THERAPEUTIC AGENT FOR AN RNA VIRUS INFECTION COMPRISING A COMBINATION OF A PYRAZINE DERIVATIVE AND A THIOPURINE DERIVATIVE

Abstract

The present invention addresses the problem of providing a therapeutic agent for RNA viral infection that includes a novel combination of a pyrazine derivative and a specific compound, the novel combination exhibiting an effect against the RNA virus. The present invention also addresses the problem of providing a therapeutic agent for RNA viral infection that includes a combination of a pyrazine derivative and a specific compound, the combination being capable of simultaneously enhancing anti-virus activities against multiple RNA viruses. The present invention provides a therapeutic agent for RNA viral infection obtained by combining a pyrazine derivative or a salt thereof and a thiopurine derivative.

Claims

1. A therapeutic agent for an RNA virus infection comprising a combination of a pyrazine derivative or a salt thereof represented by a formula [1] ##STR00015## (In the formula, R.sup.1 and R.sup.2 are the same or different and represent a hydrogen atom or a halogen atom; and R.sup.3 represents a hydrogen atom or an amino protecting group.) and a thiopurine derivative represented by a formula [2] ##STR00016## (In the formula, R.sup.4 and R.sup.5 are the same or different and represent a hydrogen atom or a hydroxyl protecting group; R.sup.6 represents a hydrogen atom, a monophosphate group which may be protected, a diphosphate group which may be protected, or a triphosphoric acid group which may be protected; R.sup.7 represents a hydrogen atom or an amino group which may be protected; and R.sup.8 represents a hydrogen atom, a C1-6 alkyl group or a group represented by formula [3] ##STR00017## (In the formula, R.sup.9 represents a hydrogen atom, a C1-6 alkyl group or a carboxy group; and R.sup.10 represents a hydrogen atom, a C1-6 alkyl group, a benzyl group or a p-nitrobenzyl group.); and X represents an oxygen atom, a sulfur atom, a carbon atom, or an imino group which may be protected.).

2. The therapeutic agent according to claim 1, wherein R.sup.1 is a hydrogen atom, R.sup.2 is a fluorine atom or a hydrogen atom, and R.sup.3 is a hydrogen atom.

3. The therapeutic agent according to claim 1, wherein R.sup.1 is a hydrogen atom, R.sup.2 is a fluorine atom, and R.sup.3 is a hydrogen atom.

4. The therapeutic agent according to claim 1, wherein R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each a hydrogen atom, R.sup.8 is a methyl group, and X is an oxygen atom.

5. A method for treating RNA virus infection comprising administering to the subject a pyrazine derivative or a salt thereof represented by a formula [1] ##STR00018## (In the formula, R.sup.1 and R.sup.2 are the same or different and represent a hydrogen atom or a halogen atom; and R.sup.3 represents a hydrogen atom or an amino protecting group.) and a thiopurine derivative represented by a formula [2] ##STR00019## (In the formula, R.sup.4 and R.sup.5 are the same or different and represent a hydrogen atom or a hydroxyl protecting group; R.sup.6 represents a hydrogen atom, a monophosphate group which may be protected, a diphosphate group which may be protected, or a triphosphoric acid group which may be protected; R.sup.7 represents a hydrogen atom or an amino group which may be protected; and R.sup.8 represents a hydrogen atom, a C1-6 alkyl group or a group represented by a formula [3]. ##STR00020## (In the formula, R.sup.9 represents a hydrogen atom, a C1-6 alkyl group or a carboxy group; and R.sup.10 represents a hydrogen atom, a C1-6 alkyl group, a benzyl group or a p-nitrobenzyl group.); and X represents an oxygen atom, a sulfur atom, a carbon atom, or an imino group which may be protected.).

6. The method for treating RNA virus infection according to claim 5, wherein R.sup.1 is a hydrogen atom, R.sup.2 is a fluorine atom or a hydrogen atom, and R.sup.3 is a hydrogen atom.

7. The method for treating RNA virus infection according to claim 5, wherein R.sup.1 is a hydrogen atom, R.sup.2 is a fluorine atom, and R.sup.3 is a hydrogen atom.

8. The method for treating RNA virus infection according to claim 5, wherein R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each a hydrogen atom, R.sup.8 is a methyl group, and X is an oxygen atom.

Description

EXAMPLES

[0089] Next, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

Test Example 1

[0090] Combination effect of pyrazine derivative and thiopurine derivative was tested by using virus cell infection model

[0091] T-705 was selected as pyrazine derivative, 6-methylmercaptopurine riboside was selected as a thiopurine derivative. Influenza viruse was selected as RNA viruses.

