Agent for preventing or treating RNA virus-related diseases

Abstract

This prophylactic or therapeutic agent for RNA virus-related diseases contains, as an active ingredient, at least one compound selected from the group consisting of a selective estrogen receptor modulator, an anti-tuberculous drug, a CysLT1 receptor antagonist, a peroxisom proliferator-activated receptor γ (PPARγ) agonist, an arachidonate 5-lipoxygenase (5-LOX) inhibitor, a derivative thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable solvate thereof.

Description

Technical Field

[0001] The present invention relates to a prophylactic or therapeutic agent for RNA virus-related diseases. This application claims priority based on U.S. Provisional Patent Application No. 63 / 017,677, filed in the United States on April 30, 2020, and U.S. Provisional Patent Application No. 63 / 124,098, filed in the United States on December 11, 2020, the contents of which are incorporated herein by reference.

Background Art

[0002] The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic due to efficient human-to-human infection and high pathogenicity, posing a threat to public health (see, for example, Non-Patent Document 1). SARS-CoV-2 is a virus belonging to the Coronaviridae family and is an enveloped virus with a single-stranded positive-sense RNA genome.

[0003] The causative viruses of emerging viral diseases such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) also belong to the Coronaviridae family. In addition, the causative viruses of highly lethal Ebola virus disease and Marburg virus disease belong to the Filoviridae family, which are enveloped viruses with a single-stranded negative-sense RNA genome.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In addition to the coronavirus and filovirus, many RNA viruses pose a threat of emerging and re-emerging infectious diseases, and it is very important to find preventive or therapeutic drugs for RNA virus-related diseases. The present invention aims to provide a preventive or therapeutic drug for RNA virus-related diseases. [Means for Solving the Problems]

[0006] The present invention includes the following aspects. [1] A preventive or therapeutic agent for RNA virus-related diseases, comprising at least one compound selected from the group consisting of a selective estrogen receptor modulator, an anti-tuberculosis drug, a CysLT1 receptor antagonist, a Peroxisome Proliferator-Activated Receptor γ (PPARγ) agonist, an Arachidonate 5-lipoxygenase (5-LOX) inhibitor, derivatives thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof as an active ingredient. [2] The preventive or therapeutic agent for RNA virus-related diseases according to [1], wherein the selective estrogen receptor modulator is raloxifene, tamoxifen, toremifene, or clomiphene. [3] The preventive or therapeutic agent for RNA virus-related diseases according to [2], wherein the tamoxifen is in the form of tamoxifen citrate. [4] The preventive or therapeutic agent for RNA virus-related diseases according to [2], wherein the toremifene is in the form of toremifene citrate. [5] The preventive or therapeutic agent for RNA virus-related diseases according to [1], wherein the anti-tuberculosis drug is rifampin. [6] The preventive or therapeutic agent for RNA virus-related diseases according to [1], wherein the CysLT1 receptor antagonist is pranlukast. [7] The preventive or therapeutic agent for RNA virus-related diseases according to [1], wherein the PPARγ agonist is pioglitazone. [8] The preventive or therapeutic agent for RNA virus-related diseases according to [1], wherein the 5-LOX inhibitor is zileuton. [9] The prophylactic or therapeutic agent for an RNA virus-related disease according to any one of [1] to [8], wherein the RNA virus is at least one virus belonging to a family selected from the group consisting of Coronaviridae, Filoviridae, and Paramyxoviridae.

[10] The prophylactic or therapeutic agent for an RNA virus-related disease according to [9], wherein the RNA virus is at least one virus selected from the group consisting of SARS-CoV-2, Ebola virus, and Sendai virus.

[11] The prophylactic or therapeutic agent for an RNA virus-related disease according to any one of [1] to

[10] , which comprises raloxifene and pioglitazone as active ingredients.

[12] The prophylactic or therapeutic agent for an RNA virus-related disease according to any one of [1] to

[10] , which further comprises remdesivir and comprises raloxifene and remdesivir as active ingredients.

[13] The prophylactic or therapeutic agent for an RNA virus-related disease according to any one of [1] to

[10] , which further comprises remdesivir and comprises pioglitazone and remdesivir as active ingredients.

[14] A method for preventing or treating an RNA virus-related disease, which comprises a step of administering an effective amount of the prophylactic or therapeutic agent for an RNA virus-related disease according to any one of [1] to

[13] to a subject in need thereof.

[15] The method for preventing or treating an RNA virus-related disease according to

[14] , which comprises a step of administering an effective amount of raloxifene and pioglitazone to a subject in need thereof.

[16] The method for preventing or treating an RNA virus-related disease according to

[14] , which comprises a step of administering an effective amount of raloxifene and remdesivir to a subject in need thereof.

[17] The method for preventing or treating an RNA virus-related disease according to

[14] , which comprises a step of administering an effective amount of pioglitazone and remdesivir to a subject in need thereof. [Effect of the Invention]

[0007] According to the present invention, a broad-spectrum drug against RNA viruses can be provided. ] [Brief Description of the Drawings]

[0008]

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[0009] [Agent for Preventing or Treating RNA Virus-Related Diseases] In one embodiment, the present invention provides an agent for preventing or treating an RNA virus-related disease, comprising at least one compound selected from the group consisting of a selective estrogen receptor modulator, an anti-tuberculosis drug, a CysLT1 receptor antagonist, a PPARγ agonist, a 5-LOX inhibitor, derivatives thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof as an active ingredient.

