阅读说明：本技术 黄病毒感染症治疗剂 (Therapeutic agent for flavivirus infection ) 是由 增田道明 石川知弘 河田聪史 富冈基康 和田康史 于 2020-04-24 设计创作，主要内容包括：本发明提供黄病毒感染症的预防和/或治疗剂、以及黄病毒感染症的预防和/或治疗方法。在黄病毒感染症的预防和/或治疗剂中使用选自5-氨基乙酰丙酸(5-ALA)、5-ALA酯、5-ALA的盐或者5-ALA酯的盐中的至少一种。作为利用5-ALA等来预防和/或治疗的对象的黄病毒,例如,可举出登革热病毒、寨卡病毒、日本脑炎病毒、蜱传脑炎病毒、西尼罗病毒、或者黄热病毒。(The present invention provides an agent for preventing and/or treating a flavivirus infection, and a method for preventing and/or treating a flavivirus infection. At least one selected from 5-aminolevulinic acid (5-ALA), a 5-ALA ester, a salt of 5-ALA, or a salt of 5-ALA ester is used in a preventive and/or therapeutic agent for a flavivirus infection. Examples of the flavivirus to be prevented and/or treated by 5-ALA and the like include dengue virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, and yellow fever virus.)

1.A preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof.

2. The preventive and/or therapeutic agent for flavivirus infection according to claim 1, wherein the flavivirus is dengue virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, or yellow fever virus.

3. The preventive and/or therapeutic agent for flavivirus infection according to claim 1, further comprising an iron compound.

4. The preventive and/or therapeutic agent for flavivirus infection according to claim 1, without requiring light irradiation.

5. A method of preventing and/or treating a flavivirus infection comprising: administering to a subject a preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from 5-aminolevulinic acid, i.e. 5-ALA or an ester thereof, or a salt thereof.

6. The method for preventing and/or treating flavivirus infection according to claim 5, wherein the flavivirus is dengue virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, or yellow fever virus.

7. The method for preventing and/or treating flavivirus infection according to claim 5, wherein the agent for preventing and/or treating flavivirus infection further comprises an iron compound.

8. The method for the prevention and/or treatment of a flavivirus infection in accordance with claim 5, wherein no light irradiation is required.

Technical Field

The present invention relates generally to agents for preventing and/or treating flavivirus infections, and methods for preventing and/or treating flavivirus infections.

Background

Flaviviridae consists of enveloped viruses with positive-stranded RNA as their genome, most of which are transmitted by blood-sucking arthropods. Examples of mosquito-borne flaviviruses include japanese encephalitis virus (see non-patent document 1), dengue virus (see non-patent document 2), zaka virus (see non-patent document 3), west nile virus, and yellow fever virus, and tick-borne flaviviruses include tick-borne encephalitis virus. Flaviviruses contain many pathogens that cause japanese encephalitis, tick-borne encephalitis, dengue hemorrhagic fever, congenital zika virus infection, and the like, which are global public health problems. Since these viruses are stored and amplified in animals other than humans, and it is difficult to completely isolate the viruses from contact with infected mosquitoes or infected ticks, the development of specific therapeutic agents for these viruses is an important issue (see non-patent document 4). In addition, along with global warming in recent years, there is a concern that the inhabitation time and inhabitation area of mosquitoes that transmit flavivirus infections are extended, and the occurrence of flavivirus infections is also extended.

In recent years, many specific inhibitors have been developed as the function and structure of flavivirus proteins become clear (see non-patent document 5). For example, nitazoxanide (see non-patent document 6) is known to inhibit replication of japanese encephalitis virus. Furthermore, niclosamide and the like (see non-patent document 7) are known to inhibit replication of the Zika virus. Hemin (see non-patent document 8) can inhibit replication of the Zika virus in vitro. In addition, ribavirin (see non-patent documents 9 and 10), Lucidone (non-patent document 11), and a specific indole derivative (non-patent document 12) are known to inhibit replication of dengue virus.

It is known that 5-aminolevulinic acid (5-ALA) is present in mitochondria of cells, is biosynthesized from mitochondria in animals, binds to an iron component to become a component essential for metabolism such as heme and a raw material of cytochrome, and is biosynthesized from chloroplasts in plants, binds to magnesium to become chlorophyll, which is an essential component for photosynthesis. Further, patent document 1 discloses a method for producing 5-ALA phosphate, and describes that a method for synthesizing 5-ALA hydrochloride is known. Further, patent document 2 discloses a method for producing 5-ALA using a microorganism.