[0092] (1) Culturing Vero cells African green monkey kidney Vero cell culture which were subcultured at 37° C. under 5% carbon dioxide conditions in a culture medium containing 10% bovine fetal serum-added eagle MEM/canamycin 60 μg/mL (eagle MEM/canamycin) were exfoliated by the trypsin method of ethylenediamine tetraacetate, and a suspension prepared to contain 2×10.sup.4 cells in 100 μL in the same medium was seeded on a 96-well plate. Under 5% carbon dioxide conditions, the cells were cultured overnight at 37° C. to obtain monolayered Vero cells.

[0093] (2) Influenza Virus Infection and Chemical Addition

[0094] As a test medium, a medium prepared by adding L-1-tosylamide-2-phenylethylchloromethylketone (TPCK)-treated trypsin to an eagle MEM/kanamycin medium at 1 μg/mL was used. The culture supernatant of Vero cells obtained in (1) was removed, and the following (A) to (C) were added to each well. After the addition of the drug, the cells were cultured at 35° C. for 2 days under 5% carbon dioxide conditions.

[0095] (A) 100 μL of Eagle MEM/Kanamycin Medium

[0096] (B) 50 μL of influenza virus (PR/8/34 (H1N1)) solution adjusted to 4.0×10.sup.3 PFU/mL with Eagle MEM/kanamycin medium containing 4 times the concentration of TPCK-treated trypsin as the test medium.

[0097] (C) 50 μL of 1% DMSO-containing Eagle MEM/kanamycin medium containing a combination of each set concentration of T-705 and 6-methylmercaptopurine riboside at 4 times the set concentration.

[0098] T-705 set concentration (μM):

[0099] 0,0.1,0.3,1,3,10,30,100,300,1000

[0100] 6-Methylmercaptopurine riboside set concentration (μM):

[0101] 0,0.1,0.3,1,3,10

[0102] (3) Determination of a Cell Modification Effect (CPE)

[0103] The CPE observed with the growth of influenza virus was determined by the following method.

[0104] After the culture was completed, 50 μL of 100% formalin solution was added to each well to inactivate the virus and fix the cells. After allowing to stand at room temperature for 2 hours or more, the aqueous solution was removed, the mixture was lightly washed with water, 50 μL/well of 0.02% methylene blue solution was added, and the mixture was allowed to stand at room temperature for 1 hour. The 0.02% methylene blue solution was removed, lightly washed with water, and then air-dried. Then, the absorbance (660 nm) was measured with a microplate reader (manufactured by Tecan, infinit M200). For non-infection control, 50 μL of Eagle MEM/canamycin medium containing TPCK-treated trypsin at a concentration 4 times that of the test medium was added instead of the influenza virus solution, and the same operation as in the test group was performed to measure the absorbance.

[0105] The test was carried out with 1/plate of cases and 2 plates (8 cases of infection control and non-infection control). The value obtained by subtracting the absorbance of the infection control from the absorbance of the non-infection control was taken as the complete suppression value of virus growth, and the CPE inhibition rate of each test was calculated from the formula shown below.

CPE inhibition rate=100×[(absorbance during single agent and combination action)−(absorbance of infection control)]/[(absorbance of non-infection control)−(absorbance of infection control)]

[0106] The FORECAST function (first-order regression method) of Microsoft Office Excel 2010 was used to calculate the 50% CPE inhibition concentration.

[0107] Table 1 shows the 50% CPE inhibitory concentration change of T-705 when 6-methylmercaptopurine riboside was added. The 50% CPE inhibitory concentration of T-705 was lower in combination with 6-methylmercaptopurine riboside as compared to T-705 alone. In this test system, 6-methylmercaptopurine riboside (10 μM) alone did not show a CPE inhibitory effect.

TABLE-US-00001 TABLE 1 The 50% CPE inhibitory concentration change of T-705 by adding 6-methylmercaptopurine riboside Concentration of 6-methyl- 0 0.1 0.3 1 3 10 mercaptopurine (μM) The 50% CPE inhibitory 183 89.4 48.9 30.6 21.9 20.2 concentration of T-705 (μM)

[0108] It was confirmed in the cell infection model that the antiviral activity was enhanced by the combination of T-705 and 6-methylmercaptopurine riboside as compared with the case of T-705 alone.