[0010] The prophylactic or therapeutic agent of the present embodiment can prevent or treat RNA virus-related diseases by suppressing the infection of RNA viruses into cells. Examples of RNA virus-related diseases include diseases caused by the infection of RNA viruses. Specifically, they include coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), common cold, Ebola hemorrhagic fever, Marburg disease, acute respiratory infection in infants, mumps (epidemic parotitis), acute respiratory infection in infants, measles, subacute sclerosing panencephalitis, Hendra virus infection, Nipah virus infection, Newcastle disease (conjunctivitis), upper respiratory tract infection in infants (common cold), and the like.

[0011] As will be described later in the examples, the inventors constructed a compound screening system using Sendai virus (SeV) and human induced pluripotent stem cells (iPSCs), and screened an FDA-approved drug library. As a result, compounds belonging to selective estrogen receptor modulators, anti-tuberculosis drugs, CysLT1 receptor antagonists, PPARγ agonists, and 5-LOX inhibitors were identified as compounds that suppress SeV infection and have low cytotoxicity. Furthermore, the antiviral effects of the selected hit compounds were evaluated using Ebola virus (EBOV) and SARS-CoV-2, and the present invention was completed.

[0012] SeV is a single-stranded negative-sense RNA virus belonging to the genus Respirovirus of the subfamily Paramyxovirinae in the family Paramyxoviridae. SeV is murine parainfluenza virus type 1 discovered in Sendai, Japan, and naturally replicates in the respiratory mucosa. A recombinant attenuated strain has been created by genetic engineering, and SeV carrying a target gene is used for human gene therapy.

[0013] Human iPSCs are widely used in disease models and drug discovery, and undifferentiated iPSCs themselves are also useful for compound screening. Human iPSCs have the advantages of possessing human genes, having a normal karyotype, and having an infinite self-renewal ability.

[0014] In the prophylactic or therapeutic agent of the present embodiment, examples of the selective estrogen receptor modulator include raloxifene (CAS No.: 84449-90-1), tamoxifen (CAS No.: 10540-29-1), toremifene (CAS No.: 89778-26-7), clomiphene (CAS No.: 911-45-5), and the like.

[0015] The chemical formula of raloxifene is shown in the following formula (1).

Chemical formula

[0016] The chemical formula of tamoxifen is shown in the following formula (2).

Chemical formula

[0017] The chemical formula of toremifene is shown in the following formula (3).

Chemical formula

[0018] The chemical formula of clomiphene is shown in the following formula (4).

Chemical formula

[0019] Tamoxifen is preferably in the form of tamoxifen citrate (CAS No.: 54965-24-1). The chemical formula of tamoxifen citrate is shown in the following formula (5).

Chemical formula

[0020] Toremifene is preferably in the form of toremifene citrate (CAS No.: 89778-27-8). The chemical formula of toremifene citrate is shown in the following formula (6).

Chemical formula

[0021] As will be described later in the examples, the inventors have found that selective estrogen receptor modulators inhibit the infection of SeV, EBOV, and SARS-CoV-2, which belong to very different families spanning both negative-sense RNA viruses and positive-sense RNA viruses. Therefore, the selective estrogen receptor modulator is an example of a broad-spectrum RNA virus inhibitor. Also, as will be described later in the examples, the inventors have clarified that the selective estrogen receptor modulator suppresses the infection of RNA viruses by inhibiting the entry of viruses into host cells via viral spike proteins.

[0022] In the prophylactic or therapeutic agent of the present embodiment, examples of the anti-tuberculosis drug include rifampin (CAS number: 13292-46-1). The chemical formula of rifampin is shown in the following formula (7).

Chemical formula

[0023] In the prophylactic or therapeutic agent of the present embodiment, examples of the CysLT1 receptor antagonist include pranlukast (CAS number: 103177-37-3). The chemical formula of pranlukast is shown in the following formula (8).

Chemical formula

[0024] In the prophylactic or therapeutic agent of the present embodiment, examples of the PPARγ agonist include pioglitazone (CAS number: 111025-46-8). The chemical formula of pioglitazone is shown in the following formula (9).

Chemical formula

[0025] In the prophylactic or therapeutic agent of this embodiment, examples of the 5-LOX inhibitor include zileuton (CAS number: 111406-87-2). The chemical formula of zileuton is shown in the following formula (10). [Chemical formula]

[0026] In the prophylactic or therapeutic agent of this embodiment, examples of the RNA virus include at least one virus belonging to a family selected from the group consisting of the Coronaviridae, Filoviridae, and Paramyxoviridae families.

[0027] Examples of the virus belonging to the Coronaviridae family include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Examples of the virus belonging to the Filoviridae family include Ebola virus. Examples of the virus belonging to the Paramyxoviridae family include Sendai virus.

[0028] The prophylactic or therapeutic agent of this embodiment may contain raloxifene as an active ingredient. Further, the prophylactic or therapeutic agent of this embodiment may contain pioglitazone or remdesivir as an additional active ingredient in addition to raloxifene.

[0029] That is, the prophylactic or therapeutic agent of this embodiment may contain raloxifene and pioglitazone as active ingredients. As will be described later in the examples, the inventors have clarified that the combined use of raloxifene and pioglitazone synergistically suppresses RNA virus infection.

