Patent Description:
RNA viruses such as influenza virus or Ebola virus cause various infectious diseases, and thus, countermeasures for these RNA viruses have been required. Meanwhile, as compounds having a wide range of antiviral activities against many RNA viruses, pyrazine derivatives such as Favipiravir (hereinafter also referred to as "T-<NUM>") have been known (Non-Patent Document <NUM>). It was known that T-<NUM> and T-<NUM> are effective against RNA viral infections (Non-Patent Document <NUM>), and that these two compounds inhibit the replication of Chikungunya virus (a RNA virus) in vitro and that they have beneficial effects on mice infected with this virus (less severe neurological effects and reduction in mortality rate) (Non-Patent Document <NUM>).

If the activities of these pyrazine derivatives were reinforced, the pyrazine derivatives would be useful as medicaments. To date, it has been reported that a combination of Favipiravir and a neuraminidase inhibitor exhibits synergistic effects against influenza virus (Patent Document <NUM>). In addition, it has also been reported that a combination of Favipiravir and Gemcitabine or Obatoclax exhibits synergistic effects against Ebola virus (Patent Document <NUM>). Further, it was known that azathioprine, <NUM>-thioguanine as well as methotrexate have either a direct anti-viral effect or have a beneficial effect when treating viral infections (Non-Patent Documents <NUM> and <NUM>; Patent Document <NUM>).

However, such RNA viruses acquire drug resistance as a result of evolution. For example, an influenza virus that is resistant to the neuraminidase inhibitor has been known (Non-Patent Document <NUM>). In order to cope with such acquisition of resistance, a novel combination of compounds, which exhibits effects against RNA viruses, has always been required.

As mentioned above, Favipiravir is characterized in that it exhibits a wide range of effects against many RNA viruses. However, a combination of a pyrazine derivative and a specific compound, which is capable of simultaneously reinforcing antiviral activities against a plurality of RNA viruses, has not yet been known.

It is an object of the present invention to provide a therapeutic agent for use in treating an RNA viral infection, comprising a novel combination of a pyrazine derivative and one or more types of a specific compounds selected from the group consisting of an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative, which exhibit effects against an RNA virus. In addition, it is another object of the present invention to provide a therapeutic agent for use in treating an RNA viral infection, comprising a combination of a pyrazine derivative and one or more types of a specific compounds selected from the group consisting of an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative, which are capable of simultaneously reinforcing antiviral activities against a plurality of RNA viruses.

Under such circumstances, the present inventor has conducted intensive studies. As a result, the present inventor has found that antiviral activities against a plurality of RNA viruses are simultaneously reinforced, when a pyrazine derivative represented by the following formula [<NUM>] or a salt thereof:
<CHM>.

Specifically, the present invention is as set out in the appended claims and provides the following features:.

A therapeutic agent for use in treating an RNA viral infection, in which a pyrazine derivative or a salt thereof is combined with one or more types of compounds selected from the group consisting of an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative which increase the amount of a pyrazine derivative ribose triphosphate in a cell, is useful for the treatment, such as therapy or prevention, of RNA viral infection.

<FIG> is a view showing a change in the amount of T-<NUM>-RTP in 293T cells caused by the combined use of T-<NUM> and <NUM>-mercaptopurine (6MP), which is shown in Test Example <NUM>.

The term "halogen atom" is used to mean a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The term "C<NUM>-<NUM> alkyl group" is used to mean linear or branched C<NUM>-<NUM> alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, <NUM>-methylbutyl, <NUM>-pentyl, <NUM>-pentyl and hexyl groups.

Examples of the amino-protecting group may include all groups that can be used as common amino-protecting groups, and examples of the amino-protecting group may include the groups described in, for example, <NPL>.

Specific examples may include an acyl group, an alkyloxycarbonyl group, an arylalkyloxycarbonyl group, an aryloxycarbonyl group, an arylalkyl group, an alkoxyalkyl group, an arylalkyloxyalkyl group, an arylthio group, an alkylsulfonyl group, an arylsulfonyl group, a dialkylaminoalkylidene group, an arylalkylidene group, a nitrogen-containing heterocyclic alkylidene group, a cycloalkylidene group, a diarylphosphoryl group, a diarylalkylphosphoryl group, an oxygen-containing heterocyclic alkyl group, and a substituted silyl group.

The salt of the compound represented by the formula [<NUM>] may be generally known salts of hydroxyl groups. Examples of such salts may include: salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, <NUM>-ephenamine, and N,N'-dibenzylethylenediamine. The salts are preferably pharmacologically acceptable salts, and are more preferably salts with sodium.

In the compound represented by the formula [<NUM>], preferably, R<NUM> is a hydrogen atom; R<NUM> is a fluorine atom; and R<NUM> is a hydrogen atom. Besides, this compound is preferably T-<NUM>.