Patent document 3 describes an agent for preventing and/or treating influenza virus infection, which contains 5-ALA. Patent document 4 describes an agent for preventing and/or treating viral infections containing 5-ALA, and the viruses to be treated include hepatitis B virus, hepatitis C virus, ibola virus, aids virus, herpes simplex virus, varicella zoster virus and smallpox virus. Patent document 5 describes a method for treating viral infection in a subject, which comprises administering 5-ALA to the subject, allowing virus-infected cells to accumulate protoporphyrin, and destroying the cells by applying red light to the cells. Patent document 6 describes the use of 5-aminolevulinic acid (5-ALA) hexyl ester or a salt thereof that can be used as a medicament in the manufacture of a composition for animal-oriented photodynamic therapy (PDT) for the treatment of viral infections in the vaginal cavity, the cervical region, or the lining of the uterus. However, in patent documents 3 to 6, no study has been made on the influence of 5-ALA on viruses of the genus Flaviviridae of the family Flaviviridae.

Non-patent documents 13, 14 and 15 disclose that the amount of heme oxygenase-1 (HO-1) in cells can be increased by using 5-ALA and Sodium Ferrous Citrate (SFC) in combination. Further, non-patent document 14 discloses that 5-ALA alone can induce HO-1 expression. Non-patent document 16 reports that HO-1 has antiviral activity against various viruses including dengue virus. Non-patent document 8 discloses that hemin having HO-1-inducing activity can reduce replication of Zika virus in vitro. Non-patent document 11 discloses that ganoderma lucidum ketone having antiviral activity against dengue virus increases intracellular HO-1.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2006 and 182753

Patent document 2: japanese patent laid-open publication No. 2005-333907

Patent document 3: international publication No. 2014/013664

Patent document 4: international publication No. 2016/163082

Patent document 5: japanese Kohyo publication No. 2000-510123

Patent document 6: japanese patent laid-open No. 2014-94963

Non-patent document

Non-patent document 1: Sang-Im Yun and Young-Min Lee, Japanese encepha litis The viruses and vacines, Human Vaccines & Immunotherapeutics,10:2,263.279; february 2014, p.263-279

Non-patent document 2: Yi-Lin Cheng, et al, Activation of Nrf2 by the dense viruses a, an increase in CLEC5A, which industries TNF-alpha production by monofacial phases, Scientific Reports,26August 2016,6: 32000; DOI 10.1038/srep32000(2016)

Non-patent document 3: oumar face, et al, Molecular Evolution of Zika Virus producing Its expression in the 20th Century, PLOS targeted genomic Diseases, January 2014, Volume 8, Issue 1, e2636

Non-patent document 4: shichuan zhihong and Xiaoxie Ying II, virus, volume 61, No. 2, 2011, p.221-238

Non-patent document 5: veaceslav Boldescu, et al, Broad-specific agents for flaviviral infections, dengue, Zika and beyond, NATURE REVIEWS,5May 2017, VOLUME 16, AUGUST 2017, p.565-586

Non-patent document 6: zixue Shi, et al, Nitazoxanide inhibitors the regenerative medicine in Japanese advanced cells and in a mouse model, Virology Journal,2014Jan 23,2014; 11:10

Non-patent document 7: miao Xu, et al, Identification of small-molecule inhibitors of Zika virus introduction and induced nuclear cell defect a drug repurposing screen, NATURE MEDICINE,29August 2016, VOLUME 22, NUMBER 10, OCTOBER 2016, p.1101-1107

Non-patent document 8: hanxia Huang, et al, Nrf2-dependent indication of input host default via enzyme-1 inhibitors Zika viruses replication, virology.2017March,503,1-5

Non-patent document 9: MARKLAND, et al, Broad-Spectrum Activity of the IMP Dehydrogenic Inhibitor VX-497, a company with Ribavirin and Demonstroration of antibiotic adsorption with Alpha Interfer, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr.2000, Vol.44, No.4, p.859-866

Non-patent document 10: ratree takhampunra, et al, Inhibition of dentistry reproduction by mycophenolic acid and ribavirin, Journal of General Virology (2006),87, p.1947-1952

Non-patent document 11: Wei-Chun Chen, et al, Lucidone suppenses densue visual reproduction of the instruction of the enzyme-1, VIRULEN CE,2018, VOL.9, NO.1, p.588-603

Non-patent document 12: dorothee Bardiot, et al, Discovery of oil Deriva properties as Novel and Point Dengue Virus Inhibitors, Journal of Medicinal Chemistry, August 27,2018, 61(18), DOI:10.1021/acs jmedch em.8b00913, p.8390-8401

Non-patent document 13: mingyi Zhao, et al, 5-aminolytic acid combined with sodium ferris citrate H2O2-induced cardiac pathophyte via activation of the MAPK/Nrf2/HO-1path, American Journal of physiolocytic cell physioiogy (2015),308: C665-C672