[0030] Alternatively, the prophylactic or therapeutic agent of this embodiment may contain raloxifene and remdesivir as active ingredients. As will be described later in the examples, the inventors have clarified that the combined use of raloxifene and remdesivir synergistically suppresses RNA virus infection. The chemical formula of remdesivir (CAS number: 1809249-37-3) is shown in the following formula (11). [Chemical formula]

[0031] In addition, the prophylactic or therapeutic agent of the present embodiment may contain pioglitazone and remdesivir as active ingredients. As will be described later in the examples, the inventors have clarified that the combined use of pioglitazone and remdesivir synergistically suppresses RNA virus infection.

[0032] In the present specification, "containing as an active ingredient" means containing as a main active ingredient and containing it to such an extent that it exhibits an effect. The "effect" referred to here means a prophylactic or therapeutic effect against a target RNA virus-related disease, and in certain embodiments, it may be an infection inhibitory effect against a target virus.

[0033] In the prophylactic or therapeutic agent of the present embodiment, pharmaceutically acceptable salts include, for example, inorganic acid salts, alkali metal salts, alkaline earth metal salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, and the like. More specifically, for example, inorganic acid salts such as hydrochloride, sulfate, hydrobromide, nitrate, phosphate; organic acid salts such as acetate, mesylate, succinate, maleate, fumarate, citrate, tartrate; alkali metal salts such as sodium salt, potassium salt; alkaline earth metal salts such as magnesium salt, calcium salt; metal salts such as aluminum salt, zinc salt; ammonium salts such as ammonium salt, tetramethylammonium salt; organic amine addition salts such as morpholine, piperidine; amino acid addition salts such as glycine, phenylalanine, lysine, aspartic acid, glutamic acid, and the like. In addition, pharmaceutically acceptable solvates include, for example, hydrates, organic solvent solvates, and the like.

[0034] The prophylactic or therapeutic agent of the present embodiment is preferably formulated as a pharmaceutical composition containing the above-described compound and a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally, for example, in the form of a solution, powder, granule, tablet, capsule, etc., or parenterally, in the form of an injection, suppository, external preparation for skin, etc. More specifically, examples of the external preparation for skin include dosage forms such as ointments and patches.

[0035] As the pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic; excipients such as starch, crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, glycerin; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. can be mentioned.

[0036] The pharmaceutical composition may contain additives. Examples of the additives include lubricants such as calcium stearate, magnesium stearate; sweeteners such as sucrose, lactose, saccharin, maltitol; flavoring agents such as peppermint, perilla oil; stabilizers such as benzyl alcohol, phenol; buffers such as phosphates, sodium acetate; solubilizing agents such as benzyl benzoate, benzyl alcohol; antioxidants such as ascorbic acid; preservatives such as paraoxybenzoic acid esters (parabens), benzalkonium chloride, chlorobutanol, cresol, etc.

[0037] The dosage of the pharmaceutical composition varies depending on the patient's symptoms, weight, age, gender, etc., and cannot be determined unconditionally. In the case of oral administration, for example, the active ingredient (the above-described compound) at 0.1 to 100 mg / kg body weight per dosage unit form may be administered once a day or divided into about 2 to 4 times a day. In the case of an injection, for example, the active ingredient at 0.01 to 50 mg per dosage unit form may be administered.

[0038] [Method for Preventing or Treating RNA Virus-Related Diseases] 1 In one embodiment, the present invention provides a method for preventing or treating an RNA virus-related disease, comprising the step of administering to a subject in need thereof an effective amount of the above-described prophylactic or therapeutic agent for an RNA virus-related disease.

[0039] As used herein, a "prophylactic or therapeutic agent in an effective amount" can refer to an agent containing an active ingredient (compound) in an effective amount. Here, the "effective amount" of an agent or compound refers to the amount of the agent or compound required to bring about a prophylactic or therapeutic effect in the treated subject, and more specifically, it may be an amount sufficient to prevent, delay, or minimize at least one or more symptoms associated with the target RNA virus-related disease.

[0040] In one aspect, the "effective amount" of a compound may be an amount sufficient to prevent, delay, or minimize one or more symptoms associated with the target RNA virus-related disease, either alone or in combination with other compounds or other treatments.

[0041] The effective amount of an agent or compound can vary depending on the target RNA virus-related disease, the route of administration, the use of excipients, and the combination with other therapeutic treatments, as recognized by those skilled in the art.

[0042] The prophylactic or therapeutic method of this embodiment may include the step of administering to a subject in need thereof an effective amount of raloxifene and pioglitazone.

[0043] Alternatively, the prophylactic or therapeutic method of this embodiment may include the step of administering to a subject in need thereof an effective amount of raloxifene and remdesivir.

[0044] Alternatively, the prophylactic or therapeutic method of this embodiment may include the step of administering to a subject in need thereof an effective amount of pioglitazone and remdesivir.

[0045] [Other Embodiments] In one embodiment, the present invention provides at least one compound selected from the group consisting of a selective estrogen receptor modulator, an anti-tuberculosis drug, a CysLT1 receptor antagonist, a PPARγ agonist, a 5-LOX inhibitor, derivatives thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof, for the prevention or treatment of RNA virus-related diseases.

