等，并且本领域技术人员可以基于本文提供的信息容易地确定精确的量。
对于给药途径而言，本发明的活性成分通过适合于待治疗病症的任何途径施用。合适的途径包括口服、直肠、鼻、肺、局部(包括口腔和舌下)、阴道和肠胃外(包括皮下、肌内、静脉内、皮内、鞘内和硬膜外)等。可以理解的是，优选途径可以根据例如接收者的状况而变化。本发明化合物的优点是它们具有口服生物利用度并可以口服给药。
活性成分的有效剂量至少取决于所治疗病症的性质、毒性、化合物是否正在预防性使用(较低剂量)或针对活性病毒感染、递送方法和药物制剂，并且将由临床医师使用常规剂量递增研究确定。
在本发明的技术方案中，ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药与其他抗病毒药物制备的组合物作为活性成分在制备预防和/或治疗冠状病毒所致疾病的药物中的应用，对于上述方案中的两种以上的活性成分以单位剂型组合同时或顺序施用于患者。联合治疗可以作为同时或顺序方案施用。当按顺序施用时，组合可以是以两次或更多次施用给予。
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方 式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1新冠病毒S蛋白修饰的假病毒颗粒构建、生产与验证
为了模拟新冠肺炎病毒(SARS-CoV-2)利用S蛋白识别ACE2受体进行宿主细胞侵染的自然过程，构建了以复制缺陷型慢病毒为核心、修饰有SARS-2-S蛋白的假病毒颗粒。
构建方法采用文章Xiuyuan Ou等.(2020年3月27日公开).Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.Nature communications,11(1),1-12.公布的方法。
首先，通过密码子优化方法，改造SARS-2-S蛋白的DNA序列而不改变其氨基酸序列，这有助于SARS-2-S在293T细胞中大量表达(图1A)。随后，利用实验室常用复制缺陷型HIV慢病毒作为假病毒颗粒的核心，在293T细胞中转染三质粒表达系统，将SARS-2-S修饰到假病毒颗粒的外膜上，形成SARS-2-S假病毒颗粒。通过免疫印迹法可以判断，此假病毒颗粒去除了原有的VSV-G包膜糖蛋白，替换成了新冠肺炎病毒的包膜糖蛋白SARS-2-S(图1B)。为了验证SARS-2-S假病毒颗粒的侵染效率，使假病毒颗粒携带有Luciferase报告基因，侵染宿主细胞293T或者ACE2/293T细胞。Luciferase细胞活性实验显示，SARS-2-S假病毒颗粒对ACE2/293T细胞具有高侵染性，侵染效率比293T细胞提高约100倍以上(图1C)。此外，也使用假病毒颗粒侵染具有内源性ACE2表达的猴肾上皮Vero-E6细胞，免疫荧光染色法发现SARS-2-S假病毒颗粒能够很好进入Vero-E6细胞；如果用siRNA将内源性ACE2受体敲降，能极大降低假病毒颗粒的侵染效率(图1D)。类似的，也使用ACE2-GFP过表达的293T作为宿主细胞观察假病毒颗粒的进入过程。免疫荧光染色结果显示，SARS-2-S能够有效进入ACE-GFP/293T细胞，而不进入无ACE2表达的293T细胞(图1E)。因此，以上结果证明，构建的SARS-2-S假病毒系统能够模拟S蛋白识别ACE2的自然过程，具有侵染活性。
通过激光共聚焦显微镜在高倍镜下进一步观察SARS-2-S假病毒颗粒的侵染过程，发现假病毒颗粒不仅定位于细胞膜上，也能够随ACE2受体进入细胞内，转运至细胞的核周区(perinuclear area)(图1F)。此外，SARS-2-S假病毒颗粒进入宿主细胞也具有时间依赖性，大约在2h后进入达到饱和期(图1G)。
实施例2细胞生物学平台筛选临床药物抑制SARS-2-S假病毒颗粒侵染
利用实施例1构建的SARS-2-S假病毒颗粒体外侵染模型(Luciferase报告系统和免疫荧光染色定位系统)，进行ITF2357体外细胞生物学验证。在96孔板中接种ACE2-GFP稳转293T细胞，用不同浓度ITF2357药物预处理细胞2h后，再加入SARS-2-S假病毒颗粒侵染 3h，随后去除上清更换为新鲜完全培养基。侵染40h后，利用Luciferase Assay System(Promega)裂解细胞并加入反应底物，用Glomax 96测定荧光素酶发光强度，此生物发光强度与病毒颗粒侵染效率成正比。
Luciferase活性检测结果显示，ITF2357具有明显的浓度依赖性效果，能够有效抑制SARS-2-S假病毒颗粒的侵染效率。ITF2357的EC50浓度约为6.8±1.0μM。
免疫荧光染色结果也显示，ITF2357在50μM浓度预处理宿主细胞2h，就能显著抑制SARS-2-S假病毒颗粒进入ACE2-GFP/293T(图2)。从图2结果来看，对照组中，假病毒颗粒大量进入细胞内，并定位于细胞核周围区，形成红色聚集区；给药组病毒颗粒大部分位于细胞膜和膜周围，说明ITF2357有效抑制了假病毒颗粒的侵入过程，将假病毒颗粒阻止在细胞膜上。