Otherwise, in the compound represented by the formula [<NUM>], preferably, R<NUM> is a hydrogen atom; R<NUM> is a hydrogen atom; and R<NUM> is a hydrogen atom. This compound is preferably T-<NUM>.

The compound represented by the formula [<NUM>] is produced by combining known methods with one another. The compound represented by the formula [<NUM>] can be produced by the production method described, for example, in International Publication <CIT>.

It has been known that the pyrazine derivative represented by the formula [<NUM>] or a salt thereof, for example, T-<NUM>, undergoes ribosyl phosphorylation in a cell, and that a pyrazine derivative ribose triphosphate generated as a result of the ribosyl phosphorylation exhibits antiviral action (<NPL>. Herein, the pyrazine derivative ribose triphosphate is a compound represented by the following formula [<NUM>]:
<CHM>
wherein R<NUM> and R<NUM>, which are the same or different, each represent a hydrogen atom or a halogen atom; and R<NUM> represents a hydrogen atom or an amino-protecting group.

The term "compound which increases the amount of a pyrazine derivative ribose triphosphate in a cell" is a compound that activates or inhibits enzymes or metabolic pathways in a cell when the compound is used in combination with the pyrazine derivative represented by the formula [<NUM>] or a salt thereof, so that the compound increases the amount of a pyrazine derivative ribose triphosphate in the cell. When compared with a case where the compound is not combined with the pyrazine derivative represented by the formula [<NUM>] or a salt thereof, the compound increases the concentration of the pyrazine derivative ribose triphosphate by preferably <NUM> times or more, and more preferably by <NUM> times or more, when it is combined with the pyrazine derivative represented by the formula [<NUM>] or a salt thereof. Such a compound is an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative. It is to be noted that the antifolate, the thiopurine antimetabolite and the tiazofurin are all antimetabolites.

The antifolate is a compound that inhibits folate metabolizing enzymes necessary for the synthesis of DNA, and thus suppresses cell proliferation. The antifolate is methotrexate and pralatrexate.

The antifolate may be produced by combining known methods with one another, or a commercially available antifolate may be used.

The thiopurine antimetabolite is an antimetabolite having a thiopurine skeleton, such as <NUM>-mercaptopurine, azathioprine, and <NUM>-thioguanine. The thiopurine antimetabolite is represented by the formula [<NUM>]. In the compound represented by the formula [<NUM>], preferably, R<NUM> is a hydrogen atom or an amino group, and R<NUM> is a hydrogen atom or a group represented by the formula [<NUM>]. In the group represented by the formula [<NUM>], preferably, R<NUM> is a hydrogen atom, and R<NUM> is a methyl group.

Examples of the particularly preferred thiopurine antimetabolite may include <NUM>-mercaptopurine, azathioprine, and <NUM>-thioguanine. Among these, <NUM>-mercaptopurine is most preferable.

The thiopurine antimetabolite may be produced by combining known methods with one another, or a commercially available thiopurine antimetabolite may be used.

The alkylating agent is a compound that interacts with DNA and inhibits cell proliferation. The alkylating agent is temozolomide.

The alkylating agent may be produced by combining known methods with one another, or a commercially available alkylating agent may be used.

The xanthine derivative is a compound having a xanthine skeleton. The xanthine derivative is theophylline.

The xanthine derivative may be produced by combining known methods with one another, or a commercially available xanthine derivative may be used.

In the present invention, a pyrazine derivative is used in combination with one or more types of compounds selected from the group consisting of an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative which increase the amount of a pyrazine derivative ribose triphosphate in a cell. The combination includes a form in which a pyrazine derivative and said compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell are administered, simultaneously, separately, or in a specific order (a combined use), and a form as a mixture (a compounding agent).

That is to say, the "combined use" does not only mean that the administration period of the pyrazine derivative or a salt thereof is identical to the administration period of the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell, but the "combined use" also includes a form in which the pyrazine derivative or a salt thereof and the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell are administered in one administration schedule. The administration route of the pyrazine derivative or a salt thereof may be either identical to or different from the administration route of the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell.

The amount ratio between the pyrazine derivative or a salt thereof and the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell may be an amount ratio, in which the antiviral activity of the pyrazine derivative or a salt thereof is reinforced. The pyrazine derivative or a salt thereof : the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell (molar ratio) is preferably <NUM> : <NUM> to <NUM> : <NUM>, more preferably <NUM> : <NUM> to <NUM> : <NUM>, further preferably <NUM> : <NUM> to <NUM> : <NUM>, and still further preferably <NUM> : <NUM> to <NUM> : <NUM>.

