CN117479940A

CN117479940A - Therapeutic agent for infectious disease against mutant virus of novel coronavirus

Info

Publication number: CN117479940A
Application number: CN202280042533.9A
Authority: CN
Inventors: 米纳孝; 古田要介
Original assignee: Toyama Chemical Co Ltd
Current assignee: Toyama Chemical Co Ltd
Priority date: 2021-05-14
Filing date: 2022-05-12
Publication date: 2024-01-30

Abstract

A therapeutic agent for coronavirus infection caused by SARS-CoV-2 mutant virus, which comprises 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or a salt thereof as an active ingredient.

Description

Therapeutic agent for infectious disease against mutant virus of novel coronavirus

Technical Field

The present invention relates to a therapeutic agent for coronavirus infection caused by a mutant virus of a novel coronavirus, which comprises 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (generic name: fampicvir, hereinafter referred to as compound A) or a salt thereof as an active ingredient.

Background

The novel coronavirus (SARS-CoV-2) first reported in China at 2019 (non-patent document 1) is an RNA virus belonging to the family Coronaviridae of the order nidoviridae (non-patent document 2). When the virus is infected, acute respiratory symptoms such as fever, cough, dyspnea, etc. occur (non-patent document 1), and if the symptoms worsen, pneumonia develops. By 2021, 5 months and 12 days, the number of SARS-CoV-2 positives in Japan has exceeded 65 ten thousand people, and the number of deaths has exceeded 1 ten thousand people (non-patent document 3).

Compound a is an antiviral drug developed by fuji film fushan chemical corporation (former fushan chemical corporation), and has been licensed for production and sales in japan in 3 months of 2014, and its efficacy and effect are limited to "new or reappeared influenza virus infection (wherein, only the other anti-influenza virus drugs are ineffective or insufficient)". The mechanism of action is that the in vivo transformed triphosphorylate (T-705 RTP) selectively inhibits viral RNA polymerase, and thus may exhibit effects on RNA viruses other than influenza viruses. In fact, it has been reported to be effective against ebola virus and RNA viruses of arenaviridae and bunyaviridae in vitro or in vivo (non-patent documents 4, 5, 6).

SARS-CoV-2 continues to accumulate genetic variation at various loci in the genome, and many variant viruses have been reportedhttps://nextstrain.org/ncov/globalHttps:// cov-lineages. Variant viruses with high infection efficiency and variant viruses that have a fear of affecting vaccine efficacy have also emerged (https:// www.cdc.gov/corenavirus/2019-ncov/cases-updates/variant-survilance/variant-info. Html).

Treatment of coronavirus infection caused by these SARS-CoV-2 variant viruses has not been established. In addition, in japan, adefovir as an antiviral drug has been approved for the treatment of covd-19, but drug resistant lesions against adefovir have been reported (non-patent documents 7, 8, 9) which may cause a decrease or inefficiency in the efficacy of the drug. Thus, new therapeutic agents are needed.

Prior art literature

Non-patent literature

Non-patent document 1: wang C, horby PW, hayden FG, gao gf. Anodel coronavirus outbreak of global health concept. Lancet.2020;395:470-3.

Non-patent document 2: coronaviridae Study Group of the International Committee on Taxonomy of viruses.the species Severe acute respiratory syndrome-related coronavirus: classification 2019-nCoV and naming it SARS-CoV-2.Nat Microbiol.2020;5:536-44.

Non-patent document 3: the national dust generator is further processed ど [ Internet ]. Tokyo, labour-saving of updated 2021May 12; citd 2021May 13. Available from https:// www.mhlw.go.jp/stf/covid-19/kokuneainesseijoukyou

Non-patent document 4: gowen BB, wong MH, jung KH, sanders AB, mendenhall M, bailey KW, et al in vitro and in vivo activities of T-705against arenavirus and bunyavirus infections.Antimicrob Agents Chemother.2007;51:3168-76.

Non-patent document 5: mendenhall M, russell A, smee DF, hall JO, skirpstunas R, furuta Y, et al effective organic favipiravir (T-705) therapy initiated after the onset of clinical disease in a model of arenavirus hemorrhagic Fever.PLoS Negl Trop Dis.2011;5:e1342.

Non-patent document 6: oesteech L, ludtke A, wurr S, rieger T, munoz-Fontela C, gunther S.Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model.Antiviral Res.2014;105:17-21.

