CN111467355B

CN111467355B - Application of foscarnet sodium in preparing medicine for preventing and treating coronavirus

Info

Publication number: CN111467355B
Application number: CN202010265267.9A
Authority: CN
Inventors: 袁曙光; 崔文强; 罗木鹏; 邹荣峰; 陈显翀; 赵金存; 孙静
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-04-23
Anticipated expiration: 2040-04-07
Also published as: WO2021203706A1; CN111467355A

Abstract

The invention relates to an application of foscarnet sodium in preparing a medicament for preventing and treating coronavirus, and particularly discloses an application of foscarnet sodium in preparing a medicament for preventing and treating coronavirus. The application of foscarnet or a pharmaceutically acceptable salt, isotope, stereoisomer, a mixture of stereoisomers, tautomer, ester, amide or prodrug thereof in preparing a medicament for preventing and/or treating diseases caused by coronavirus. The coronavirus is novel coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. The half effective concentration of foscarnet sodium on the novel coronavirus of the new coronopneumonia is 0.89 mu M, and the therapeutic index is 449.43, which indicates that the foscarnet sodium has a good therapeutic window.

Description

Application of foscarnet sodium in preparing medicine for preventing and treating coronavirus

Technical Field

The invention belongs to the field of antiviral drugs, and particularly relates to application of foscarnet sodium in preparation of drugs for preventing and treating coronavirus.

Background

New coronary pneumonia (COVID-19) is an infectious disease caused by The infection of a human body by a novel coronavirus (SARS-Cov-2), and The symptoms mainly comprise fever, hypodynamia, dry cough, dyspnea, renal failure and The like [ The Lancet,2020,395(10223): 507-513; the Lancet,2020,395(10223) 497-506.

Coronaviruses belong to the phylogenetic group of Coronaviridae (Coronaviridae) coronaviruses (Corona viruses). The coronavirus is a positive strand single strand RNA virus with an outer mantle (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. SARS-Cov-2 was found to be similar to SARS-Cov (79.5%) [ Nature,2020] and bat coronavirus (96%) [ bioRxiv,2020,2020.01.22.914952 ] by comparison with gene sequences of other virus samples, and it was speculated that the virus might originate from bat [ bioRxiv,2020,2020.01.24.915157; nature,2020 ]. The SARS-Cov-2 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. Global spread indicates that the new coronavirus pneumonia epidemic situation still has the possibility of spreading. However, no specific medicine can cure the novel coronavirus pneumonia until now. The development of effective antiviral drugs and vaccines is becoming the most urgent task. At present, the treatment of COVID-19 mainly depends on symptomatic adjuvant therapy, and effective specific drugs and vaccines are not available. However, the development of new drugs and the preparation of vaccines are time-consuming processes, and not only the development cycle is long, but also the mass production after approval on the market is a time-consuming project. The search for "old medicine" with the effect of resisting SARS-Cov-2 virus infection from the marketed medicines is an effective strategy for treating and preventing the explosive infection disease of COVID-19. The anti-SARS-Cov-2 virus drugs currently under study mainly include RNA polymerase inhibitors, interferon-beta, monoclonal antibodies, vaccines, etc. However, to date, there are no truly effective antiviral drugs and vaccines. From an analysis of the in vitro antiviral activity data for these drugs, the antiviral IC of most drugs₅₀At low micromolarThe therapeutic effect in vivo is still observed between mols and medium micromoles. Foscarnet inhibits viral RNA polymerase, reverse transcriptase, DNA polymerase by noncompetitive inhibition of dNTPs to inhibit viral replication. Foscarnet is effective in treating viral infectious diseases such as skin infections, herpetic keratitis, genital infections, or systemic and intracerebral infections. In view of the action mechanism of the medicine, the invention develops the application research of foscarnet in the antiviral efficacy of coronavirus.

The development of effective specific drugs for treating coronavirus pneumonia is an urgent problem to be solved. In the aspect of research and development of a novel coronavirus (SARS-Cov-2) virus medicament, the invention researches the antiviral effect of foscarnet on coronavirus, and foscarnet can block the host cell infected by the novel coronavirus and can be used for treating diseases in the aspect of resisting the infection of the novel coronavirus.

Disclosure of Invention

The invention aims to provide application of foscarnet sodium in preparing a medicament for preventing or treating diseases caused by coronavirus.

Specifically, in order to solve the technical problem of the present invention, the following technical scheme is adopted:

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

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 infectious disease or complication thereof caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV; preferred are respiratory tract infectious diseases, such as severe acute respiratory syndrome, severe acute respiratory syndrome coronavirus type 2, and middle east respiratory syndrome.

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

In the technical scheme of the invention, foscarnet or pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs thereof are used as the only active ingredient in the preparation of drugs for preventing and/or treating diseases caused by coronavirus.

In the technical scheme of the invention, foscarnet or a composition prepared from foscarnet or pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or prodrug thereof and other antiviral drugs is used as an active ingredient for preparing 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, rimantadine, oseltamivir, abacavir, acemenan, acyclovir sodium, adefovir, alovudine, avsunotol, amantadine hydrochloride, amadordine, alitame, ativudine mesylate, alfvudine, cidofovir, simetrexedine, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desciclovir, didanosine, dioxaxaline, edexuridine, emivirin, itracetirizine, emviran, engixime, greevudine, famciclovir, chlorphenisoquine hydrochloride, fosetyl, feuridine, fossilide, foscamet sodium, ganciclovir sodium, idoxuridine, indinavir, ethoxybutanedione, lamivudine, labracavir, lotvir adenosine, lopinavir, 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.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing diseases caused by viruses of the family coronaviridae, comprising foscarnet 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, pills, tablets, capsules or oral liquid.