[0046] The selective estrogen receptor modulator, anti-tuberculosis drug, CysLT1 receptor antagonist, PPARγ agonist, 5-LOX inhibitor, derivatives thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof are the same as those described above.

[0047] In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of RNA virus-related diseases, which contains raloxifene and pioglitazone as active ingredients.

[0048] In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of RNA virus-related diseases, which contains raloxifene and remdesivir as active ingredients.

[0049] In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of RNA virus-related diseases, which contains pioglitazone and remdesivir as active ingredients.

[0050] In one embodiment, the present invention provides the use of at least one compound selected from the group consisting of a selective estrogen receptor modulator, an anti-tuberculosis drug, a CysLT1 receptor antagonist, a PPARγ agonist, a 5-LOX inhibitor, derivatives thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof, for the manufacture of a preventive or therapeutic agent for RNA virus-related diseases.

[0051] In one embodiment, the present invention provides the use of raloxifene and pioglitazone for the manufacture of a prophylactic or therapeutic agent for RNA virus-related diseases.

[0052] In one embodiment, the present invention provides the use of raloxifene and remdesivir for the manufacture of a prophylactic or therapeutic agent for RNA virus-related diseases.

[0053] In one embodiment, the present invention provides the use of pioglitazone and remdesivir for the manufacture of a prophylactic or therapeutic agent for RNA virus-related diseases.

Examples

[0054] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0055] [Materials and Methods] (Generation of iPS cells) Human iPSCs were generated from peripheral blood mononuclear cells (PBMCs) using a previously reported episomal vector (Sox2, Klf4, Oct3 / 4, L-Myc, Lin28, p53-shRNA) and cultured in a feeder-free culture system using StemFit (Ajinomoto) medium. Karyotype analysis of iPSCs was performed by LSI Medience Corporation.

[0056] (Compounds) For high-throughput screening, a US Food and Drug Administration (FDA)-approved drug library (Enzo Life Sciences) was used. Compounds used for RNA quantification assays were purchased from Selleck Chemicals.

[0057] (Compound screening using Sendai virus and human iPS cells) For high-throughput compound screening, human iPSCs were dissociated into single cells with TrypLE Express (Thermo Fisher Scientific), and seeded onto 96-well plates coated with iMatrix using StemFit containing 10 μM Y-27632 (Nacalai Tesque). After 24 hours, the medium was replaced with fresh StemFit containing the compound and incubated for 3 hours. Subsequently, the iPSCs were exposed to Sendai virus (SeV) carrying the EGFP gene. The multiplicity of infection (MOI) was estimated to be 1.

[0058] After incubation for 48 hours, the cells were washed twice with PBS and then fixed in 4% paraformaldehyde (PFA) at room temperature for 10 minutes. 4′,6-Diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) was used for nuclear labeling. Cell images were acquired using an IN CELL Analyzer 6000 (GE Healthcare) or an IN CELL Analyzer 2000 (GE Healthcare), and the number of EGFP-positive cells was quantified using IN CELL Developer Toolbox software 1.92 (GE Healthcare).

[0059] (Quantitative RT-PCR) Human iPSCs were seeded onto 24-well plates coated with iMatrix in StemFit (Ajinomoto) and exposed to SeV. After washing the cells twice with PBS, total RNA of the cultured human iPSCs was extracted using a miRNeasy Mini Kit (Qiagen). Subsequently, 500 ng of RNA was reverse-transcribed using ReverTra Ace (Toyobo). The mRNA level was measured by reverse transcription using oligo dT, and the viral genomic RNA level was measured by reverse transcription using random primers. Quantitative PCR analysis was performed using SYBR Premix Ex Taq II (Takara Bio) and a StepOnePlus system (Thermo Fisher Scientific). PCR primers for detecting viral genomic RNA were designed between the NP gene and the P gene. The nucleotide sequences of the primers used are shown in Table 1 below.

[0060]

Table 1

[0061] (Cell line) 293T cells, Huh7 cells and Vero E6 cells (provided by Dr. Reito Takada, Hokkaido University) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution.

[0062] (Production and infection assay of Ebola trVLP) To evaluate the anti-Ebola virus activity of the compounds, Ebola trVLP expressing a GFP reporter was prepared using 293T cells (T. Hoenen, et. al., Modeling the lifecycle of Ebola virus under biosafety level 2 conditions with virus-like particles containing tetracistronic minigenomes. J Vis Exp, 52381, 2014.). Plasmids pCAGGS expressing EBOV-NP, EBOV-VP35, EBOV-VP30, EBOV-L, and T7-polymerase, and a plasmid expressing a trVLP tetracistronic minigenome encoding EBOV-GP, EBOV-VP40, EBOV-VP24, and the GFP reporter gene were transfected into 293T cells using the calcium phosphate method. One day after transfection, the culture supernatant was replaced with fresh medium. After an additional 3 days, the supernatant was collected and clarified by centrifugation at 2,000 × g for 15 minutes to obtain Ebola trVLP. The Ebola trVLP was stored at -80°C.