When the inventive pyrazine derivative or a salt thereof and the inventive compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell are used, in general, pharmaceutical aids used in formulation, such as excipients, carriers, and diluents, may be mixed, as appropriate, into the pyrazine derivative or a salt thereof and the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell. The thus obtained mixtures may be processed into tablets, capsules, powder agents, syrups, granules, pills, suspending agents, emulsions, liquid agents, powdery preparations, suppositories, eye drops, nasal drops, ear drops, patches, ointments, injections, etc. according to ordinary methods.

In the case of using as a compounding agent, the pyrazine derivative or a salt thereof may be mixed with the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell in the above-described formulation process, and they may be homogenized, and may be then processed into a suitable preparation.

The administration route of the therapeutic agent for use in treating an RNA viral infection of the present invention is not particularly limited, and the present therapeutic agent for use in treating an RNA viral infection may be administered via intravenous, oral, intramuscular, subcutaneous, inhalation, spraying or other administration routes. Moreover, the pyrazine derivative or a salt thereof and the compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell may be administered, simultaneously or in a specific order.

The administration method, the applied dose, and the number of administrations can be selected, as appropriate, depending on the age, body weight, and symptoms of a patient. In general, the pyrazine derivative or a salt thereof used as an active ingredient may be administered to an adult at a dose of <NUM> to <NUM>/kg, preferably <NUM> to <NUM>/kg, via oral administration or parenteral administration (for example, injection, drip infusion, administration to a rectal site, etc.), once a day or divided over several administrations.

The therapeutic agent for use in treating an RNA viral infection of the present invention is useful for the treatment, such as therapy or prevention, of RNA viral infection.

The RNA viral infection is an infectious disease caused by RNA viruses such as influenza virus, parainfluenza virus, bunyavirus (Crimean-Congo fever virus, Rift Valley fever virus, Lacrosse encephalitis virus, Dobrava virus, Maporal virus, Prospect Hill virus, Andes virus, Sand fly fever virus, Heartland virus, Puntatrovirus, Severe febrile thrombocytopenia virus, etc.), Arenavirus (Funin virus, Pitinde virus, Tacaribe virus, Guanarito virus, Machupo virus, Lymphocytic choriomeningitis virus, Lassa fever virus, etc.), filovirus (Ebola virus, Marburg virus, etc.), rabies virus, human metapneumovirus, RS virus, Nipah virus, Hendra virus, measles virus, hepatitis A virus, hepatitis C virus, hepatitis E virus, Chikungunya virus, Western equine encephalitis virus, Venezuelan encephalitis virus, Eastern equine encephalitis virus, Norovirus, Poliovirus, Echovirus, Coxsackie virus, Enterovirus, Rhinovirus, Rotavirus, Newcastle disease virus, Mumps virus, vesicular stomatitis virus, Japanese encephalitis virus, tick-borne flavivirus, yellow fever virus, dengue virus, West Nile virus, or Zika virus. The present invention can be used preferably as a therapeutic agent for infectious diseases caused by influenza virus, rabies virus, Lassa fever virus, bunyavirus (Crimea-Congo fever virus, Rift Valley fever virus, Severe febrile thrombocytopenia virus, etc.) and filovirus (Ebola virus, Marburg virus, etc.), and particularly preferably as a therapeutic agent for influenza infection.

The therapeutic agent for use in treating an RNA viral infection of the present invention can be further used in combination with other therapeutic agents for RNA viral infection or drugs which exhibit RNA virus inhibitory action, for the purpose of reinforcing the action thereof. Such other therapeutic agents for RNA viral infection are therapeutic agents for RNA viral infection, and examples thereof may include a therapeutic agent for influenza, a therapeutic agent for hepatitis C, and a therapeutic agent for filovirus infection. Examples of the therapeutic agent for influenza may include Amantadine, Rimantadine, Oseltamivir, Zanamivir, Peramivir, Laninamivir, and Baloxavir. Examples of the therapeutic agent for hepatitis C may include Ribavirin, pegylated interferon, Telaprevir, and Boceprevir. Examples of the therapeutic agent for filovirus infection may include Ribavirin, Palivizumab, Motabizumab, RSV-IGIV, MEDI-<NUM>, A-<NUM>, MDT-<NUM>, BMS-<NUM>, Amiodarone, Dronedarone, Verapamil, Ebola convalescent plasma, TKM-<NUM>, BCX4430, FGI-<NUM>, TKM-Ebola, ZMapp, rNAPc2, OS-<NUM>, MVA-BN filo, Brincidofovir, and Ad26-ZEBOV. Examples of such drugs which exhibit RNA virus inhibitory action may include mycophenolic acid, Daptomycin, Nicorosamide, Azithromycin, Novobiocin, Chloroquine, Memantine, Prochlorperazine, Chlorcyclizine, Manidipine, GS-<NUM>, Imatinib, Chlorpromazine, and Nitazoxanide (<NPL>. It is preferable to combine the therapeutic agent for use in treating an RNA viral infection of the present invention with Ribavirin.

