CN114432305A

CN114432305A - Application of CAY10603 in preparing medicine for preventing and treating coronavirus

Info

Publication number: CN114432305A
Application number: CN202011189026.7A
Authority: CN
Inventors: 袁曙光; 李红昌; 刘科; 崔文强; 陈显翀; 李红春; 邹荣峰
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2022-05-06

Abstract

The invention relates to an application of CAY10603 in preparing a medicament for preventing and treating coronavirus. In particular discloses an application of CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, a mixture of stereoisomers, tautomer, ester, amide or prodrug thereof in preparing medicaments for preventing and treating diseases caused by coronavirus. The coronavirus is novel coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. The disease caused by coronavirus is pneumonia or its complication caused by any one of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV. The invention proves the inhibiting effect of CAY10603 on the novel coronavirus of the new coronary pneumonia for the first time, and the CAY10603 has low half effective concentration and great potential.

Description

Application of CAY10603 in preparing medicine for preventing and treating coronavirus

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of CAY10603 in preparation of a medicine for preventing and treating coronavirus.

Background

The new type coronavirus pneumonia (Corona Virus Disease 2019) is an infectious Disease caused by The new type coronavirus (SARS-Cov-2) infecting human body, and The symptoms mainly comprise fever, hypodynamia, dry cough, dyspnea, renal failure and The like [ The Lancet,2020,395(10223): 507-); the Lancet,2020,395(10223) 497-506. Coronaviruses belong to the genus coronaviruses (Coronaviridae) of the family Coronaviridae (Corona viruses) on a systematic classification. The coronavirus is a positive strand single strand RNA virus with an outer envelope (envelope), the diameter of the coronavirus is about 80-120 nm, the genetic material of the coronavirus is the largest of all RNA viruses, and the coronavirus can only infect human, mouse, pig, cat, dog and poultry vertebrates generally. Coronavirus was first isolated from chickens in 1937. The coronavirus particles are irregular in shape and have a diameter of about 60 to 220 nm. Viruses have an envelope structure with three proteins: spike glycoprotein (S, Spike Protein), small Envelope glycoprotein (E, Envelope Protein) and Membrane glycoprotein (M, Membrane Protein), a few also hemagglutinin glycoprotein (HE Protein) [ Nederlands Tijdschrift Voor Genesenkend, 2014,158(158): A8119-A8119 ].

The diameter of SARS-Cov-2 virus particle is between 60-140 nm, 9-12 nm of spine is outside the envelope, and the shape is similar to corolla. Genome sequencing shows that SARS-Cov-2 is a single-stranded RNA coronavirus. The 2019-nCoV virus belongs to the β CoV, and is the 7 th member of the HCoV family that is different from SARS-CoV and MERS-CoV [ New England Journal of Medicine, 2020], and the remaining 6 members include HCoV 229E, NL63, OC43, HKU1, SARS-CoV and MERS-CoV.

The new type of coronavirus pneumonia is a new type of coronavirus, which is the same genus coronavirus as SARS-CoV known to cause atypical pneumonia, but the type is different, and the fatality rate is lower than SARS-CoV but the infectivity is much higher than SARS-CoV.

Until now, no specific medicine can cure the novel coronavirus pneumonia. The existing treatment methods are mostly symptomatic treatment, and particularly have poor curative effect on some severe patients. Therefore, the development of effective specific drugs for treating coronavirus pneumonia is an urgent problem to be solved.

Disclosure of Invention

The invention aims to provide application of CAY10603 in preparing a medicament for preventing or treating novel coronavirus resistant to new coronary pneumonia.

Specifically, to solve the technical problem of the present invention, the following technical solutions are adopted:

the invention provides an application of CAY10603 or its pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug in preparing a medicament for preventing and/or treating diseases caused by coronavirus.

In another aspect, the invention provides the use of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof, for the preparation of a medicament for preventing entry of coronaviruses into the perinuclear zone.

In the technical scheme of the invention, the coronavirus is novel coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.

In the technical scheme of the invention, the disease caused by the coronavirus is pneumonia or a complication thereof caused by any one of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV.

In the technical scheme of the invention, CAY10603 is shown as a structural formula (1)

In the technical scheme of the invention, CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, a mixture of stereoisomers, tautomer, ester, amide or prodrug thereof is used as the only active ingredient in the preparation of medicaments for preventing and/or treating diseases caused by coronavirus.

In the technical scheme of the invention, CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, a mixture of stereoisomers, tautomer, ester, amide or prodrug thereof and other antiviral drugs are used for preparing a composition which is used as an active ingredient in the preparation of drugs for preventing and/or treating diseases caused by coronavirus.