[0063] To increase the titer of the Ebola trVLP, the Ebola trVLP was passaged 2 to 3 times as follows. pCAGGS expression plasmids encoding EBOV-NP, EBOV-VP35, EBOV-VP30, EBOV-L, and the host factor Tim-1 were transfected into 293T cells using the calcium phosphate method. One day after transfection, the cells were exposed to trVLP for 1 day and further cultured for 3 days. Then, the culture supernatant was collected to obtain high-titer Ebola trVLP. In the experimental examples of this application, the high-titer Ebola trVLP was used.

[0064] To evaluate the antiviral activity of the compound, pCAGGS expression plasmids encoding EBOV-NP, EBOV-VP35, EBOV-VP30, and EBOV-L were transfected into Huh7 cells using the TransIT LT1 transfection reagent (Mirus). Two days after transfection, the cells were pretreated with each compound at an appropriate concentration, exposed to the passaged trVLP, and incubated for 2 days in the presence of the compound.

[0065] Subsequently, the cells exposed to trVLP were fixed overnight with 10% formalin, stained with Hoechst 33342 dye for nuclear staining, and imaged with a Cytation 5 imaging plate reader (BioTek Instruments) equipped with a 4× lens. The counting of cell nuclei and infected cells was performed using the Cell Profiler image analysis software (Broad Institute, USA) and a customized analysis pipeline.

[0066] (SARS-CoV-2 Propagation and Infection Assay) A SARS-CoV-2 strain (accession number: EPI-ISL-481251, GISAID) isolated from a Japanese patient was propagated in Vero E6 cells. Four days after infection, the culture supernatant was collected, clarified by centrifugation at 2,000×g for 15 minutes, and stored at -80°C until use.

[0067] To evaluate the antiviral activity of the compounds, Vero E6 cells were seeded in 96-well plates and incubated for 1 hour in the presence of each compound at an appropriate concentration. The cells were then exposed to SARS-CoV-2 at an MOI of 0.002 and incubated at 37 °C in the presence of the compounds. Two days later, they were fixed overnight using 4% PFA.

[0068] Cells infected with SARS-CoV-2 were detected by fluorescence immunostaining using rabbit anti-SARS-CoV N antibody as the primary antibody and Alexa Fluor 488-labeled goat anti-rabbit IgG antibody as the secondary antibody. Detection of the total number of cells was performed by nuclear staining using Hoechst33342 dye. The ratio of the number of SARS-CoV-2-infected cells (Alexa Fluor 488-positive cells) to the total number of cells (Hoechst33342-positive cells) was calculated as the SARS-CoV-2 infection rate (%).

[0069] The cells were imaged with a Cytation5 imaging plate reader equipped with a 4× lens. Counting of cell nuclei and infected cells was performed using Cell Proffiler image analysis software and a customized analysis pipeline. All experiments using replication-competent SARS-CoV-2 were conducted in a biosafety level 3 (BSL3) laboratory at Nagasaki University.

[0070] (Combination analysis of drugs) To examine the efficacy of drug combination, using SynergyFinder (https: / / synergyfinder.fimm.fi / ), the inhibition rate (%) against viral infection was calculated for each combination of doses of raloxifene and remdesivir or pioglitazone, compared with each drug alone. The synergy score for the combination of compounds was calculated using the Zero Interaction Potency (ZIP) model. When the synergy score is (i) less than -10, the interaction between the two drugs is likely to be antagonistic; (ii) between -10 and 10, the interaction between the two drugs is likely to be additive; and (iii) greater than 10, the interaction between the two drugs is likely to be synergistic.

[0071] (Production and infection assay of pseudotyped VSV) To analyze the mechanism of action of the drugs, vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein or the SARS-CoV spike protein and in which the VSV-G gene was replaced with the firefly luciferase reporter gene (sometimes referred to as VSVΔG-SARS2-S and VSVΔG-SARS-S, respectively) was produced.

[0072] The codon-optimized SARS-CoV-2 S gene with a 19-amino acid residue deletion at the C-terminus and the codon-optimized SARS-CoV S gene with a 19-amino acid residue deletion at the C-terminus were synthesized and inserted into the pCAGGS expression plasmid using In-Fusion HD (Clontech). The pCAGGS plasmid encoding the SARS-CoV-2 S gene or the SARS-CoV-S gene was transfected into 293T cells by the calcium phosphate method. One day after transfection, the cells were infected for 1 hour with a recombinant VSV in which the VSV-G gene was replaced with the firefly luciferase reporter gene (hereinafter sometimes referred to as VSVΔG-VSV-G). One day after infection, the supernatant was collected and clarified by centrifugation at 2,000×g for 15 minutes to obtain VSVΔG-SARS2-S or VSVΔG-SARS-S. It was stored at -80°C until use.

[0073] As a control for the contamination of the infected virus, VSV having VSV glycoprotein (G) on its envelope (hereinafter sometimes referred to as VSV-G) was produced. A pCAGGS plasmid encoding the G gene was transfected into 293T cells, exposed to VSVΔG-VSV-G for 1 hour, and the culture supernatant was collected 1 day later to obtain VSV-G.

[0074] To evaluate the effect of the compound on pseudotype VSV infection, Vero E6 cells were seeded in a 96-well plate and incubated for 1 hour in the presence of each compound at an appropriate concentration. Subsequently, the cells were exposed to each pseudotype VSV and incubated at 37 °C in the presence of the compound. After 20 hours, the cells were lysed and luciferase activity was measured using a SpectraMax iD5 microplate reader (Molecular Devices).