When the therapeutic agent for use in treating an RNA viral infection of the present invention is used in combination with other therapeutic agents for RNA viral infection or drugs which exhibit RNA virus inhibitory action, the amount ratio between the pyrazine derivative or a salt thereof and the one or more types of compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell is not particularly limited, as long as it is an amount ratio, in which the antiviral activity of the pyrazine derivative or a salt thereof is reinforced. The pyrazine derivative or a salt thereof : the one or more types of compounds which increase the amount of a pyrazine derivative ribose triphosphate in a cell (molar ratio) is preferably <NUM> : <NUM> to <NUM> : <NUM>, more preferably <NUM> : <NUM> to <NUM> : <NUM>, and further preferably <NUM> : <NUM> to <NUM> : <NUM>.

The amount ratio between the therapeutic agent for use in treating an RNA viral infection of the present invention and other therapeutic agents for RNA viral infection or drugs which exhibit RNA virus inhibitory action is not particularly limited, as long as it is an amount ratio, in which the antiviral activity of the therapeutic agent for use in treating an RNA viral infection of the present invention is reinforced. The therapeutic agent for use in treating an RNA viral infection of the present invention : other therapeutic agents for RNA viral infection or drugs which exhibit RNA virus inhibitory action (molar ratio) is preferably <NUM> : <NUM> to <NUM> : <NUM>, more preferably <NUM> : <NUM> to <NUM> : <NUM>, further preferably <NUM> : <NUM> to <NUM> : <NUM>, and still further preferably <NUM> : <NUM> to <NUM> : <NUM>.

Next, the present invention will be described in the following examples. However, these examples are not intended to limit the scope of the present invention.

The effects of the combination of a pyrazine derivative and an antimetabolite against an RNA virus were examined using the replicon activity of a complex of the virus with RNA-dependent RNA polymerase (RdRp). For evaluation of the replicon activity, a reporter assay system was constructed using cells, with reference to a known method (<NPL>)). In this assay system, the replicon activity was evaluated using the activity of luciferase (Luc) serving as a reporter protein.

<NUM>-Fluoro-<NUM>-hydroxy-<NUM>-pyrazinecarboxamide (T-<NUM>) was selected as a pyrazine derivative. Regarding antimetabolites, methotrexate and pralatrexate were selected as antifolates, and <NUM>-mercaptopurine, azathioprine, and thioguanosine were selected as thiopurine antimetabolites. As an RNA virus, an influenza virus was selected.

In accordance with the method described in the above-described known publication, genes encoding the viral proteins PA, PB1, PB2, and NP were amplified from the Influenza A/PR/<NUM>/<NUM> (H1N1) strain by an RT-PCR method, and were then cloned into a pcDNA3. <NUM> vector. The obtained pcDNA3. <NUM>/PR8_PA plasmid DNA, pcDNA3. <NUM>/PR8_PB1 plasmid DNA, pcDNA3. <NUM>/PR8_PB2 plasmid DNA, and pcDNA3. <NUM>/PR8_NP plasmid DNA were denominated as wild-type PA DNA, wild-type PB1 DNA, wild-type PB2 DNA, and wild-type NP DNA, respectively.

Subsequently, a plasmid DNA comprising a reporter gene was produced. Specifically, a DNA was produced by connecting, from the <NUM>'-terminus to the <NUM>'-terminus, a region encoding a human-derived polymerase I promoter (<NPL>)), a Luc gene sandwiched with the NTRs of the Influenza A/PR/<NUM>/<NUM> (H1N1) strain, and a region encoding a mouse-derived polymerase I terminator (<NPL>)), and the produced DNA was then cloned into a pcDNA3. <NUM> vector to obtain the reporter plasmid pcDNA3. <NUM>/polI_NTR_RhLuc.

As cells used herein, human embryonic kidney cells 293T expressing SV40 large T antigen (hereinafter referred to as "293T cells") were used. 293T cells, which had been sub-cultured in a DMEM medium supplemented with <NUM>% fetal bovine serum under conditions of <NUM>% CO<NUM> at <NUM>, were removed by a trypsin ethylenediaminetetraacetate method, and were then seeded in a flask to result in an amount of <NUM> × <NUM><NUM> cells/<NUM><NUM> flask. Thereafter, the cells were cultured overnight under conditions of <NUM>% CO<NUM> at <NUM>, so as to obtain a single layer of 293T cells.