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

In another aspect of the invention, a disinfectant for eliminating contamination by viruses of the family Coronaviridae is provided, the disinfectant comprising foscarnet.

Another aspect of the present 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 foscarnet, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide 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 a therapeutically effective amount of foscarnet, or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester or prodrug thereof, prior to infection.

In the technical scheme of the invention, the medicine or the medicine composition does not contain glucocorticoid or mineralocorticoid.

Advantageous effects

The invention discovers for the first time that foscarnet has antiviral effect on the novel coronavirus, can block the novel coronavirus from infecting host cells, and can be used for treating diseases in the aspect of resisting coronavirus infection.

Evaluation of drug efficacy of Phosphosodium formate against New coronavirus of pneumonia by CCK8 method, half Effective Concentration (EC) of New coronavirus of pneumonia (SARS-Cov-2)₅₀) 0.89 mu M, low toxicity and good therapeutic window.

Detailed Description

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

Example 1 viral amplification

VeroE6 Vero cells were grown at 3X 10⁵One well, inoculated into a 96-well plate, and placed at 37 ℃ in a minimum Eagle' medium (MEM; Gibco Invitrogen) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) with 5% CO₂Culturing until the monolayer grows. Diluting new coronavirus clinical isolate 100 times, inoculating to 96-well plate full of monolayer cells, placing at 37 deg.C and 5% CO₂Two days of culture (containing normal control).

After two days, the pathological change degree reaches over 75 percent, the mixture is placed in an ultra-low temperature refrigerator at minus 80 ℃, freeze thawing is carried out repeatedly for one time, virus liquid amplified by cells is collected, centrifuged for 30 minutes at 3000r/min, precipitates are removed, and the mixture is subpackaged into small tubes to be placed in the ultra-low temperature refrigerator at minus 80 ℃ for long-term storage.

Example 2 Phosphosodium formate drug toxicity evaluation

Dissolving foscarnet sodium powder by DMSO, adding culture solution to dilute to 20mg/mL, wherein the final concentration of DMSO is 1%, filtering by a 0.22 μm filter membrane, and storing at 4 ℃; filtering, and storing at 4 deg.C. About 2.5X 10 per hole⁴Inoculating the cells to a 96-well plate, removing culture solution after the cells grow into a monolayer after 24-48 h, adding medicaments L00 mu L/well with different dilutions, adding MEM 00 mu L/well into a normal cell control well, and adding 5% CO at 37 DEG C₂Continuously culturing for 2-5 days, adding 20 μ L of CCK8 method solution (5mg/mL) per well, and placing at 37 deg.C with 5% CO₂Incubation in the incubator was continued for 4 hours. The culture supernatant was aspirated off, 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was shaken at a low speed for 10 minutes to sufficiently melt the crystals. The 490nm wavelength is selected, and the absorbance of each pore is measured on an enzyme linked immunosorbent instrument. Toxicity evaluation shows that when the concentration of the added drug reaches 400 mu M, obvious cytotoxicity still does not appear, which indicates that the drug has low cytotoxicity and a good treatment window.

Example 3 evaluation of the drug efficacy of foscarnet sodium against the novel coronavirus of New Copyneumoniae

To evaluate the antiviral efficacy of the drugs, VeroE6 cells were grown at a density of 5X 10⁴Cells/well in 48-well cell culture dishes overnight. The virus (MOI 0.05) was added and allowed to infect for 2 hours. Then 2-fold gradient diluted drug was added, 4 multiple wells were set for each concentration, 200 μ M was used as the initial concentration of drug, 5% CO at 34 deg.C₂Incubate in incubator for 2 days. Cytopathic Effect (CPE) was recorded, CPE was recorded in the cells according to 6-grade criteria, after CPE was recorded, staining was performed by CCK8 method, OD was measured, and half effective concentration of drug (EC) was calculated by Reed-Muench method₅₀) Half maximal Effective Concentration (EC)₅₀) It was 0.89. mu.M. The experiment result shows that sodium foscarnet has low antiviral half effective concentration and good antiviral effect.

Example 4 immunofluorescence assay

Immunofluorescence microscopy: to detect expression of viral proteins in VeroE6 cells, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Cells were then blocked with 5% Bovine Serum Albumin (BSA) for 2 hours at room temperature. The cells are incubated with a primary antibody (polyclonal antibody to viral nucleocapsid protein of bat SARS-associated coronavirus, Anti-NP at a dilution of 1: 1000) for 2 hours, and then incubated with a secondary antibody (488 affinity assay Donkey Anti-Rabbit IgG (H + L). cell nuclei are stained with Hoechst33258 dye (Beyotime, China). Observation through a fluorescence microscope shows that foscarnet can effectively kill viruses in the cells, has little influence on the cells, and has a good therapeutic window.

Claims

1. The application of foscarnet sodium in preparing medicine for preventing and/or treating coronavirus caused diseases; the disease caused by coronavirus is infectious disease caused by SARS-Cov-2 virus or its complication.

2. The use according to claim 1, wherein the infectious disease is a respiratory infectious disease.

3. Use according to claim 1, of foscarnet sodium as the sole active ingredient in the preparation of a medicament for the prevention and/or treatment of diseases caused by coronaviruses.

4. Use of foscarnet sodium in the preparation of a disinfectant for eliminating coronavirus virus contamination; wherein the coronavirus is novel coronavirus SARS-Cov-2.