CORONAVIRUS INFECTION THERAPEUTIC AGENT FORMED THROUGH COMBINATION OF PYRAZINE DERIVATIVE AND ANOTHER CORONAVIRUS INFECTION THERAPEUTIC DRUG

Abstract

An object of the present invention is to provide a novel combination of substances showing effects against coronavirus. The present invention provides a therapeutic agent for coronavirus infection comprising a combination of a pyrazine derivative or a salt thereof and another therapeutic agent for coronavirus infection.

Claims

1. A pharmaceutical composition for treating coronavirus infection, comprising a pyrazine derivative represented by the following general formula: ##STR00006## (wherein R.sup.1 and R.sup.2, identical or different, each represent a hydrogen atom or a halogen atom; and R.sup.3 represents a hydrogen atom or an amino protecting group) or a salt thereof and one or more therapeutic agents for coronavirus infection selected from the following (1) to (19): (1) Interferon; (2) Nucleic acid analogue; (3) Protease inhibitor; (4) Furin convertase cleavage inhibitor; (5) Reverse transcriptase inhibitor and/or other RNA polymerase inhibitor; (6) Neuraminidase inhibitor; (7) Angiotensin II receptor blocker; (8) Endosome fusion inhibitor and/or endosome alkalinizer; (9) AAK1, GAK, or clathrin A,B,C (endocytosis) inhibitor; (10) Hemagglutinin esterase inhibitor; (11) Cytokine or inflammation inhibitor or modifier; (12) Cathepsin B inhibitor; (13) Cathepsin L inhibitor; (14) Helicase nsp13 inhibitor; (15) MBL2 gene agonist; (16) TP53 inhibitor; (17) Selective estrogen receptor modifier; (18) Corticosteroid; and (19) Amantadine, foscarnet, triazavirin, umifenovir, rapamycin, everolimus, nitazoxanide, tizoxanide, caraphenol A, ivermectin, VIR-2703, tocilizumab, bamlanivimab, etesebimab, kasiribimab/imdebimab, AZD7422, VIR-7831, VIR-7832, or BI 767551.

2. The pharmaceutical composition according to claim 1, wherein the interferon is interferon α-2A, interferon α-2B, interferon α-n1, interferon α-n3, interferon β-1a, or interferon β-1b.

3. The pharmaceutical composition according to claim 1, wherein the nucleic acid analogue is acyclovir, ganciclovir, ribavirin or taribavirin.

4. The pharmaceutical composition according to claim 1, wherein the protease inhibitor is indinavir, nelfinavir, saquinavir, camostat, lopinavir/ritonavir combination (brand name, Kaletra), epigallocatechin gallate, kaempferol-7-glucoside, mycophenolic acid, darunavir, mercaptopurine, disulfiram, nafamostat, or PF-07321332.

5. The pharmaceutical composition according to claim 1, wherein the furin convertase cleavage inhibitor is tenofovir disoproxil, dolutegravir, boceprevir, andrographolide, luteolin, or baicalein.

6. The pharmaceutical composition according to claim 1, wherein the reverse transcriptase inhibitor and/or other RNA polymerase inhibitor is remdesivir, sofosbuvir, dactinomycin, galidesivir, baloxavir marboxil, molnupiravir, sangibamycin, or AT-527.

7. The pharmaceutical composition according to claim 1, wherein the neuraminidase inhibitor is oseltamivir or zanamivir.

8. The pharmaceutical composition according to claim 1, wherein the angiotensin II receptor blocker is valsartan, telmisartan, losartan, irbesartan, azilsartan, olmesartan, or emodin.

9. The pharmaceutical composition according to claim 1, wherein the endosome fusion inhibitor and/or endosome alkalinizer is baicalin, chloroquine, hydroxychloroquine, griffithsin, quinine, or lactoferrin.

10. The pharmaceutical composition according to claim 1, wherein the AAK1, GAK, or clathrin A, B, C (endocytosis) inhibitor is baricitinib, sunitinib, erlotinib, fedratinib, gefitinib, or silibinin.

11. The pharmaceutical composition according to claim 1, wherein the hemagglutinin esterase inhibitor is 3,4-dichloroisocoumarin.