To <NUM> of Opti-MEM (manufactured by Thermo Fisher Scientific), <NUM>µg of wild-type PA DNA, <NUM>µg of wild-type PB1 DNA, <NUM>µg of wild-type PB2 DNA, <NUM>µg of wild-type NP DNA, and <NUM>µg of two types of reporter plasmids pcDNA3. <NUM>/polI_NTR_RhLuc, and <NUM>µg of pGL4. <NUM> (manufactured by Promega, #E5061) (for confirmation of the presence or absence of DNA introduction) were added, and further, <NUM>µL of PLUS Reagent included with Lipofectamine LTX Reagent with PLUS Reagent (manufactured by Thermo Fisher Scientific) was added. At the same time, <NUM> of Lipofectamine LTX Reagent was added to <NUM> of Opti-MEM, and was then left for <NUM> minutes. Thereafter, the two liquids were mixed with each other to a volume ratio of <NUM> : <NUM>, and the obtained mixture was then left at room temperature for <NUM> minutes. The above mixed solution was added to a culture flask containing 293T cells to result in an amount of <NUM>/<NUM><NUM> flask, and was then left for approximately <NUM> hours, so that the RdRp of the influenza virus, NP, and the reporter plasmid were introduced into the cells.

The transduced 293T cells were removed by a trypsin ethylenediaminetetraacetate method, and were then seeded on a <NUM>-well plate (manufactured by Corning, #<NUM>) to which any one of the following test solutions (A) to (D) had been added, so as to achieve <NUM> × <NUM><NUM> cells/<NUM>µL/well. At the same time, as blanks to which no cells were added, the following solutions (A) to (D) were added to wells, so as to prepare blank wells. Thereafter, the obtained mixtures were each cultured under conditions of <NUM>% CO<NUM> at <NUM> for approximately <NUM> hours.

All of the solutions (A) to (D) were diluted and mixed with a DMEM medium supplemented with <NUM>% fetal bovine serum.

For the measurement of Luc activity, Dual-Glo Luciferase Assay System (manufactured by Promega) was used. After completion of the culture for approximately <NUM> hours, Dual-Glo Reagent included with the aforementioned System was added in an amount of <NUM>µL/well to the plate, and the obtained mixture was then stirred at room temperature for <NUM> minutes or more, so that the cells were dissolved. Thereafter, using a microplate reader (manufactured by PerkinElmer, <NUM> EnVision), fluorescence intensity was measured. Subsequently, Stop & Glo Reagent included with the System was added in an amount of <NUM>µL/well to the plate, and the obtained mixture was then stirred at room temperature for <NUM> minutes or more. Thereafter, using the microplate reader (as described above), fluorescence intensity was measured. The operations were carried out with the number of cases of <NUM>. According to the following equation, a replicon reaction percentage and a replicon inhibiting percentage were calculated. <MAT> <MAT> <MAT>.

As a result, as shown in Table <NUM>, it was found that the replicon inhibiting percentage of T-<NUM> is improved by addition of an antifolate or a thiopurine preparation, although a single T-<NUM> agent has an uninhibitable concentration. It is to be noted that thioguanosine is a compound formed by binding a ribose ring to <NUM>-thioguanine. Since it is considered that <NUM>-thioguanine is metabolized to thioguanosine (or a metabolite of thioguanosine) in cells, <NUM>-thioguanine is also considered to improve the replicon inhibiting percentage of T-<NUM>, as with thioguanosine.

Using the following test solutions (E) to (H), addition of the drugs to the cells and the culture of the cells were carried out in the same manner as that in (<NUM>-<NUM>) above. Thereafter, the replicon inhibiting activity was evaluated in the same manner as that in (<NUM>-<NUM>) above.

For calculation of the <NUM>% inhibitory concentration, Dose Response One Site (Sigmoidal Dose-Response Model; [fit = (A + ((B-A) / (<NUM> + ((C/x)^D))))]) of XLfit (version <NUM>. <NUM>) was used. The <NUM>% inhibitory concentration of T-<NUM> used in combination with each antimetabolite is shown in Table <NUM>. It was found that the combination of T-<NUM> and each antimetabolite shows a lower <NUM>% inhibitory concentration than a single T-<NUM> agent.

In the present test system, a single <NUM>-mercaptopurine agent (<<NUM>), a single methotrexate agent (<<NUM>), and a single pralatrexate agent (<<NUM>) have a replicon inhibiting percentage of less than <NUM>%, and no inhibitory activity was found. The replicon inhibiting activity was significantly reinforced by the combination of T-<NUM> and an antimetabolite, compared with the single use of T-<NUM>.

Regarding the effects of the combined use of a pyrazine derivative and an antimetabolite, a test was further performed using virus-infected cell models.

T705 was selected as a pyrazine derivative. <NUM>-Mercaptopurine was selected as an antimetabolite. As an RNA virus, an influenza virus was selected.