In the technical scheme of the invention, the other antiviral drugs are selected from ganciclovir, acyclovir, amantadine, oseltamivir, abacavir, acemannan, acyclovir sodium, adefovir, alovudine, avsunotol, tricyclodecylamine hydrochloride, alaudine, alitame, atidine mesylate, alfvudine, cidofovir, simetrexene, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desciclovir, didanosine, dioxaxaline, edexuridine, amivir, itracin, emviran, engixime, greeting, famciclovir, chlophenylisoquin, noncitabine, fexuridine, fossilizid, foscarnet sodium, foscamet sodium, ganciclovir sodium, idoxuridine, indinavir, ethoxybutaneal, lamivudine, labrocavir, lofuadenin, lovudine, Lopinavir, memantine hydrochloride, methylthioninium chloride, nelfinavir, nevirapine, penciclovir, pirodapvir, ribavirin, saquinavir mesylate, ritonavir, sotalol hydrochloride, solivudine, penicillin, stavudine, tenofovir, troglonide hydrochloride, trifluridine, valacyclovir hydrochloride, vidarabine phosphate, vidarabine sodium phosphate, tipranavir, virdoxime, zalcitabine, zidovudine, neat weximate.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing diseases caused by viruses of the family coronaviridae, which comprises CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof.

In the technical scheme of the invention, the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

In the technical scheme of the invention, the dosage form of the pharmaceutical composition is oral preparation, lung inhalation preparation, mucosa administration preparation, eye preparation or injection.

In the technical scheme of the invention, the oral preparation is selected from granules, powder grinding agents, pills, tablets, capsules or oral liquid.

In another aspect, the invention provides the use of CAY10603 as a disinfectant against viruses of the family coronaviridae.

Another aspect of the invention provides a method for treating a disease caused by a virus of the family coronaviridae, comprising administering to a subject a therapeutically effective amount of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester or prodrug thereof.

Another aspect of the invention provides a method for preventing infection of a subject with a virus of the family coronaviridae, comprising administering to the subject prior to infection a therapeutically effective amount of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester or prodrug thereof.

Advantageous effects

The invention proves the inhibiting effect of CAY10603 on the novel coronavirus of the new coronary pneumonia for the first time, the therapeutic index is high, and the half effective concentration is low; and the CAY10603 is applied before infection to effectively increase the anti-virus effect. CAY10603 is used as an effective drug for treating the infection of the novel coronavirus resistant to new coronary pneumonia.

Drawings

FIG. 1 shows the production and validation of a novel coronavirus particle (SARS-2-S pseudotype particle).

Wherein (A) SARS-2-S protein can be successfully expressed in mammalian cells. (B) SARS-2-S pseudovirions have S protein modification. (C) SARS-2-S pseudovirions can successfully infect host cells and integrate a reporter gene. (D) SARS-2-S pseudovirions enter susceptible cells by recognizing the ACE2 receptor. (E) SARS-2-S pseudovirus particles are able to infect ACE2-GFP expressing human 293T cells, but not into 293T cells without ACE2 expression. ACE2-GFP, green; flag tag, red, showing S protein; (F) SARS-2-S pseudovirions are capable of binding to the ACE2 receptor and have co-localization signals at different locations (cell membrane and cytoplasm) within the cell. (G) The entry of SARS-2-S pseudovirus particles into ACE2-GFP/293T cells is time-dependent.

FIG. 2 shows that CAY10603 is effective in inhibiting infection by pseudovirions. The immunofluorescence staining method determines the inhibition effect of the screened clinical medicine on the infection of the SARS-2-S pseudovirus particles on host cells. After infecting 293T cells expressed by ACE2-GFP by pseudovirus particles, the intracellular localization of the pseudovirus particles is observed by fixed staining. Blue, DAPI; red, Flag antibody identifies S protein; green, ACE2-GFP signal.

Detailed Description

In the present invention, the term treating as used herein refers to reversing, alleviating, inhibiting the progression of, or preventing the disease or disorder to which the term applies or one or more symptoms of the disease or disorder.

The term "therapeutically effective amount" as used herein is the amount of compound 1 or a pharmaceutically acceptable salt thereof present in the compositions described herein, which, when such compositions are administered by a selected route of administration, provides the desired level of drug in the secretions and tissues of the airways and lungs, or alternatively in the blood stream of the subject to be treated, to produce the desired physiological response or the desired biological effect. The exact amount will depend upon a number of factors, such as the specific activity of the composition, the delivery device used, the physical characteristics of the composition, its intended use, and animal considerations such as the severity of the disease state and the like, and can be readily determined by one skilled in the art based on the information provided herein.

For the route of administration, the active ingredients of the 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 on, for example, the condition of the recipient. The compounds of the present invention have the advantage that they are orally bioavailable and can be administered orally.