Non-patent document 7: szemiel et al In vitro evolution of Remdesivir resistance reveals genome plasticity of SARS-CoV-2.bioRxiv 2021[https:// doi.org/10.1101/2021.02.01.429199]

Non-patent document 8: martinot M, jary A, fafi-Kremer S, leducq V, delagreverie H, garnier M et al Remdeivir failure with SARS-CoV-2RNA-dependent RNA-polymerase mutation in a B-cell immunodeficient patient with protracted Covid-19.Clin Infect Dis.2020[doi:10.1093/cid/ciaa1474]

Non-patent document 9: agroctini ML, andres EL, sims AC, graham RL, sheahan TP, lu X et al Coronavir Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribanuclease. MBio.2018;9:e00221-18.

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing an agent for treating infectious diseases, which exhibits an effect on a mutant virus of a novel coronavirus.

Technical scheme for solving problems

Under such circumstances, the present inventors have found that an infectious disease caused by a mutant virus of a novel coronavirus can be treated by the compound A or a salt thereof, and have completed the present invention.

According to the present invention, the following invention is provided.

[1]

[2]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variant viruses has a mutation in genetic information of Wuhan-Hu-1 strain produced at a gene encoding S protein.

[3]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at the gene encoding the M protein.

[4]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at the gene encoding the E protein.

[5]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at the gene encoding the N protein.

[6]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding an RNA-dependent RNA polymerase.

[7]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding exonuclease.

[8]

The therapeutic agent for coronavirus infection according to [1], wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding a protease.

[9]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is a virulent or attenuated virus as compared to SARS-CoV-2.

[10]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is a virus having an increased or decreased human-human infection efficiency as compared to SARS-CoV-2.

[11]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is a virus having an increased immunological escape ability as compared with SARS-CoV-2.

[12]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is a virus having an increased or decreased resistance to the therapeutic agent for coronavirus infection as compared to SARS-CoV-2.

[13]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is a virus having increased or decreased effectiveness of a novel coronavirus vaccine as compared to SARS-CoV-2.

[14]

The therapeutic agent for coronavirus infection according to [1], wherein the mutant virus is B.1.1.7 (501Y.V1), B.1.351 (501Y.V2), P.1 (501Y.V3), B.1.427 (20C/S: 452R), B.1.429 (20C/S: 452R), B.1.526 (20C/S: 484K), B.1.526.1 (20C), B.1.525 (20A/S: 484K), P.2 (20J), B.1.617 (20A), B.1.617.1 (20A/S: 154K), B.1.617.2 (20A/S: 478K), B.1.617.3 (20A), B.1.1.316, B.1.617+S: V382L or a clinically isolated Swissimnier resistant coronavirus.

Effects of the invention

The therapeutic agent for infectious diseases of the present invention is useful for treatment such as treatment or prevention of infectious diseases of coronaviruses caused by mutant viruses of new coronaviruses.

Detailed Description

The present invention will be described in detail below.

In the present specification, unless otherwise specified, each term has the following meaning.

In the present specification, the numerical range indicated by the term "to" means a range including numerical values described before and after the term "to" as a minimum value and a maximum value, respectively.

The compound A refers to 6-fluoro-3-hydroxy-2-pyrazinecarboxamide.

The salt of the compound a includes a salt of a generally known basic group such as an amino group or an acidic group such as a hydroxyl group or a carboxyl group.

Examples of the salt of the basic group include: salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and the like; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylene sulfonic acid, and naphthalenesulfonic acid.

Examples of the salt of the acidic group include: salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; an ammonium salt; salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-beta-phenylethylamine, 1-efenamine, N' -dibenzylethylenediamine, meglumine and the like.

Among the above salts, pharmacologically acceptable salts are preferable, and salts with sodium or meglumine are more preferable.

In the case where isomers (for example, optical isomers, geometric isomers, tautomers, and the like) are present in the compound a or a salt thereof, the present invention includes all of them, and further includes hydrates, solvates, and all of the crystal forms.

The term "coronavirus" as used in the present specification is an RNA virus belonging to the family Coronaviridae of the order nidoviridae. The infection caused by coronavirus is a coronavirus infection.

"New coronavirus" refers to SARS-CoV-2 reported in coronaviruses at the end of 2019. Coronavirus infection caused by SARS-CoV-2 is also known as COVID-19.