Madin-Darby Caine Kidney cells (hereinafter referred to as "MDCK"), which had been sub-cultured in a <NUM>% fetal bovine serum-added Eagle's MEM medium used as a culture medium under conditions of <NUM>% CO<NUM> at <NUM>, were removed according to a trypsin ethylenediaminetetraacetate method. A suspension prepared by allowing <NUM>µL of the same medium as described above to comprise the <NUM> × <NUM><NUM> cells was seeded on a <NUM>-well plate. The suspension was cultured overnight under conditions of <NUM>% CO<NUM> at <NUM>, so as to obtain MDCK cells that were in the form of a single layer.

As a test medium, a medium, which was prepared by adding L-<NUM>-tosylamide-<NUM>-phenylethylchloromethylketone (TPCK)-treated trypsin to an Eagle's MEM medium supplemented with <NUM>µg/mL kanamycin to a concentration of <NUM>µg/mL, was used. A culture supernatant of the MDCK cells obtained in (<NUM>) above was removed, and the plate was then rinsed with an Eagle's MEM medium, and the following (A) to (C) were then added to each well. After addition of the drug, the obtained mixture was cultured under conditions of <NUM>% CO<NUM> at <NUM> for <NUM> to <NUM> days.

Cytopathic effect (CPE) found with proliferation of the influenza virus was judged by the following method.

After completion of the culture, <NUM>µL of a <NUM>% formalin solution was added to each well, and thereby, the virus was inactivated and the cells were immobilized. The reaction mixture was left at rest at room temperature for <NUM> hours or more, and a <NUM>% methylene blue solution was then added in an amount of <NUM>µL/well to each well, followed by leaving at rest at room temperature for <NUM> hour. Thereafter, the <NUM>% methylene blue solution was removed, and the reaction mixture was lightly washed with water and was then air-dried. Thereafter, using a microplate reader (manufactured by Tecan; infinit M200), the absorbance (<NUM>) was measured. As a non-infection control, <NUM>µL of an Eagle's MEM medium comprising TPCK-treated trypsin having <NUM> times the concentration of the test medium was added to the wells, instead of the influenza virus solution, and the same operations as those for the test group were then carried out, followed by the measurement of the absorbance. As a blank, the same operations as those for the non-infection control were carried out on wells, into which MDCK cells had not been seeded, and the absorbance was then measured.

The test was carried out three times with the number of cases of <NUM> (the number of cases was <NUM> in the infection control). Using a mean value, the numerical value obtained by subtracting the blank value from the mean value was defined as an absorbance. The value obtained by subtracting the absorbance of the infection control from the absorbance of the non-infection control was defined as a complete suppression value of virus proliferation. The CPE inhibition percentage in each test was calculated according to the following equation.

For calculation of <NUM>% inhibitory concentration, FORECAST function (linear regression method) of Microsoft Office Excel <NUM> was used.

A change in the <NUM>% inhibitory concentration of T-<NUM> upon addition of <NUM>-mercaptopurine is shown in Table <NUM>. When compared with a single T-<NUM> agent, the <NUM>% inhibitory concentration of T-<NUM> that was used in combination with <NUM>-mercaptopurine became a low value. Besides, in the present test system, a single <NUM>-mercaptopurine (<NUM>) agent did not exhibit CPE inhibitory effects.

It could be confirmed even by using infected cell models that antiviral activity is reinforced by a combination of T-<NUM> and an antimetabolite, compared with a single T-<NUM> agent.

Using the same influenza virus-infected cell models as those in Test Example <NUM>, the combined use effects of a pyrazine derivative, an antimetabolite and another therapeutic agent for use in treating an RNA viral infection were further examined.

As a pyrazine derivative, T-<NUM> was selected. As an antimetabolite, <NUM>-mercaptopurine was selected. As another therapeutic agent for use in treating an RNA viral infection, Ribavirin was selected. The set concentrations of the drugs and the judgment of CPE were determined as follows, and the test was then carried out.

The set concentrations of the drugs were determined as follows.

With regard to the judgment of CPE, a plate stained with a methylene blue solution was confirmed by visual observation, so that a minimal drug concentration which exhibits a complete CPE suppression effect was determined.

When T-<NUM> and Ribavirin were used each alone, the minimum drug concentrations which exhibit complete CPE suppression effects were <NUM> and <NUM> (µM), respectively. In addition, the minimum drug concentration of T-<NUM> which exhibits a complete CPE suppression effect was <NUM> (µM), when <NUM>-mercaptopurine (<NUM>) was added. Moreover, the minimum drug concentration of T-<NUM> which exhibits a complete CPE suppression effect was <NUM> (µM), when <NUM>-mercaptopurine (<NUM>) and Ribavirin (<NUM>) were added.

It could be confirmed using infected cell models that antiviral activity is further reinforced by a combination of T-<NUM>, an antimetabolite and another therapeutic agent for use in treating an RNA viral infection, when compared with a single T-<NUM> agent, or a combination of T-<NUM> and an antimetabolite.