Non-patent document 14: kei Saito, et al, Dynamics of aberration, metablism, and evolution of 5-aminolevulinic acid in human endogenous Caco-2cells, Biochemistry and Biophysics Reports,13July 2017,11:105-

Non-patent document 15: hidenori Ito, et al, Oral administration of 5-amino levivulinic acid indexes of enzymes-1 expression in topical blood mono cells of health human subjects in combination with microorganisms, European Journal of pharmacy, 10May 2018,833:25-33

Non-patent document 16: espoza JA, Gonzalez PA, Kalergis AM.modulation of Antiviral by Heme ozone-1. Am J Pathol.2017 Mar; 187(3):487-493

Disclosure of Invention

With the increasing importance of flavivirus infection countermeasures, further preventive and/or therapeutic agents for flavivirus infection are needed. In particular, there is a need for further preventive and/or therapeutic agents against flavivirus infections, which have stronger anti-flavivirus effects than existing compounds such as chirotkatone. And there is a need for a preventive and/or Therapeutic agent for flavivirus infections that exhibits a wider difference in concentration between cytotoxicity and drug efficacy (i.e., Therapeutic Window) than existing compounds such as lucidone.

The present inventors have intensively studied further preventive and/or therapeutic agents for flavivirus infections, and as a result, they have found that 5-ALA, an ester thereof, or a salt thereof is extremely useful, and the present invention has been completed based on this finding.

That is, several embodiments of the present invention are as follows.

[1] A preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof.

[2] The preventive and/or therapeutic agent for flavivirus infection according to [1], wherein the flavivirus is dengue virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, or yellow fever virus.

[3] The preventive and/or therapeutic agent for flavivirus infection according to [1], further comprising an iron compound.

[4] The preventive and/or therapeutic agent for flavivirus infection according to [1], which does not require light irradiation.

[5] A method of preventing and/or treating a flavivirus infection comprising: administering to a subject a preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof.

[6] The method for preventing and/or treating flavivirus infection according to [5], wherein the flavivirus is dengue virus, Zika virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, or yellow fever virus.

[7] The method for the prevention and/or treatment of flavivirus infection according to [5], wherein the agent for the prevention and/or treatment of flavivirus infection further contains an iron compound.

[8] The method for preventing and/or treating flavivirus infection according to [5], wherein light irradiation is not required.

The preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof according to an embodiment of the present invention can inhibit replication of flavivirus, thereby having an effect of preventing and/or treating flavivirus infection. The preventive and/or therapeutic agent for flavivirus infection comprising at least one member selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof according to one embodiment of the present invention exerts a stronger anti-flavivirus effect than that of columbin which is considered to exert an antiviral effect via HO-1. In addition, the preventive and/or therapeutic agent for flavivirus infections comprising at least one member selected from the group consisting of 5-aminolevulinic acid (5-ALA) and esters thereof, and salts thereof according to one embodiment of the present invention has an advantageous effect of exhibiting a wider difference in concentration between cytotoxicity and drug efficacy (i.e., a wider therapeutic window) as compared with ganoderma lucidum ketone.

Drawings

FIG. 1 is a graph showing the effect of 5-ALA in inhibiting dengue virus replication when cells were treated with 5-ALA prior to infection.

FIG. 2 is a graph showing the effect of 5-ALA in inhibiting dengue virus replication when cells were treated with 5-ALA simultaneously with infection.

FIG. 3 is a graph showing the inhibitory effect of 5-ALA on dengue virus replication in the case of cells treated with 5-ALA 1 day after infection.

FIG. 4 is a graph showing the concentration-dependent inhibitory effect of 5-ALA on dengue virus replication in the case of cells treated with 5-ALA 1 day after infection.

FIG. 5 is a graph showing the concentration-dependent inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the case of cells treated with 5-ALA 1 day after infection.

FIG. 6 is a graph showing the inhibitory effect of 5-ALA on dengue virus replication in the case of cells treated with 5-ALA 7 days after infection.

FIG. 7 is a graph showing the inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the case of cells treated with 5-ALA 7 days after infection.

FIG. 8 is a graph showing the inhibitory effect of 5-ALA on dengue virus replication in the case of cells treated with 5-ALA 4 days after infection.

FIG. 9 is a graph showing the inhibitory effect of 5-ALA on dengue virus replication in the case of cells treated with 5-ALA 2 days after infection.

FIG. 10 is a graph showing the inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the case of cells treated with 5-ALA 4 days after infection.

FIG. 11 is a graph showing the inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the case of cells treated with 5-ALA 2 days after infection.

FIG. 12 is a graph showing the cytotoxicity of 5-ALA on non-infected cells treated with a 5-ALA solution for 2 days.