"mutant virus of a novel coronavirus" refers to a virus that has been mutated to at least a portion of the genetic information of the Wuhan-Hu-1 strain (Accession No. nc_ 045512) defined as Reference strain on the NCBI website (https:// www.ncbi.nlm.nih.gov/sars-cov-2 /). The term "mutant virus" is a term including "mutant strain" and "variant virus".

Variant viruses of the novel coronavirus include those published on the national institute for infectious diseases website for the novel variant of the novel coronavirus (SARS-CoV-2) that may be increased in infection/transmission and may be changed in antigenicity (https:// www.niid.go.jp/nid/ja/2019-ncov/2551-cepr/10743-covid 19-62. Html). Examples include: alpha strain, beta strain, gamma strain, delta strain, (old) kappa strain, lambda strain, mu strain, AY.4.2.

The genetic information can be mutated at any site in the genome of SARS-CoV-2, and the mutation occurring in the gene encoding a structural protein or a non-structural protein can be exemplified. As the structural protein, there may be mentioned: s protein, M protein, E protein, N protein. As the non-structural proteins, there may be mentioned: primer enzymes, RNA-dependent RNA polymerases, helicases, exonucleases, ribonucleases, methyltransferases, proteases, and the like.

Examples of the mutant virus having a mutation in the gene encoding the S protein of SARS-CoV-2 include: b.1.1.7 (501 Y.V1), B.1.351 (501 Y.V2), P.1 (501 Y.V3), B.1.427 (20C/S: 452R), B.1.429 (20C/S: 452R).

Examples of the mutant virus that has a mutation in a gene encoding the M protein or E protein of SARS-CoV-2, which is another structural protein, include: viruses having the variants described in the literature (Jakhmola S, indri O, kashyap D, varshney N, das A, manivannan et al, mutional analysis of structural proteins of SARS-CoV-2.Heliyon.2021; 7:e06572.).

The mutant virus of the novel coronavirus is a virus whose properties are altered compared to SARS-CoV-2. Here, the change in the properties of the virus can be exemplified by: the method comprises the steps of virulence or attenuation, increase or decrease of immune escape capacity, increase or decrease of human-human infection efficiency, increase or decrease of drug resistance of a therapeutic drug for coronavirus infection, increase or decrease of effectiveness of a novel coronavirus vaccine, and increase or decrease of virus proliferation.

A virulent or attenuated variant virus refers to a virus that produces a change in the pathogenicity of the host. Viruses with increased immune escape ability represent viruses that have been altered in such a way that they are not easily recognized as foreign bodies by the host and are not easily eliminated by immunization. The virus having an increased or decreased human-to-human infection efficiency means a virus having a change in infection efficiency due to a change in the binding capacity of ACE2 to host cells, or the like. Viruses with increased or decreased resistance to a therapeutic agent for coronavirus infection represent viruses that have a change in the structural or non-structural proteins of the virus at the site of action of the therapeutic agent. The virus with increased or decreased effectiveness of the novel coronavirus vaccine means a virus with increased or decreased responsiveness to neutralizing antibodies obtained by vaccination of the host. Viruses with increased or decreased viral proliferation indicate viruses with increased or decreased viral proliferation efficiency within the host cell.

The identified variant viruses of the novel coronavirus include the following viruses.

B.1.1.7 (501 Y.V1), B.1.351 (501 Y.V2), P.1 (501 Y.V3), B.1.427 (20C/S: 452R), B.1.429 (20C/S: 452R), B.1.526 (20C/S: 484K), B.1.526.1 (20C), B.1.525 (20A/S: 484K), P.2 (20J), B.1.617 (20A), B.1.617.1 (20A/S: 154K), B.1.617.2 (20A/S: 478K), B.1.617.3 (20A), B.1.1.316, B.1.617+S: V382L, experimentally or clinically isolated adefovir-resistant coronavirus

The major amino acid mutation positions identified in the above-listed mutant viruses of the novel coronaviruses are shown in Table 1 below.

[ Table 1]

The mutant viruses of the novel coronavirus also include unidentified mutant viruses, and unknown mutant viruses such as non-emerging mutant viruses. Unknown variant viruses may be identified, for example, on the site of the CDC that updates over time (https:// www.cdc.gov/corenavirus/2019-ncov/cases-updates/variant-survivinlan/variant-info. Html).