12. The pharmaceutical composition according to claim 1, wherein the cytokine or inflammation inhibitor or modifier is ligustrazine, statin, melatonin, eplerenone, or methylprednisolone.

13. The pharmaceutical composition according to claim 1, wherein the cathepsin B inhibitor is salvianolic acid B.

14. The pharmaceutical composition according to claim 1, wherein the cathepsin L inhibitor is MOL736, chelidocystatin, astaxanthin, curcumin, or vitamin D.

15. The pharmaceutical composition according to claim 1, wherein the helicase nsp13 inhibitor is valsartan, bananin, iodobananin, vanillinbananin, eubananin, or silvestrol.

16. The pharmaceutical composition according to claim 1, wherein the MBL2 gene agonist is β-glucan or vitamin A.

17. The pharmaceutical composition according to claim 1, wherein the TP53 inhibitor is vitexin or gossypol.

18. The pharmaceutical composition according to claim 1, wherein the selective estrogen receptor modifier is toremifene or equilin.

19. The pharmaceutical composition according to claim 1, wherein the corticosteroid is ciclesonide or dexamethasone.

20. The pharmaceutical composition according to claim 1, wherein the one or more therapeutic agent for coronavirus infection is a reverse transcriptase inhibitor and/or other RNA polymerase inhibitor.

Description

EXAMPLES

[0088] The present invention will be described using examples, but the present invention is not limited to these.

Test Example 1

[0089] A test was performed using a viral infection cell model to evaluate the effect of combination use of a pyrazine derivative and remdesivir. Specifically, the antiviral effect was evaluated by determining the cytopathic effect (CPE). In addition to the CPE assay, the antiviral effect can be evaluated by a viral antigen-antibody method or by real-time polymerase chain reaction (PCR) of virus RNA extracted from a virus culture supernatant.

[0090] T-705 was selected as a pyrazine derivative. Remdesivir was selected as another therapeutic agent for coronavirus infection. Novel coronavirus (SARS-CoV-2) was selected as an RNA virus.

(1) Vero E6 Cell Culture

[0091] African green monkey kidney Vero E6 cells that were subcultured in a 10% fetal bovine serum-added Eagle minimum essential medium (MEM) containing 60 μg/mL kanamycin (Eagle MEM/kanamycin medium) at 37° C. under the 5% carbon dioxide condition were removed from the culture broth by the trypsin-ethylenediaminetetraacetic acid method, and a suspension containing 2×10.sup.4 cells per 100 μL of the medium was prepared and seeded on a 96 well plate. The Vero E6 cells were cultured overnight at 37° C. under the 5% carbon dioxide condition and obtained as a monolayer.

(2) SARS-CoV-2 Infection and Addition of Drugs

[0092] A 2% fetal bovine serum-added Eagle MEM/kanamycin medium was used as a 30 test medium. The culture supernatant containing Vero E6 cells obtained in (1) was removed, the following (A) or (B) was added to each well, and the mixture was cultured at 37° C. for 2 hours under the 5% carbon dioxide condition:

(A) 100 μL of SARS-CoV-2 solution prepared with the test medium to obtain a final multiplicity of infection of 0.02
(B) 100 μL of 0.5% DMSO-containing test medium that contained a combination of T-705 and remdesivir at concentrations two-fold the specified concentrations for each combination of the specified concentrations: [0093] Specified concentrations (μM) of T-705: [0094] 0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 [0095] Specified concentrations (μM) of remdesivir: [0096] 0, 0.1, 0.3, 1, 3, 10, 30, 100

[0097] At 2 hours after infection, the culture supernatant was removed from each well, and 100 μL of 0.5% DMSO-containing test medium that contained a combination of T-705 and remdesivir at concentrations one-fold the specified concentration for each combination of the specified concentrations were added. After the drugs were added, the cells were cultured at 37° C. for 2 days under the 5% carbon dioxide condition.

(3) Determination of Cytopathic Effect (CPE)

[0098] The CPE associated with proliferation of SARS-CoV-2 was determinated by the following method.