The effective dose of the active ingredient will depend at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower dose) or against an active viral infection, method of delivery and pharmaceutical formulation, and will be determined by the clinician using routine dose escalation studies.

In the technical scheme of the invention, the application of a composition prepared by CAY10603 or pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs thereof and other antiviral drugs as an active ingredient in the preparation of a medicament for preventing and/or treating diseases caused by coronavirus is characterized in that more than two active ingredients in the scheme are combined in a unit dosage form and are simultaneously or sequentially administered to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be given in two or more administrations.

In the context of the present invention, a cell is any cell, such as a eukaryotic or prokaryotic cell, in particular a host cell capable of functioning as a coronavirus, in particular a cell comprising the ACE2 receptor.

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments of the present invention are described in detail below, but the present invention is not to be construed as limiting the implementable range of the present invention.

Example 1 construction, production and validation of novel coronavirus S protein-modified pseudovirions

In order to simulate the natural process of host cell infection of new coronavirus (SARS-CoV-2) by using S protein to recognize ACE2 receptor, pseudovirus particles modified with SARS-2-S protein and using replication defective lentivirus as core are constructed. The construction method adopts a method published by article evaluation segment access resource recovery 2infectivity by pseudo viral particles, J Med Virol.2020, 08/10.

First, the DNA sequence of SARS-2-S protein was modified without changing its amino acid sequence by a codon optimization method, which facilitates the high-level expression of SARS-2-S in 293T cells (FIG. 1A). Subsequently, a three-plasmid expression system is transfected in 293T cells by using the replication-defective HIV lentivirus commonly used in laboratories as the core of the pseudovirion, and SARS-2-S is modified onto the outer membrane of the pseudovirion to form the SARS-2-S pseudovirion. It was confirmed by immunoblotting that the pseudovirion had been removed from the original VSV-G envelope glycoprotein and replaced with the envelope glycoprotein SARS-2-S of a novel coronavirus (FIG. 1B). In order to verify the infection efficiency of SARS-2-S pseudovirus particles, the pseudovirus particles carry a Luciferase reporter gene and infect a host cell 293T or ACE2/293T cell. Luciferase cell activity experiments show that SARS-2-S pseudovirus particles have high infectivity on ACE2/293T cells, and the infection efficiency is improved by about 100 times than that of 293T cells (FIG. 1C). In addition, the monkey kidney epithelium Vero-E6 cells with endogenous ACE2 expression are infected by the pseudovirion, and the immunofluorescence staining method finds that the SARS-2-S pseudovirion can well enter Vero-E6 cells; if the endogenous ACE2 receptor is knocked down by siRNA, the infection efficiency of pseudovirions can be greatly reduced (FIG. 1D). Similarly, the entry process of pseudoviral particles was also observed using ACE2-GFP over-expressed 293T as host cell. Immunofluorescent staining results showed that SARS-2-S was able to efficiently enter ACE-GFP/293T cells, but not 293T cells without ACE2 expression (FIG. 1E). Therefore, the results prove that the constructed SARS-2-S pseudovirus system can simulate the natural process of recognizing ACE2 by S protein and has infection activity.

Further observation of infection process of SARS-2-S pseudovirion under high power microscope by confocal laser microscopy revealed that the pseudovirion was not only localized on the cell membrane, but also entered into the cell with ACE2 receptor and transported to the perinuclear region of the cell (FIG. 1F). Furthermore, the entry of SARS-2-S pseudovirions into the host cells was also time-dependent, entering to reach the saturation phase after approximately 2h (FIG. 1G).

EXAMPLE 2 cell biology platform screening of clinical drugs to inhibit SARS-2-S pseudovirion infection

CAY10603 in vitro cell biological assay was performed using the SARS-2-S pseudovirion in vitro infection model (Luciferase reporter System and immunofluorescence staining mapping System) constructed in example 1. ACE2-GFP stably transformed 293T cells were inoculated in a 96-well plate, the cells were pretreated with different concentrations of CAY10603 drug for 2h, then SARS-2-S pseudovirus particles were added for infection for 3h, and then the supernatant was removed and replaced with fresh complete medium. After 40h of infection, the cells were lysed using Luciferase Assay System (Promega) and the reaction substrate was added, and the Luciferase luminescence intensity was measured using Glomax 96, which is proportional to the efficiency of infection with viral particles.

The result of the Luciferase activity test shows that CAY10603 has obvious concentration dependence effect and can effectively inhibit the infection efficiency of SARS-2-S pseudovirion. The EC50 concentration for CAY10603 was about 13.5. + -. 9.4. mu.M.