Treatment refers to the alleviation or amelioration of one or more symptoms caused by a particular disorder, as well as the delay of progression of the disorder, to a subject suffering from the disorder. In the present embodiment, for example, the present invention refers to a method for alleviating or improving symptoms such as fever, cough, and pneumonia in a patient suffering from an infection with a mutant virus of a new coronavirus, and for example, to a method for improving body temperature, percutaneous arterial oxygen saturation (SpO 2), and chest imaging examination results or for turning negative of a mutant virus of a new coronavirus.

The compound a or a salt thereof used in the present invention can be produced by a method known per se or by appropriately combining these methods. For example, the product can be produced by the method described in International publication No. 00/10569. In the compound a, 6-fluoro-3-oxo-3, 4-dihydro-2-pyrazinecarboxamide exists as a tautomer.

The compound a or a salt thereof used in the present invention can be formulated into pharmaceutical preparations such as oral preparations (tablets, capsules, powders, granules, fine granules, pills, suspensions, emulsions, solutions, syrups, etc.), injections, eye drops, nasal or transdermal preparations by blending various pharmaceutical additives such as excipients, binders, disintegrants, disintegration inhibitors, anti-caking/adhesion agents, lubricants, absorption/adsorption carriers, solvents, fillers, isotonic agents, solubilizers, emulsifiers, suspending agents, thickening agents, coating agents, absorption promoters, gelation/coagulation promoters, photostabilizers, preservatives, moisture-proofing agents, emulsifying/suspension/dispersion stabilizers, coloring agents, oxygen-removing/antioxidant agents, flavoring/odor-proofing agents, colorants, foaming agents, antifoaming agents, analgesics, antistatic agents, buffers, pH adjusters, etc. The administration preparation for patients suffering from infection caused by a novel coronavirus variant virus is preferably an oral preparation or an injection, more preferably an oral preparation, and still more preferably a tablet.

The above-mentioned medicines are formulated into preparations by ordinary methods.

The method of administration of compound a is not particularly limited, and is appropriately determined according to the form of the preparation, other conditions such as age and sex of the patient, and the degree of symptoms of the patient.

The amount of compound a to be administered is appropriately selected depending on the use, age, sex, disease form, other conditions, etc., and for example, as compound a, 10 to 5000mg, or preferably 200 to 2400mg can be administered to an adult once or in several times per day. Compound a may be administered in multiple doses or in a single dose until the desired therapeutic effect is achieved. Administration can generally be monitored and repeated as needed.

In the present invention, as compound a, 1000 to 2400mg (day 1) and 400 to 1200mg (day 2 and later) may be administered to an adult 1 day 2 and 1 day 2. Preferably, 1600mg (day 1) and 600mg (after day 2) are administered to an adult 1 day 2 and 1 day 2 as compound a; and 1800mg (day 1) and 800mg (after day 2) for 1 day 2 administration to an adult as compound a, more preferably 1800mg (day 1) and 800mg (after day 2) for 1 day 2 administration to an adult as compound a.

The interval between administrations of 2 times a day is preferably at least 4 hours after the first administration and then the second administration is performed, more preferably at least 6 hours after the first administration.

The administration period is appropriately determined according to the lapse of symptoms, and may be selected from, for example, up to 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and 22 days. Preferably up to 10 days, 13 days, 14 days, 22 days. More preferably for a maximum of 13 days, 14 days.

In the present invention, administration of compound a or a salt thereof may include combination drug and/or combination therapy. As the combination drug and/or the combination therapy, a therapeutic agent for coronavirus infection can be used. Examples of the therapeutic agent for coronavirus infection include the following agents.

(1) Interferon

(2) Nucleic acid analogues

(3) Protease inhibitors

(4) Furin converting enzyme cleavage inhibitor

(5) Reverse transcriptase inhibitors and/or other RNA polymerase inhibitors

(6) Neuraminidase inhibitors

(7) Angiotensin II receptor blockers

(8) Endosomal fusion inhibitors and/or endosomal alkalizers

(9) AAK1, GAK and clathrin A, B, C (endocytosis) inhibitors

(10) Hemagglutinin esterase inhibitors

(11) Cytokine or inflammation inhibitor and regulator

(12) Cathepsin B inhibitors

(13) Cathepsin L inhibitors

(14) Inhibitors of helicase nsp13

(15) MBL2 gene agonists

(16) TP53 inhibitors

(17) Selective estrogen receptor modulators

(18) Steroid medicine

(19) Other medicaments

Examples of the interferon include: interferon alpha-2A, interferon alpha-2B, interferon alpha-n 1, interferon alpha-n 3, interferon beta-1 a, and interferon beta-1B. The interferon may be produced by combining methods known per se, or may be commercially available.