值得一提的是，由于免疫荧光染色需要对病毒颗粒进行成像，所以侵染使用的假病毒颗粒用量远远高于Luciferase报告实验，而后者更接近与生理状态下病毒在感染人体细胞时的用量。因此，Luciferase报告系统更能反应临床药物在生理条件下的抗病毒效果。这说明ITF2357具有较大实际抗新冠病毒感染潜力。该临床药物的作用通路和靶点在抑制新冠肺炎病毒侵染宿主细胞中的明确步骤，有极大的研究价值和药物筛选潜力。

Claims (11)

  1. ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药在制备预防和/或治疗冠状病毒所致疾病的药物中的应用。
  2. ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药在制备阻止冠状病毒进入细胞的药物中的应用。
  3. 根据权利要求1或2所述的应用，其中所述的冠状病毒为新型冠状病毒SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1和MERS-CoV。
  4. 根据权利要求1或2所述的应用，冠状病毒所致疾病为SARS-Cov-2、SARS-CoV、HCoV 229E、NL63、OC43、HKU1或MERS-CoV任一病毒引起的肺炎或其并发症。
  5. 根据权利要求1或2所述的应用，ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药作为唯一活性成分。
  6. 根据权利要求1或2所述的应用，ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药与其他抗病毒药物制备的组合物作为活性成分。
  7. 根据权利要求6所述的应用，其他抗病毒药物选自更昔洛韦、阿昔洛韦、金刚烷胺、金刚乙胺、奥司他韦、阿巴卡韦、醋孟南、阿昔洛韦钠、阿德福韦、阿洛夫定、阿韦舒托、盐酸三环癸胺、阿拉诺丁、阿立酮、阿替韦啶甲磺酸酯、阿夫立定、西多福韦、西潘茶碱、恩曲他滨、盐酸阿糖胞苷、甲磺酸地拉韦啶、地昔洛韦、去羟肌苷、二噁沙利、依度尿苷、乙米韦林、依曲西他平、恩韦拉登、恩韦肟、贺普丁、泛昔洛韦、盐酸氯苯氢异喹、非西他滨、非阿尿苷、磷利酯、膦甲酸钠、膦乙酸钠、甘西洛维钠、碘苷、茚地那韦、乙氧丁酮醛、拉米夫定、洛布卡韦、洛德腺苷、洛匹那韦、盐酸美莫汀、甲红硫脲、那非那韦、奈韦拉平、喷昔洛韦、吡罗达韦、利巴韦林、甲磺酸沙奎那韦、利托那韦、盐酸索金刚胺、索立夫定、匍枝青霉菌素、司他夫定、替诺福韦、盐酸梯络龙、曲氟尿苷、盐酸伐昔洛韦、阿糖腺苷、磷酸阿糖腺苷、阿糖腺苷磷酸钠、替拉那韦、韦罗肟、扎西他滨、齐多夫定、净韦肟。
  8. 一种治疗或预防冠状病毒科病毒所致疾病的药物组合物，其包括ITF2357或其可药用的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯、酰胺或前药作为活性成分；
    优选地，药物组合物的还包括药学上可接受的辅料；
    优选地，药物组合物的剂型为口服制剂、肺部吸入制剂、粘膜给药制剂、眼用制剂或注射剂。
  9. [根据细则91更正 27.11.2020]
    根据权利要求8所述的药物组合物，药物组合物中还包括其他抗病毒药物；
    优选地，其他抗病毒药物选自更昔洛韦、阿昔洛韦、金刚烷胺、金刚乙胺、奥司他韦、阿巴卡韦、醋孟南、阿昔洛韦钠、阿德福韦、阿洛夫定、阿韦舒托、盐酸三环癸胺、阿拉诺丁、阿立酮、阿替韦啶甲磺酸酯、阿夫立定、西多福韦、西潘茶碱、恩曲他滨、盐酸阿糖胞苷、甲磺酸地拉韦啶、地昔洛韦、去羟肌苷、二噁沙利、依度尿苷、乙米韦林、依曲西他平、恩韦拉登、恩韦肟、贺普丁、泛昔洛韦、盐酸氯苯氢异喹、非西他滨、非阿尿苷、磷利酯、膦甲酸钠、膦乙酸钠、甘西洛维钠、碘苷、茚地那韦、乙氧丁酮醛、拉米夫定、洛布卡韦、洛德腺苷、洛匹那韦、盐酸美莫汀、甲红硫脲、那非那韦、奈韦拉平、喷昔洛韦、吡罗达韦、利巴韦林、甲磺酸沙奎那韦、利托那韦、盐酸索金刚胺、索立夫定、匍枝青霉菌素、司他夫定、替诺福韦、盐酸梯络龙、曲氟尿苷、盐酸伐昔洛韦、阿糖腺苷、磷酸阿糖腺苷、阿糖腺苷磷酸钠、替拉那韦、韦罗肟、扎西他滨、齐多夫定、净韦肟。
  10. 一种用于治疗冠状病毒科病毒所致疾病的方法，包括将治疗有效量的ITF2357或其药物学上可接受的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯或前药给药于受试者。
  11. 一种用于预防受试者感染冠状病毒科病毒的方法，包括将治疗有效量的ITF2357或其药物学上可接受的盐、同位素、立体异构体、立体异构体的混合物、互变异构体、酯或前药在感染冠状病毒前给予受试者。
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