Immunofluorescent staining results also showed that CAY10603 pretreated host cells at 50. mu.M concentration for 2h significantly inhibited the entry of SARS-2-S pseudovirus particles into ACE2-GFP/293T (FIG. 2). From the results of FIG. 2, in the control group, pseudovirus particles entered the cells in large amounts and localized to the perikaryocyte region, forming red aggregate regions; the administered viral particles are mostly located on and around the cell membrane, which indicates that CAY10603 effectively inhibits the invasion process of the pseudovirion, and the pseudovirion is prevented from being on the cell membrane.

It is worth mentioning that since immunofluorescent staining requires imaging of viral particles, the amount of pseudoviral particles used for infection is much higher than in the Luciferase report experiment, which is closer to the amount of virus used in the infection of human cells under physiological conditions. Therefore, the Luciferase report system can better reflect the antiviral effect of clinical drugs under physiological conditions. This indicates that CAY10603 has a greater potential for actual protection against infection by the new coronavirus. The action path and the target point of the clinical medicine are clear steps in inhibiting the new coronaviridae virus from infecting host cells, and the clinical medicine has great research value and medicine screening potential.

Claims

Use of CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof for the preparation of a medicament for the prophylaxis and/or treatment of diseases caused by coronaviruses.
Use of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof, for the preparation of a medicament for preventing entry of a coronavirus into a cell.
3. The use according to claim 1 or 2, wherein the coronavirus is the novel coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.
4. The use according to claim 1 or 2, wherein the disease caused by coronavirus is pneumonia or its complications caused by any one of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV.
5. The use according to claim 1 or 2, CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof, as the only active ingredient.
6. The use according to claim 1 or 2, CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof, in combination with other antiviral agents, as active ingredient.
7. The use according to claim 6, wherein the other antiviral agent is selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemenan, acyclovir sodium, adefovir, alovudine, avisultor, tricyclodecylamine hydrochloride, alaudine, thalidomide, altividine mesylate, alfutidine, cidofovir, cidofophylline, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desciclovir, didanosine, dioxazali, edaridine, emivirin, itravirine, itracetapine, emviran, emvir oxime, hoplatin, famciclopirox, clophenylisoquine hydrochloride, fexotabine, fexuridine, foscarnitin sodium, foscarnet sodium, ganciclovir sodium, idovir, indinavir, ethoxybutaneal, lamivudine, lobecavir, lopinavir hydrochloride, Memantine hydrochloride, methylthioninium chloride, nelfinavir, nevirapine, penciclovir, pirodapvir, ribavirin, saquinavir mesylate, ritonavir, sotalomide hydrochloride, solivudine, penicillin, stavudine, tenofovir, trovudine hydrochloride, valacyclovir hydrochloride, vidarabine phosphate, vidarabine sodium phosphate, tipranavir, viroxime, zalcitabine, zidovudine, and neat oxime.
8. A pharmaceutical composition for treating or preventing diseases caused by viruses of the family coronaviridae, which comprises CAY10603 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof as an active ingredient;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials;

preferably, the pharmaceutical composition is in the form of an oral preparation, a pulmonary inhalation preparation, a mucosal administration preparation, an ophthalmic preparation or an injection.
9. The pharmaceutical composition of claim 8, further comprising other antiviral drugs;

preferably, the other antiviral drug is selected from the group consisting of ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemenan, acyclovir sodium, adefovir, alovudine, avrinol, amantadine hydrochloride, amantadine, aliquodine mesylate, avridine, cidofovir, cidofophylline, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desciclovir, didanosine, dioxazoline, edexuridine, emivirin, itracitabine, emviraden, enviroxime, hoplatin, famciclovir, cloquine hydrochloride, decitabine, fexitabine, fexiuridine, foscarnet sodium, fosamivir sodium, idovir sodium, idoside, indinavir, ethoxybutovidone aldehyde, lamivudine, lobbucavir, lodenafine hydrochloride, lopinavir, mavir hydrochloride, mavir sodium acetate, trexate, trematodine hydrochloride, tretinomycin hydrochloride, and sodium acetate, Nelfinavir, nevirapine, penciclovir, pirodavir, ribavirin, saquinavir mesylate, ritonavir, hydrochloric acid of sodamide, solivudine, bractenocillin, stavudine, tenofovir, hydrochloric acid of trovudine, valacyclovir hydrochloride, vidarabine, adenosine phosphate, vidarabine sodium phosphate, tipranavir, viroxim, zalcitabine, zidovudine, net oxime.
10. A method for treating a disease caused by a virus of the family coronaviridae, comprising administering to a subject a therapeutically effective amount of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester or prodrug thereof.
11. A method for preventing infection of a subject with a virus of the family coronaviridae, comprising administering to the subject a therapeutically effective amount of CAY10603, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester or prodrug thereof, prior to infection with the coronavirus.