Examples of the nucleic acid analog include: acyclovir, ganciclovir, ribavirin and talepstein. The method may be carried out by combining methods known per se, or by using commercially available products. The nucleic acid analog may be produced by combining methods known per se, or may be commercially available.

Examples of protease inhibitors include: indinavir, nelfinavir, saquinavir, camostat, lopinavir/ritonavir complex (product name: kaltra), epigallocatechin gallate, kaempferol-7-glucoside, mycophenolic acid, darunavir, mercaptopurine, disulfiram, nafamostat and PF-07321332. The protease inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of inhibitors of furin converting enzyme cleavage include: tenofovir disoproxil, dorame Weibo privir, andrographolide, luteolin and baicalein. The furin converting enzyme cleavage inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of reverse transcriptase inhibitors and/or RNA polymerase inhibitors include: adefovir, sofosbuvir, actinomycin D, ganaxan Li Xiwei, balo Sha Weima Boxib, mo Nuola pyrroside, sanguinarine and AT-527. The reverse transcriptase inhibitor and/or the RNA polymerase inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of neuraminidase inhibitors include: oseltamivir and zanamivir. The neuraminidase inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of the angiotensin II receptor blocker include: valsartan, telmisartan, losartan, irbesartan, azilsartan, olmesartan, and emodin. The angiotensin II receptor blocker may be produced by combining methods known per se, or may be commercially available.

Examples of the endosomal fusion inhibitor and/or the endosomal alkalizing agent include: baicalin, chloroquine, hydroxychloroquine, griffithsin, quinine and lactoferrin. The endosomal fusion inhibitor and/or the endosomal alkalizing agent may be produced by combining methods known per se, or may be commercially available.

Examples of inhibitors of AAK1, GAK and clathrins a, B, C (endocytosis) include: baroretinib, sunitinib, erlotinib, fei Dela tinib, gefitinib and silibinin. The AAK1, GAK and clathrin a, B, C (endocytosis) inhibitors may be produced by combining methods known per se or may be commercially available.

Examples of the hemagglutinin esterase inhibitor include 3, 4-dichloroisocoumarin. The hemagglutinin esterase inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of cytokines, inflammation inhibitors and modulators include: ligustrazine, statins, melatonin, li Pu epothilone, and methylprednisolone. The cytokine or the inflammation inhibitor and the regulator may be produced by combining methods known per se, or may be commercially available.

Examples of the cathepsin B inhibitor include salvianolic acid B. The cathepsin B inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of the cathepsin L inhibitor include: MOL736, chelidostatin, astaxanthin, curcumin and vitamin D. The cathepsin L inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of the helicase nsp13 inhibitor include: valsartan, bananin, iodobananin, vanillinbananin, eubananin and silverstrol. The helicase nsp13 inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of MBL2 gene agonists include: beta-glucan and vitamin a. MBL2 gene agonists may be produced by combining methods known per se or may be commercially available.

Examples of TP53 inhibitors include: vitexin and gossypol. The TP53 inhibitor may be produced by combining methods known per se, or may be commercially available.

Examples of selective estrogen receptor modulators include: toremifene and equilin. The selective estrogen receptor modulator may be produced by combining methods known per se, or may be commercially available.

Examples of the steroid drug include: ciclesonide and dexamethasone. The steroid drug may be produced by combining methods known per se, or may be commercially available.

Examples of the other agents include: amantadine, sodium foscarnet, ribavirin, wu Mi Feinuo, rapamycin, everolimus, nitazoxanide, tizoxanide, carabinol a, ivermectin, VIR-2703, tolizumab, bamlanizumab, etlizumab, casirivimab/imdevimab, AZD7422, VIR-7831, VIR-7832 and BI 767551. Amantadine, sodium phosphonate, ribavirin, wu Mi Feinuo, rapamycin, everolimus, nitazoxanide, tizoxanide, carabinol a, ivermectin, VIR-2703, tolizumab, bamlanizumab, etlizumab, casirivimab/imdevimab, AZD7422, VIR-7831, VIR-7832 and BI 767551 are manufactured by combining methods known per se or commercially available products are used.