[0099] After completion of culture, 25 μL of 100% formalin solution was added to each well to inactivate virus and immobilize cells. The mixture was left stand at room temperature for 2 hours or longer, the aqueous solution was removed, cells were gently washed with water, and then 50 μL of 0.02% methylene blue solution was added to each well, and the mixture was left stand at room temperature for one hour. The aqueous solution was removed, and cells were gently washed with water and then air-dried. Then, absorbance (660 nm) was measured using a microplate reader (Tecan). For anon-infected control, 100 μL of the test medium was added instead of the SARS-CoV-2 solution, the same operations as for the test group were performed, and the absorbance was measured.

[0100] A test was performed for two plates (one patient per plate) (eight patients for infected controls and non-infected controls). The value obtained by subtracting the absorbance of the infected control from the absorbance of the non-infected control was used as the value indicating complete inhibition of viral proliferation, and the CPE inhibition rate was calculated for each test by the following formula:

CPE inhibition rate=100×([absorbance after monotherapy or combination use)−(absorbance of infected control])/([absorbance of non-infected control)−(absorbance of infected control])

[0101] Microsoft Office Excel 2016 FORECAST function (linear regression) was used to calculate 50% CPE inhibitory concentration.

[0102] The 50% CPE inhibitory concentration was confirmed when T-705 and remdesivir were used in combination at concentrations lower than their respective 50% inhibitory concentrations (IC.sub.50) of monotherapy.

[0103] In addition to remdesivir, the same test as described above was performed for the therapeutic agents for coronavirus infection listed in Table 1. The IC.sub.50 was confirmed when T-705 and remdesivir were used in combination at concentrations lower than their respective IC.sub.50 of monotherapy.

TABLE-US-00001 TABLE 1-1 No. Name 1 Interferon α-2A 2 Interferon α-2B 3 Interferon α-n1 4 Interferon α-n3 5 Interferon β-1a 6 Interferon β-1b 7 Acyclovir 8 Ganciclovir 9 Ribavirin 10 Taribavirin 11 Indinavir 12 Nelfinavir 13 Saquinavir 14 Camostat 15 Lopinavir/ritonavir combination (brand name, Kaletra) 16 Epigallocatechin gallate 17 Kaempferol-7-glucoside 18 Mycophenolic acid 19 Darunavir 20 Mercaptopurine 21 Disulfiram 22 Tenofovir disoproxil 23 Dolutegravir 24 Boceprevir 25 Andrographolide 26 Luteolin 27 Baicalein 28 Sofosbuvir 29 Dactinomycin 30 Galidesivir 31 Baloxavir marboxil 32 Oseltamivir 33 Zanamivir 34 Valsartan 35 Telmisartan

TABLE-US-00002 TABLE 1-2 36 Losartan 37 Irbesartan 38 Azilsartan 39 Olmesartan 40 Emodin 41 Baicalin 42 Chloroquine 43 Hydroxychloroquine 44 Griffithsin 45 Quinine 46 Lactoferrin 47 Baricitinib 48 Sunitinib 49 Erlotinib 50 Fedratinib 51 Gefitinib 52 Silibinin 53 3,4-Dichloroisocoumarin 54 Ligustrazine 55 Statin 56 Melatonin 57 Eplerenone 58 Methylprednisolone 59 Salvianolic acid B 60 MOL736 61 Chelidocystatin 62 Astaxanthin 63 Curcumin 64 Vitamin D 65 Valsartan 66 Bananin 67 Iodobananin 68 Vanillinbananin 69 Eubananin 70 Silvestrol 71 β-glucan

TABLE-US-00003 TABLE 1-3 72 Vitamin A 73 Vitexin 74 Gossypol 75 Toremifene 76 Equilin 77 Ciclesonide 78 Amantadine 79 Foscarnet 80 Triazavirin 81 Umifenovir 82 Rapamycin 83 Everolimus 84 Nitazoxanide 85 Tizoxanide 86 Caraphenol A

TABLE-US-00004 TABLE 1-4 87 Dexamethasone 88 Nafamostat 89 Ivermectin 90 Molnupiravir 91 Sangibamycin 92 AT-527 93 PF-07321332 94 VIR-2703 95 Tocilizumab 96 Bamlanivimab 97 Etesebimab 98 Kasiribimab/Imdebimab 99 AZD7422 100 VIR-7831 101 VIR-7832 102 BI 767551

Test Example 2

[0104] T-705 was selected as a pyrazine derivative. Remdesivir was selected as another therapeutic agent for coronavirus infection. Human coronavirus (HCoV-OC43) responsible for common cold, which belongs to the genus betacoronavirus as with the novel coronavirus, was selected as an RNA virus.