Test Example 2

[0109] As mentioned above, pyrazine derivatives or salts thereof undergo intracellular ribosyl phosphorylation. For example, T-705 is known to exhibit antiviral activity by inhibiting the viral RdRp protein by T-705-4-ribofuranosyll-5-triphosphate (T-705RTP) produced by ribosyl phosphorylation. In order to confirm whether the activity-enhancing effect of T-705 by 6-methylmercaptopurine riboside seen in Test Example 1 is due to a change in the amount of T-705RPP, the amount of T-705RTP below was measured under 6-Methylmercaptopurine riboside treatment in Vero cells.

[0110] (1) Measurement of T-705RTP Amount in Vero Cells Under Treatment with 6-Methylmercaptopurineriboside

[0111] (1-1) Extraction of Intracellular T-705RPP

[0112] Vero cells which were seeded in each well of a 6-well plate at 2×10.sup.5 cells were cultured in vehicle (0.0125% DMSO) or various concentrations of 6-methylmercaptopurineriboside, 100 μM T-705 or both agents under 5% carbon dioxide conditions at 37° C. for 24 hours (Example 3).

[0113] After culturing, the cells were washed with PBS, 300 μL trypsin was added, and the cells were allowed to stand at 37° C. for 5 minutes to peel off the cells. 700 μL of Eagle's MEM medium supplemented with 10% fetal bovine serum was added, suspended, and collected in a 1.5 mL tube. Of that, 10 μL was used for cell number measurement.

[0114] All remaining cell suspensions were centrifuged at 1,000 rpm, 4° C. for 5 minutes (MX-207. TOMY) to remove the supernatant. Next, 1 mL PBS was added to the cell pellet, the cells were resuspended, and the cells were centrifuged again at 1,000 rpm at 4° C. for 5 minutes, and the supernatant was removed. Then, 300 μL of 70% methanol was added and suspended well. Samples were then measured using a high performance liquid chromatograph (HPLC-FL) with a fluorescence detector.

[0115] (1-2) Measurement of Cell Number and HPLC-FL Analysis

[0116] The cell number was measured and HPLC-FL analysis was performed, and the amount of T-705RTP in each sample was measured. 20 μL of 6-bromo-3-hydroxy-2-pyrazinecarboxamide solution and 180 μL of distilled water as internal standards were added to 50 μL of a well-stirred cell sample (prepared in (1-1) above) under ice cooling and the mixture was centrifuged (about 1600 rpm, room temperature, 5 minutes, MICRO SINKER) and the supernatant was analyzed by HPLC. A C18 column was used for HPLC analysis, and T-705RTP was retained in the column by adding an ion pair reagent (tetrabutylammonium bromide) to the weakly acidic mobile phase. The excitation wavelength for fluorescence detection was 370 nm, and the fluorescence wavelength was 445 nm. From the HPLC area ratio (T-705RTP/internal standard) of each cell sample, the T-705RTP concentration was determined by the reverse regression of the calibration curve. Table 2 shows changes in the amount of T-705RTP. It was found that 6-methylmercaptopurine riboside increased the intracellular T-705RTP amount after T-705 treatment.

TABLE-US-00002 TABLE 2 The change of T-705RTP amount in Vero cells Concentration of 6-methylmercaptopurine (μM) 0 1 10 T-705RTP concentration (pmol/10.sup.6 cells) 88.3 148 167

[0117] The mechanism of action of T-705, that is, that it exhibits antiviral activity after being converted to T-705RTP in cells, is applicable to any virus as long as T-705 is effective. Since the intracellular T-705RTP amount was increased by the thiopurine derivative, it can be said that the combination of T-705 and the thiopurine derivative simultaneously enhances the antiviral activity against a plurality of RNA viruses for which T-705 is effective.

[0118] This mechanism applies not only to T-705 but also to a pyrazine derivative represented by the formula [1] or a salt thereof. For example, in addition to T-705, it is suggested that the compound (3-hydroxy-pyrazincarboxamide) of formula [1] in which R.sup.1 is a hydrogen atom; R.sup.2 is a hydrogen atom; and R.sup.3 is a hydrogen atom shows antivirus activity (Antival Res. 2009; 82 (3): 95-102.) and is intracellularly transformed into a ribose triphosphate (Mol Pharmacol. 2013; 84 (4): 615-29.). That is, by combining a pyrazine derivative or a salt thereof with a thiopurine derivative, the antiviral activity against a plurality of RNA viruses to which the pyrazine derivative or the salt thereof is effective can be simultaneously enhanced.

INDUSTRIAL APPLICABILITY

[0119] An RNA virus infection therapeutic agent comprising a combination of a pyrazine derivative or a salt thereof and a thiopurine derivative exhibits enhanced antiviral activity and is useful in the field of pharmaceutical industry.