[ example ]

The present invention will be described below with reference to test examples, but the present invention is not limited thereto.

Test example 1

For compound a, experiments were performed by a cell infection model of the virus. As the cells used, vero E6 cells were used. In particular, antiviral effects were evaluated by detecting cytopathic effects (cytopathic effect; CPE). In addition to Vero E6, other cells may be used, and evaluation may be performed using Vero, vero76, caCo-2, hep G2, calu-3, etc. that exhibit viral susceptibility. The antiviral effect may be evaluated by a viral antigen-antibody method, a real-time PCR (polymerase chain reaction) method after extracting viral RNA from a viral culture supernatant, or the like, in addition to the CPE method.

The variant strains B.1.1.7 (501 Y.V1) and B.1.351 (501 Y.V2) of the novel coronavirus were selected as RNA viruses.

(1) Culture of Vero E6 cells

Vero E6 cells of African green monkey kidney subcultured at 37℃under 5% carbon dioxide in Eagle MEM/kanamycin (Eagle MEM/kanamycin) medium supplemented with 10% fetal bovine serum were detached by the trypsin method of ethylenediamine tetraacetic acid, and 100. Mu.L of the medium was prepared to contain 2X 10 ⁴ The individual cell suspensions were seeded into 96-well plates. After one night of incubation at 37℃under 5% carbon dioxide, vero E6 cells were obtained as monolayers.

(2) Infection of mutant virus of novel coronavirus and drug addition

As test medium, eagle MEM/kanamycin medium supplemented with 2% fetal bovine serum was used. The culture supernatant of Vero E6 cells obtained in (1) was removed, and the following (A) and (B) were added to each well, followed by culturing at 37℃under 5% carbon dioxide for 1 to 2 hours.

(A) 100 mu L of a novel coronavirus variant virus solution prepared by a test medium in a manner that the final infection severity reaches 0.1-10

(B) Compound a at a concentration 2 times the set concentration, test medium (drug) containing 0.5% dmso comprising the combination of each set concentration, 100 μl

Set concentration of compound a (μm):

0、0.1、0.3、1、3、10、30、100、300、1000、3000、5000、8000、10000

after 1 to 2 hours of infection, the culture supernatant of each well was removed, and 100. Mu.L of a test medium (drug) containing 0.5% DMSO at each of the set concentrations and containing 1-fold concentration of Compound A was added. After adding the reagent, the culture was carried out at 37℃under 5% carbon dioxide for 2 to 3 days.

(3) Determination of cytopathic effect (CPE)

CPE identified with proliferation of SARS-CoV-2 was determined by the following method.

After completion of the culture, 25. Mu.L of 100% formalin was added to each well, and virus inactivation and cell fixation were performed. After leaving at room temperature for 2 hours or more, the aqueous solution was removed, and after washing with water gently, 0.02% methylene blue solution was added at 50. Mu.L/well, and left at room temperature for 1 hour. The aqueous solution was removed, gently rinsed with water and air dried. Then, absorbance (660 nm) was measured by a microplate reader. In the non-infectious control, 100. Mu.L of the test medium was added in place of SARS-CoV-2 solution, and the absorbance was measured in the same manner as in the test group.

Two-plate (8 for both infected and non-infected controls) experiments were performed as per 1/plate. The CPE inhibition rate of each test was calculated according to the following equation, using the value obtained by subtracting the absorbance of the infected control from the absorbance of the non-infected control as the complete inhibition value of virus proliferation.

CPE inhibition = 100× [ (absorbance when compound a was used) - (absorbance of infected control) ]/[ (absorbance of non-infected control) - (absorbance of infected control) ]

The 50% cpe inhibition concentration was calculated using the forect function of Microsoft Office Excel 2016 (linear regression method).

The 50% inhibitory concentration of compound a was calculated for mutant virus b.1.1.7 (501y.v1) and b.1.351 (501y.v2) strains of the new coronavirus.

Compound a was confirmed to show sensitivity to mutant virus strains b.1.1.7 (501y.v1) and b.1.351 (501y.v2) of the novel coronavirus.