(1) Culture of BHK-21 cells

[0105] Hamster kidney BHK-21 cells that were subcultured in a 10% fetal bovine serum-added EMEM containing 60 μg/mL kanamycin (EMEM/kanamycin medium) at 37° C. under the 5% carbon dioxide condition were removed from the culture broth by the trypsin-ethylenediaminetetraacetic acid method, then a suspension containing 4×10.sup.4 cells per 100 μL in the medium was prepared and seeded on a 96 well plate. The BHK-21 cells were cultured overnight at 37° C. under the 5% carbon dioxide condition and obtained as a monolayer.

(2) HCoV-OC43 Infection and Addition of Drugs

[0106] A 2% fetal bovine serum-added EMEM/kanamycin medium was used as a test medium. The culture supernatant containing BHK-21 cells obtained in (1) was removed, the following (A) to (C) were added:

(A) 100 μL of the test medium
(B) 50 μL of HCoV-OC43 solution prepared with the test medium to obtain a final infectivity titer of 4.0×10.sup.3 TCID.sub.50
(C) 50 μL of 1% DMSO containing test medium that contained a combination of T-705 and remdesivir at concentrations four-fold the specified concentrations for each combination of the specified concentrations:

TABLE-US-00005 Specified concentration (μM) of T-705: 0, 1, 10, 100, 1000 Specified concentration (μM) of remdesivir: 0, 0.1, 0.3

[0107] After the drugs were added, the cells were cultured at 33° C. for 3 to 4 days under the 5% carbon dioxide condition.

(3) Determination of Cytopathic Effect (CPE)

[0108] The CPE associated with proliferation of HCoV-OC43 was assessed by the following method.

[0109] After completion of culture, 50 μL of 100% formalin solution was added to each well to inactivate virus and immobilized cells. The mixture was left stand at room temperature for 2 hours or longer, the aqueous solution was removed, and cells were gently washed with water, and then 50 μL of 0.02% methylene blue solution was added to each well, and the mixture was left stand at room temperature for one hour. The aqueous solution was removed, and cells were gently washed with water and then air-dried. Then, absorbance at 660 nm was measured using a microplate reader (Tecan). For anon-infected control, 50 μL of the test medium was added instead of the HCoV-OC43 solution, the same operations as for the test group were performed, and the absorbance was measured.

[0110] A test was performed for two plates (one patient per plate) (six patients for infected controls and non-infected controls). The value obtained by subtracting the absorbance of the infected control from the absorbance of the non-infected control was used as the value indicating complete inhibition of viral proliferation, and the CPE inhibition rate was calculated for each test by the following formula:

CPE inhibition rate=100×([absorbance after monotherapy or combination use)(absorbance of infected control])/([absorbance of non-infected control)−(absorbance of infected control])

[0111] Microsoft Office Excel 2016 FORECAST function (linear regression) was used to calculate 50% CPE inhibitory concentration.

[0112] The CPE inhibition rates on single use or combination use of drug(s) were compared. A CPE inhibition rate on single use of 1000 μM of T-705 was 79%, and a CPE inhibition rate on each of the single use of 0.1 μM and 0.3 μM of remdesivir was 51% and 80%, respectively. In contrast, a CPE inhibition rate on each of combination use of 0.1 μM o T-705 and remdesivir and 0.3 μM of those was 92% and 102%, respectively.

[0113] The combination use increased the CPE inhibition rate compared to each of single use of T-705 and remdesivir, and almost 100% of CPE inhibition effect thereof was confirmed. Also, on the basis of this result, it is considered that drugs having a mechanism of action similar to remdesivir, namely reverse transcriptase inhibitors and/or RNA polymerase inhibitors (for example, molnupiravir) are also effective in combination with T-705.

INDUSTRIAL APPLICABILITY

[0114] A therapeutic agent for coronavirus infection comprising a combination of a pyrazine derivative or a salt thereof and another therapeutic agent for coronavirus infection is useful in the field of pharmaceutical industry.