[0075] (Statistical analysis) The results were analyzed using one-way ANOVA following Dunnett's post hoc test to determine statistical significance. A difference with p < 0.05 was considered significant. The analysis was performed using GraphPad Prism software version 8.0 for Windows (GraphPad Software).

[0076] [Experimental Example 1] (Screening of anti-RNA virus drugs using human iPS cells and SeV) Compound screening for identifying therapeutic drugs against RNA viruses was performed using Sendai virus (SeV) and human iPSCs.

[0077] The SeV genome contains a 3' leader sequence and a 5' trailer sequence and encodes six genes that are transcribed in the order of nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large polymerase (L).

[0078] To eliminate the ability to produce progeny viruses capable of infecting other cells, the F gene was removed, and SeV having an EGFP gene in the 3' region of the viral genomic RNA was produced. Figure 1 is a schematic diagram showing the genomic structure of the produced SeV.

[0079] In addition, human iPSCs were generated from healthy individuals. Figure 2 is a photograph showing the results of karyotype analysis of the generated iPSCs. An assay system was constructed to measure virus replication by infecting these iPSCs with SeV and measuring the number of EGFP-positive iPSCs.

[0080] Figure 3 is a schematic diagram showing the timeline of compound screening. Figure 4 is a schematic diagram showing the outline of compound screening. iPSCs were seeded in a 96-well plate, and after 24 hours, 10 μM of the compound was added and incubated for 3 hours, followed by exposure to SeV. At 48 hours after virus infection, the number of EGFP-positive cells was quantified, and compounds that resulted in a decrease in the number of EGFP-positive cells compared to the negative control (no compound added) were extracted. In addition, the number of DAPI-positive cells (total cell number) was also quantified, and compounds that resulted in a significant decrease compared to the negative control were excluded as being highly cytotoxic. Then, the top 30 compounds with a low number of EGFP-positive cells were designated as hit compounds.

[0081] Figures 5 and 6 are graphs showing the results of the first-round screening. Approximately 500 types of compounds were evaluated. In Figures 5 and 6, white squares indicate the negative control (DMSO). White circles indicate hit compounds, including raloxifene, rifampin, pranlukast, zileuton, and pioglitazone. The list of hit compounds is shown in Table 2 below. In Table 2, the score indicates the number of EGFP-positive cells (corrected value between batches).

[0082] [Table 2]

[0083] [Experimental Example 2] (Inhibition of Virus Replication by FDA-Approved Drugs) From among the hit compounds, drugs with few effects on cardiovascular circulation and the central nervous system were selected, and for the next analysis, five drugs with different targets were focused on: raloxifene, a selective estrogen receptor modulator (SERM); rifampin, an antituberculosis drug; pranlukast, a CysLT1 antagonist; pioglitazone, a PPARγ agonist; and zileuton, a 5-LOX inhibitor.

[0084] iPSCs were seeded in 24-well plates and incubated with 10 μM of each compound for 3 hours, and then infected with SeV. Twenty-four hours after virus infection, RNA was extracted from the iPSCs, and the mRNA amount of EGFP was quantified using quantitative RT-PCR.

[0085] Figure 7(a) shows the measurement results of cells to which raloxifene, rifampin, pranlukast, pioglitazone, or zileuton was added (all at 10 μM, n = 3), and Figure 7(b) shows the measurement results of cells to which remdesivir, an RNA-dependent RNA polymerase inhibitor approved by the FDA for the treatment of COVID-19, was added (1 μM, n = 3). In Figures 7(a) and (b), "vehicle" indicates the results in the presence of DMSO, which is a negative control. The graphs show the mean ± standard error, and "*" indicates a significant difference with p < 0.005.

[0086] First, as shown in Figure 7(b), in the cells to which remdesivir was added, the mRNA of EGFP was not substantially detected. This indicates that in the assay system using the iPSCs and SeV, the effect of the drug on RNA-dependent RNA polymerase can be detected with high sensitivity. Next, from Figure 7(a), it was also shown that the addition of raloxifene, rifampin, pranlukast, pioglitazone, or zileuton significantly and substantially decreased the mRNA amount of EGFP. This result indicates that these hit compounds may inhibit the viral RNA synthesis activity or steps in the viral life cycle upstream of it.

[0087] Subsequently, the dose-dependence of these drugs against SeV infection was evaluated. Figures 8(a) to (f) are graphs showing the dose-dependence of remdesivir, raloxifene, rifampin, pranlukast, pioglitazone, and zileuton, respectively.

[0088] As a result, remdesivir reduced the SeV infection rate at an IC 50 value of 0.077 μM. This indicated that this assay system is useful as an evaluation system for the inhibitory effect of drugs on RNA virus infection. In addition, raloxifene, rifampin, pranlukast, pioglitazone, and zileuton showed IC 50 values of 4.3 μM, 3.9 μM, 5.0 μM, 4.9 μM, and 4.5 μM, respectively, and it was revealed that they dose-dependently reduced the SeV infection rate.

[0089] Therefore, similar to remdesivir, raloxifene, rifampin, pranlukast, pioglitazone, and zileuton were also shown to be able to effectively inhibit the infection of Sendai virus, which is a type of RNA virus, into cells.