Test example 2

As RNA viruses, mutant viruses P.1 (501 Y.V3), B.1.427 (20C/S: 452R), B.1.429 (20C/S: 452R), B.1.526 (20C/S: 484K), B.1.526.1 (20C), B.1.525 (20A/S: 484K), P.2 (20J), B.1.617 (20A), B.1.617.1 (20A/S: 154K), B.1.617.2 (20A/S: 478K), B.1.617.3 (20A), B.1.1.316, B.1.617+S: V382L, and experimentally or clinically isolated Swisswell-resistant coronaviruses of Compound A were selected, and 50% inhibitory concentrations of Compound A were calculated in the same manner as in test example 1 above, thereby confirming that Compound A exhibited sensitivity to various mutant viruses of novel coronaviruses.

Test example 3

< determination of drug sensitivity >

The drug susceptibility of compound A to a mutant virus of a novel coronavirus was determined by the yield reduction (yield reduction) method. As a wild strain of the novel coronavirus, USA_WA1/2020 (strain A) WAs selected as a variant of the novel coronavirusThe virus selected from hCoV-19/England/204820464/2020 (strain B.1.1.7), hCoV-19/Japan/TY7-503/2021 (strain P.1). The virus was propagated in the presence of 3.2 to 1000. Mu.M (public ratio 3) of Compound A, and then the amount of the virus at each concentration was measured. From the resulting viral load, 90% effective concentration was calculated (90%effective concentration;EC ₉₀ )。

< proliferation of virus >

The day before virus infection, vero76 cells suspended in cell culture medium were plated. At 5% CO ₂ The Vero76 cells, which became monolayers, were used for the next day of culture at 37 ℃. Virus culture medium containing Compound A was added at 5% CO ₂ Pretreatment was carried out at 37℃for 1 hour. After the medium was aspirated, a medium containing Compound A and a virus solution (complex of infection (Multiplicity of Infection) 0.01) were added to each well so that the final concentration reached the predetermined concentration, and the concentration was 5% CO ₂ Culturing at 37 deg.c for 3-4 days. After the end of the culture, the culture supernatant from each well was transferred to a new plate and stored at-80℃until virus quantification.

The day before virus infection, vero76 cells suspended in cell culture medium were plated. Will be<Viral propagation>After serial dilution of the virus culture broth obtained in (1) at a public ratio of 10, the diluted virus broth was added to Vero76 cells (n=4) at 5% co ₂ Culturing at 37 deg.c for 3-4 days. After completion of the culture, the presence or absence of CPE in each well was confirmed, and then the concentration of infection in 50% cell culture was calculated by the Reed-Muench method (CCID) ₅₀ /mL(Log ₁₀ )). In addition, log of the concentration of Compound A ₁₀ Value and viral load (CCID) ₅₀ /mL(Log ₁₀ ) Regression analysis to calculate EC ₉₀ Values.

EC of Compound A against USA_WA1/2020 (line A), hCoV-19/England/204820464/2020 (line B.1.1.7), hCoV-19/Japan/TY7-503/2021 (line P.1) strains ₉₀ The values were 68. Mu.M, 83. Mu.M, 190. Mu.M, respectively.

Compound A was used in combination with hCoV-19/England/204820464/2020 (line B.1.1.7) and hCoV-EC of 19/Japan/TY7-503/2021 (P.1 strain) strain ₉₀ The values showed a range of 1.2 to 2.8 times as large as that of USA_WA1/2020 (strain A), and it WAs found that compound A also showed a drug effect on the mutant strain to the same extent as that of the wild strain of the novel coronavirus.

Further, the drug sensitivity of Compound A was measured with respect to the strain hCoV-19/Japan/QK002/2020 (B.1.1.7 strain), hCoV-19/Japan/TY8-612/2021 (B.1.351 strain), hCoV-19/Japan/TY7-501/2021 (P.1 strain), hCoV-19/Japan/TY11-927-P1/2021 (B.1.617 strain), and hCoV-19/Japan/11-330-P1/2021 (B.1.617.1 strain) which are mutant viruses of the novel coronaviruses, by the same methods as described above. Wherein the compound A was present at a concentration of 100 to 1000. Mu.M (public ratio 3.3) during virus propagation, the pretreatment time after adding the compound A was 3 hours, the multiplicity of infection (Multiplicity of Infection) of the added virus solution was 0.001, and the culture time after adding the virus solution was 40 hours, and Vero E6/TMPRSS2 cells were used. In addition, the test was performed in two times.