Cell models infected with virus other than the influenza virus were created using Punta Toro virus as a bunyavirus, and the same test as that in Test Example <NUM> was then carried out using the cell models. T705 was selected as a pyrazine derivative. As compounds, <NUM>-mercaptopurine, azathioprine, temozolomide, theophylline, and tiazofurin were selected.

As cells used herein, 293T cells were used. 293T cells, which had been sub-cultured in a DMEM medium supplemented with <NUM>% fetal bovine serum under conditions of <NUM>% CO<NUM> at <NUM>, were removed by a trypsin ethylenediaminetetraacetate method, and a suspension was then prepared with the same medium as described above, such that <NUM> of the medium comprises <NUM> × <NUM><NUM> cells. The prepared suspension was seeded on a <NUM>-well plate, and was then cultured overnight under conditions of <NUM>% CO<NUM> at <NUM>.

A culture supernatant of the 293T cells obtained in (<NUM>) above was removed, and the plate was then rinsed with an Eagle's MEM medium. Thereafter, <NUM> of a Punta Toro virus solution adjusted to <NUM> × <NUM><NUM> PFU/mL was added to each well, and the obtained mixture was then cultured under conditions of <NUM>% CO<NUM> at <NUM> for <NUM> hours. Thereafter, the virus solution was removed, and a <NUM>% fetal bovine serum-added Eagle's MEM medium comprising a single T-<NUM> agent (final concentration: <NUM>), an antimetabolite (final concentration: <NUM>), or a mixture of a single T-<NUM> agent (final concentration: <NUM>) and an antimetabolite (final concentration: <NUM>), was added to each well. The thus obtained mixture was cultured under conditions of <NUM>% CO<NUM> at <NUM> for <NUM> days. In addition, as an infection control, a <NUM>% fetal bovine serum-added Eagle's MEM medium was added, instead of the medium comprising the drugs.

Monkey kidney Vero cells, which had been sub-cultured in a <NUM>% fetal bovine serum-added Eagle's MEM medium used as a culture medium under conditions of <NUM>% CO<NUM> at <NUM>, were removed according to a trypsin ethylenediaminetetraacetate method. A suspension prepared by allowing <NUM>µL of a <NUM>% fetal bovine serum-added Eagle's MEM medium to comprise the <NUM> × <NUM><NUM> cells was seeded on a <NUM>-well plate. The suspension was cultured overnight under conditions of <NUM>% CO<NUM> at <NUM>, so as to obtain Vero cells that were in the form of a single layer.

The virus culture solution obtained in (<NUM>) above was serially diluted up to <NUM> times with an Eagle's MEM medium at a common ratio of <NUM>. The stock solution and diluted solutions were each added in an amount of <NUM>µL each to the Vero cells obtained in (<NUM>-<NUM>) above (n = <NUM>). The obtained mixture was cultured under conditions of <NUM>% CO<NUM> at <NUM> for <NUM> to <NUM> days.

After completion of the culture, <NUM>µL of a <NUM>% formalin solution was added to each well, and thereby, the virus was inactivated and the cells were immobilized. The reaction mixture was left at rest at room temperature for <NUM> hours or more, and a <NUM>% methylene blue solution was then added in an amount of <NUM>µL/well to each well, followed by leaving at rest at room temperature for <NUM> hour. Thereafter, the <NUM>% methylene blue solution was removed, and the resultant was lightly washed with water and was then air-dried. Thereafter, the logarithmic value of the median infective dose of each sample (LogTCID<NUM>) was calculated using the following equation of a Bohrens-Karber method. The effect of reducing the virus amount with respect to the infection control was indicated with <IMG>LogTCID<NUM>.

The results are shown in Table <NUM>. It could be confirmed that<IMG> LogTCID<NUM> was increased by using T-<NUM> in combination with the compound.

It could be confirmed that antiviral activity is reinforced by the combined use of T-<NUM> and an antimetabolite, not only against the influenza virus of Test Example <NUM>, but also against the Punta Toro virus. In addition, it was found that temozolomide, theophylline and tiazofurin also exhibit an excellent effect of reducing the virus amount, when they are used in combination with T-<NUM>.

T-<NUM> was selected as a pyrazine derivative, <NUM>-mercaptopurine was selected as a compound, and the same test as that in Test Example <NUM> was carried out. The results are shown in Table <NUM>.

It could be confirmed that, as with T-<NUM>, the antiviral effects of T-<NUM> are also reinforced by being used in combination with the antimetabolite.

As described above, the pyrazine derivative or a salt thereof undergoes ribosyl phosphorylation in a cell. For example, it has been known that T-<NUM>-<NUM>-ribofuranosyl-<NUM>-triphosphate (T-705RTP) generated as a result of the ribosyl phosphorylation of T-<NUM> inhibits the RdRp protein of virus and thereby exhibits antiviral activity. In order to examine whether or not the effect of <NUM>-mercaptopurine to reinforce the activity of T-<NUM>, which was shown in Test Example <NUM>, is caused by a change in the amount of T-705RTP, the amount of T-705RTP in 293T cells was measured under the treatment with <NUM>-mercaptopurine.