[0090] [Experimental Example 3] (Evaluation of Compounds Using an Ebola Virus Life Cycle Modeling System) To evaluate the antiviral activity of the selected compounds, unlike the wild-type virus, an antiviral activity was evaluated using a virus-like particle (trVLP) system with transcription and replication ability that does not require a biosafety level 4 (BSL4) laboratory.

[0091] In the presence of various concentrations of each compound, Huh7 cells derived from human liver were exposed to Ebola trVLP expressing a GFP reporter, and the number of infected cells and cell viability were measured.

[0092] Figures 9(a) to (f) are graphs showing the dose-dependence of remdesivir, raloxifene, rifampicin, pranlukast, pioglitazone, and zileuton, respectively. As a result, remdesivir, a nucleotide analog developed as a therapeutic agent for Ebola virus infection, strongly inhibited Ebola trVLP infection, and the IC 50 value was 0.12 μM, indicating the validity of this assay system.

[0093] In addition, raloxifene also dose-dependently decreased the infection rate of Ebola trVLP, and the IC 50 value was 0.88 μM. Furthermore, since a significant divergence was observed between the infection inhibitory activity and cytotoxicity, it was shown that there was a specific antiviral effect. In contrast, rifampicin, pranlukast, pioglitazone, and zileuton did not show significant antiviral activity.

[0094] From the above results, it was shown that raloxifene could be a prophylactic or therapeutic agent for Ebola virus infection.

[0095] [Experimental Example 4] (Evaluation of Compounds against SARS-CoV-2) The antiviral activity of the selected compounds was evaluated using SARS-CoV-2. Vero E6 cells were exposed to wild-type SARS-CoV-2 in the presence of various concentrations of each compound, and then the number of infected cells was quantified using an immunostaining method, and the cell viability was determined.

[0096] Figures 10(a) to (f) are graphs showing the dose-dependence of remdesivir, raloxifene, rifampicin, pranlukast, pioglitazone, and zileuton, respectively. As a result, remdesivir, which has been approved by the FDA for application to COVID-19, decreased the infection rate of SARS-CoV-2 at an IC 50 value of 0.89 μM.

[0097] As shown in Figure 10(b), raloxifene had an IC 50The value dose-dependently decreased the infection rate of SARS-CoV-2. In addition, pioglitazone partially suppressed viral infection at high concentrations. In contrast, rifampin and pranlukast tended to decrease the infection rate of SARS-CoV-2 but not significantly, and ziltong did not show significant antiviral activity.

[0098] From the above results, it was shown that raloxifene and pioglitazone could be preventive or therapeutic agents for SARS-CoV-2 infection.

[0099] Subsequently, using SynergyFinder, the combined effects of raloxifene and remdesivir, raloxifene and pioglitazone, and pioglitazone and remdesivir were examined (Ianevski A, Giri AK and Aittokallio T (2020) SynergyFinder 2.0: visual analytics of multi-drug combination synergies. Nucleic Acids Res 48, W488-W493, 2020.).

[0100] For each combination of two compounds, namely raloxifene and remdesivir, raloxifene and pioglitazone, and pioglitazone and remdesivir, Vero E6 cells were exposed to wild-type SARS-CoV-2 in the presence of various concentrations of each compound. Then, the number of infected cells was quantified using immunostaining, the cell viability was determined, and the combined effect was examined.

[0101] Figure 11 shows the dose-response matrix and three-dimensional synergy map for the combination of raloxifene and remdesivir. When the synergy score is (i) less than -10, the interaction between the two drugs is likely to be antagonistic; (ii) between -10 and 10, the interaction between the two drugs is likely to be additive; and (iii) greater than 10, the interaction between the two drugs is likely to be synergistic.

[0102] As a result, it was revealed that raloxifene and remdesivir exhibited a synergistic antiviral effect at specific concentrations. The ZIP synergy score was 6.91, and the highest synergy score was 30.09.

[0103] Figure 12 shows the dose-response matrix and three-dimensional synergy map for the combination of raloxifene and pioglitazone. As a result, it was also revealed that raloxifene and pioglitazone also exhibited a synergistic antiviral effect at specific concentrations. The ZIP synergy score was 4.49, and the highest synergy score was 12.42.

[0104] Figure 13 shows the dose-response matrix and three-dimensional synergy map for the combination of pioglitazone and remdesivir. As a result, it was also revealed that pioglitazone and remdesivir also exhibited a synergistic antiviral effect at specific concentrations. The ZIP synergy score was 8.85, and the highest synergy score was 30.42.

[0105] From the above results, it was strongly suggested that when raloxifene and remdesivir, raloxifene and pioglitazone, and pioglitazone and remdesivir were combined, respectively, a synergistic effect occurred and a very high antiviral effect was exerted.

[0106] [Experimental Example 5] (Effect of SERM on SARS-CoV-2 Infection) As described above, among the selected compounds, raloxifene exhibited antiviral activity against both Ebola virus and SARS-CoV-2. Raloxifene is a type of cancer therapeutic drug approved by the FDA and is a type of selective estrogen receptor modulator (SERM).