EC of Compound A against the above Standard strain and variant strain ₉₀ The values were 302. Mu.M (first test) or 466. Mu.M (second test), 359. Mu.M (first test), 223. Mu.M (second test), 306. Mu.M (second test), 294. Mu.M (first test), 491. Mu.M (second test), respectively, and the EC of Compound A relative to the variant ₉₀ The compound A was found to have a range of 0.48 to 1.2 times the range of variation compared with the standard strain, and thus the compound A showed the same level of efficacy as the standard strain.

Test example 4

< cell culture >

Vero E6 cells were seeded on 6-well assay plates and incubated for about 4 hours prior to treatment with compound a.

< pretreatment and titre test >

Cells from each well were pretreated with a compound A solution serially diluted at the concentration described below for about 4 hours, and then infected with a novel coronavirus. The standard strain WAs SARS-2WA1/2020, and the mutant virus of the novel coronavirus WAs SARS-2-9017 (delta) and SARS-2-Omicron. The titer of compound a was evaluated by setting a concentration of 7.81 to 1000 μm (public ratio 2) in the wells on the plate. The multiplicity of infection (Multiplicity of Infection) of the added virus solution was 0.01, and the incubation time for the added virus solution was 72 hours.

Culture supernatants were collected from each well after infection, serially diluted at a public ratio of 10, and added to VeroE6 cells (n=4) as monolayers for culture. After clear CPE was observed, 50% cell culture infection concentration was calculated by Reed-Muench method (CCID ₅₀ /mL(Log ₁₀ )). In addition, log of the concentration of Compound A ₁₀ Value and viral load (CCID) ₅₀ /mL(Log ₁₀ ) Regression analysis to calculate EC ₉₀ Values.

EC of Compound A against the above Standard strain and variant strain ₉₀ The values were 125 to 250. Mu.M (SARS-2 WA 1/2020), 125 to 250. Mu.M (SARS-2-9017 (delta)), and 62.5 to 125. Mu.M (SARS-2-Omicron), respectively, and it WAs found that Compound A also showed a drug effect on the mutant strain to the same extent as that of the standard strain.

Based on the results, compound a is considered effective against a variant virus of the new coronavirus.

Industrial applicability

Therapeutic agents against a mutant virus of a novel coronavirus, which contain 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or a salt thereof as an active ingredient, are useful in the field of pharmaceutical industry.

Claims

1. A therapeutic agent for coronavirus infection caused by SARS-CoV-2 mutant virus, which comprises 6-fluoro-3-hydroxy-2-pyrazinecarboxamide or a salt thereof as an active ingredient.

2. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced at a gene encoding S protein in the variant virus.

3. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of the genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding M protein.

4. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of genetic information of Wuhan-Hu-1 strain is produced at a gene encoding E protein in the variant virus.

5. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of the genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding the N protein.

6. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of the genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding an RNA-dependent RNA polymerase.

7. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of the genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding an exonuclease.

8. The therapeutic agent for coronavirus infection according to claim 1, wherein at least one of the variants of the genetic information of Wuhan-Hu-1 strain is produced in the mutant virus at a gene encoding a protease.

9. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is a virulent or attenuated virus compared to SARS-CoV-2.

10. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is a virus having an increased or decreased human-human infection efficiency as compared to SARS-CoV-2.

11. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is a virus having an increased immunological escape ability as compared to SARS-CoV-2.

12. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is a virus having an increased or decreased resistance to the therapeutic agent for coronavirus infection as compared to SARS-CoV-2.

13. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is a virus having increased or decreased effectiveness of a novel coronavirus vaccine as compared to SARS-CoV-2.

14. The therapeutic agent for coronavirus infection according to claim 1, wherein the mutant virus is b.1.1.7 (501y.v1), b.1.351 (501y.v2), p.1 (501y.v3), b.1.427 (20C/S: 452R), b.1.429 (20C/S: 452R), b.1.526 (20C/S: 484K), b.1.526.1 (20C), b.1.525 (20A/S: 484K), p.2 (20J), b.1.617 (20A), b.1.617.1 (20A/S: 154K), b.1.617.2 (20A/S: 478K), b.1.617.3 (20A), b.1.1.316, b.1.617+s: V382L, or a adefovir resistance coronavirus isolated experimentally or clinically.