293T cells (<NUM> × <NUM><NUM> cells) seeded in each well of a <NUM>-well plate were cultured with a vehicle (<NUM>% DMSO) or with <NUM>-mercaptopurine, T-<NUM>, or the two drugs in various concentrations, under conditions of <NUM>% CO<NUM> at <NUM> for <NUM> hours.

After completion of the culture, the reaction mixture was washed with <NUM> of PBS, and <NUM>µl of trypsin was then added thereto. The obtained mixture was left at rest at <NUM> for <NUM> minutes, and the cells were then removed. Subsequently, <NUM>µl of a trypsin inhibitor was added and suspended into the cells, and the cell suspension was then recovered in a <NUM>-ml tube. The cell suspension was centrifuged at <NUM>,<NUM> rpm at <NUM> for <NUM> minutes (MX-<NUM>, TOMY), and <NUM>µl of a supernatant was then removed. Subsequently, <NUM> of PBS was added and re-suspended into the cells, and <NUM>µl of the suspension was then distributed in a <NUM>-ml tube and was used as a sample for the measurement of the number of distributed cells. Then, <NUM> of the remaining cell suspension was distributed in a <NUM>-ml tube, and was then used for the extraction of T-705RTP. The distributed cell suspension was centrifuged at <NUM>,<NUM> × g at <NUM> for <NUM> minutes, and <NUM>µl of a supernatant was then removed. Subsequently, for the extraction of T-705RTP, <NUM>µl of methanol was added to the suspension, and was fully suspended. Using a centrifugal evaporator (CVE-<NUM>, EYELA), the obtained suspension was evaporated and dried at <NUM>. The sample was then used in an LC/MS/MS analysis.

The measurement of the number of cells and an LC/MS/MS analysis were carried out, and the amount of T-705RTP in each sample was then measured. The test method was applied in accordance with <NPL>). A change in the amount of T-705RTP is shown in <FIG>. It was found that the amount of T-705RTP in the cells after the treatment of T-<NUM> was increased by <NUM>-mercaptopurine.

The mechanism of action of T-<NUM>, namely, the mechanism by which after T-<NUM> is converted to T705RTP in a cell, it exhibits antiviral activity, is applied to all species of viruses, which are affected by T-<NUM>. Since the amount of T-705RTP in a cell has been increased by an antimetabolite, it is said that the combination of T-<NUM> and an antimetabolite simultaneously reinforces antiviral activities against a plurality of RNA viruses that are affected by T-<NUM>.

This mechanism is broadly applied, not only to T-<NUM>, but also to the pyrazine derivative represented by the formula [<NUM>] or a salt thereof. For example, it is suggested that a compound represented by the formula [<NUM>] other than T-<NUM>, wherein R<NUM> is a hydrogen atom, R<NUM> is a hydrogen atom and R<NUM> is a hydrogen atom (i.e., <NUM>-hydroxy-pyrazinecarboxamide), exhibits antiviral activity (<NPL>. ), and that this compound is converted to a ribose triphosphate in a cell (<NPL>. That is to say, by combining the pyrazine derivative or a salt thereof with one or more types of compounds which increase the amount of a pyrazine derivative ribose triphosphate, antiviral activities against a plurality of RNA viruses that are affected by the pyrazine derivative or a salt thereof can be simultaneously reinforced.

Claim 1:
A therapeutic agent for use in treating an RNA viral infection, which comprises a combination of a pyrazine derivative represented by the following formula [<NUM>] or a salt thereof and one or more types of compounds selected from the group consisting of an antifolate, a thiopurine antimetabolite, thiazofurin, an alkylating agent, and a xanthine derivative:
<CHM>
wherein R<NUM> and R<NUM>, which are the same or different, each represent a hydrogen atom or a halogen atom; and R<NUM> represents a hydrogen atom or an amino-protecting group, and
wherein
the antifolate is methotrexate or pralatrexate, the thiopurine antimetabolite is a mercaptopurine derivative represented by the following formula [<NUM>]:
<CHM>
wherein R<NUM> represents a hydrogen atom or an optionally protected amino group; and R<NUM> represents a hydrogen atom or the following formula [<NUM>]:
<CHM>
<CHM>
(wherein R<NUM> represents a hydrogen atom, a C<NUM>-<NUM> alkyl group, or a carboxy group; and R<NUM> represents a hydrogen atom, a C<NUM>-<NUM> alkyl group, a benzyl group, or a p-nitrobenzyl group), the alkylating agent is temozolomide, and the xanthine derivative is theophylline.