[0107] Therefore, first, using the trVLP system, we examined whether tamoxifen, toremifene, and clomiphene, which are SERMs other than raloxifene, inhibit Ebola trVLP infection. In the presence of various concentrations of each compound, Huh7 cells derived from human liver were exposed to Ebola trVLPs expressing a GFP reporter, and the number of infected cells and cell viability were measured. Figures 14(a) to (c) are graphs showing the dose-dependence of tamoxifen, toremifene, and clomiphene, respectively. As a result, it was confirmed that tamoxifen, toremifene, and clomiphene all inhibit Ebola trVLP infection.

[0108] Subsequently, the antiviral activities of tamoxifen, toremifene, and clomiphene were evaluated using SARS-CoV-2. In the presence of various concentrations of each compound, Vero E6 cells were exposed to wild-type SARS-CoV-2, and then the number of infected cells was quantified using immunostaining, and cell viability was determined.

[0109] Figures 15(a) to (c) are graphs showing the dose-dependence of tamoxifen, toremifene, and clomiphene, respectively. As a result, it was revealed that tamoxifen, toremifene, and clomiphene show the effect of suppressing SARS-CoV-2 infection in Vero E6 cells at IC 50 values of 10.9 μM, 10.0 μM, and 6.7 μM, respectively.

[0110] From the above results, it was shown that raloxifene, tamoxifen, toremifene, and clomiphene can all be used as preventive or therapeutic drugs for SARS-CoV-2 infection. Furthermore, it was strongly suggested that SERMs can be used as preventive or therapeutic drugs for SARS-CoV-2 infection.

[0111] [Experimental Example 6] (Analysis of the mechanism of the antiviral effect of SERMs) The mechanism of the antiviral effect of SERM was analyzed. Coronaviruses such as SARS-CoV-2 are enclosed by an envelope (outer membrane) consisting of a lipid bilayer and outer membrane proteins, with a viral core containing viral genomic RNA. For SARS-CoV-2, one of the outer membrane proteins, the spike protein, binds to a receptor on the cell membrane of the host cell. After entering the cell by endocytosis, the envelope fuses with the endosome membrane and the viral genome is released into the cell, or 2) the spike protein is activated by a host protease present on the cell membrane, and the envelope fuses with the cell membrane, releasing the viral genome into the cell and establishing infection.

[0112] Therefore, a pseudotyped vesicular stomatitis virus (VSVΔG-SARS2-S) having a luciferase gene and further having the spike protein of SARS-CoV-2 or a pseudotyped vesicular stomatitis virus (VSVΔG-SARS-S) having the spike protein of SARS-CoV was prepared (Figure 16), and it was examined whether SERM inhibits the entry of the pseudotyped VSV into host cells. Also, for comparison, a similar examination was conducted using a vesicular stomatitis virus (VSV-G) having a luciferase gene and having the VSV glycoprotein (G) on the envelope.

[0113] Figures 17(a) to (c) show the results of incubating Vero E6 cells in the presence of raloxifene, tamoxifen or clomiphene for 1 hour, then exposing them to the pseudotyped VSV and measuring the luciferase activity after cell lysis. The horizontal axis represents the treatment concentration of each drug, and the vertical axis represents the relative value with respect to the luciferase activity obtained in the drug-untreated group. As shown in Figures 17(a) to (c), raloxifene, tamoxifen, and clomiphene inhibited the infection of VSVΔG-SARS2-S at ICs of 3.9 μM, 5.3 μM, and 4.4 μM, respectively. 50Inhibited by the value. Also, raloxifene, tamoxifen, and clomiphene inhibited the infection of pseudotyped VSV having the SARS-CoV spike protein (SARS-CoV-S) at IC 50 values of 4.1 μM, 7.3 μM, and 5.8 μM, respectively. However, no inhibitory effect on VSV-G was observed.

[0114] Therefore, it was shown that vesicular stomatitis virus is not originally inhibited from entering host cells by any of raloxifene, tamoxifen, and clomiphene, but when SARS-CoV-2 or the spike protein of SARS-CoV-2 is expressed on the envelope, the entry into host cells is effectively inhibited by the said drug.

[0115] From the above results, it was suggested that SERM exerts an antiviral effect by inhibiting the entry of pathogenic coronaviruses into host cells via the viral spike protein. Figure 18 is a schematic diagram showing (part of) the mechanism of the antiviral effect of SERM. It is conceivable that SERM disrupts the ion homeostasis in endosomes containing virus particles and inhibits the membrane fusion for releasing the virus core into the cytoplasm of host cells.

[0116] Table 3 below summarizes the effects of each compound on the virus. In Table 3, “+” indicates that virus infection was inhibited, “−” indicates that virus infection was not inhibited, “N.S.” indicates that a tendency of infection inhibition was observed but there was no significance (Not Significant), and “N.D.” indicates that it was not tested (Not Determined).

[0117]

Table 3

Industrial Applicability

[0118] According to the present invention, a prophylactic or therapeutic agent for RNA virus-related diseases can be provided.

Claims

Claim 1 A prophylactic or therapeutic agent for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising as an active ingredient raloxifene and pioglitazone, raloxifene and remdesivir, or pioglitazone and remdesivir. Claim 2 A prophylactic or therapeutic agent for SARS-CoV-2 infection, comprising as an active ingredient raloxifene, clomiphene or pioglitazone. Claim 3 A prophylactic or therapeutic agent for Sendai virus infection, comprising as an active ingredient raloxifene, rifampin, pranlukast, ziltong or pioglitazone.

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