FIG. 13 is a graph showing the cytotoxicity of 5-ALA on non-infected cells treated with a 5-ALA solution for 4 days.

FIG. 14 is a graph showing the cytotoxicity of 5-ALA when non-infected cells were treated with a 5-ALA solution for 7 days.

FIG. 15 is a graph showing cytotoxicity of ganoderma lucidum ketone when non-infected cells were treated with ganoderma lucidum ketone solution for 7 days.

FIG. 16 is a graph showing the inhibitory effect of 5-ALA or columbin on dengue virus replication in the case of cells treated with 5-ALA or 40. mu.M columbin 7 days after infection.

FIG. 17 is a graph showing the inhibitory effect of 5-ALA or Ganoderma lucidum ketone on Japanese encephalitis virus replication in the case of cells treated with 5-ALA or 40. mu.M Ganoderma lucidum ketone 7 days after infection.

FIG. 18 is a photograph showing an optical microscope photograph of cells treated with 0.2mM 5-ALA or 40. mu.M columbin for 2 days in the experiments shown in FIGS. 16 and 17.

FIG. 19 is a graph showing the inhibitory effect of 5-ALA or columbin on dengue virus replication in the case of cells treated with 5-ALA or low dose of columbin 7 days after infection.

FIG. 20 is a graph showing the inhibitory effect of 5-ALA or columbin on Japanese encephalitis virus replication in the case of cells treated with 5-ALA or low dose of columbin 7 days after infection.

Fig. 21 is a schematic diagram showing timings of drug treatment, virus infection treatment, and the like in each example.

FIG. 22 is a graph showing the inhibitory effect of 5-ALA on dengue virus type 1 replication in the case of cells treated with 5-ALA 7 days after infection.

FIG. 23 is a graph showing the inhibitory effect of 5-ALA on dengue virus type 3 replication in the case of cells treated with 5-ALA 7 days post infection.

FIG. 24 is a graph showing the inhibitory effect of 5-ALA on dengue virus type 4 replication in the case of cells treated with 5-ALA 7 days after infection.

FIG. 25 is a graph showing the inhibitory effect of 5-ALA on Zika virus replication when cells were treated with 5-ALA 7 days after infection.

Detailed Description

One embodiment of the present invention is an agent for preventing and/or treating flavivirus infection, comprising at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof. Further, an embodiment of the present invention is use of at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, in a preventive and/or therapeutic agent for a flavivirus infection. In addition, another embodiment of the present invention is a composition for preventing and/or treating flavivirus infection comprising at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof.

In the present invention, 5-aminolevulinic acid (5-ALA) is a compound also called delta-aminolevulinic acid. In the present invention, "5-ALA or an ester thereof" means "5-ALA or a 5-ALA ester", and is represented by the following formula (I). In the present invention, the phrase "a salt thereof" in the phrase "5-ALA, an ester thereof, or a salt thereof" means a salt of 5-ALA or a salt of 5-ALA ester. Examples of the salt include, but are not limited to, acid addition salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, methylphosphoric acid, ethylphosphoric acid, phosphite, hypophosphite, nitrate, sulfate, acetate, propionate, tosylate, succinate, oxalate, lactate, tartrate, glycolate, mesylate, butyrate, valerate, citrate, fumarate, maleate, malate and the like, metal salts such as sodium salt, potassium salt, calcium salt and the like, ammonium salt, alkylammonium salt and the like.

In the above formula (I), R₁Is a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group. R₁When hydrogen is used, the formula (I) represents 5-ALA. R₁In the case of a straight-chain or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group, the above formula (I) represents a 5-ALA ester.

R₁The straight-chain or branched alkyl group shown in (1) is preferably an alkyl group having 1 to 18 carbon atoms, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an isopropyl group, a tert-butyl group, a n-pentyl group, a methyl group, an isopropyl group, a n-pentyl group, a methyl group, a n-pentyl group, a tert-pentyl group, and the like,Isopentyl, neopentyl, tert-pentyl, 2-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, ethylbutyl, n-heptyl, 2-methylhexyl, n-octyl, isooctyl, tert-octyl, 2-ethylhexyl, 3-methylheptyl, n-nonyl, isononyl, 1-methyloctyl, ethylheptyl, n-decyl, 1-methylnonyl, n-undecyl, 1-dimethylnonyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, and the like. Examples of the cycloalkyl group include, in addition to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, cycloalkyl groups having an alkyl substituent, for example, cycloalkyl groups having an alkyl substituent having 1 to 6 carbon atoms, for example, 3-methylcyclohexyl, 4-ethylcyclohexyl, and 2-methylcyclooctyl. The straight-chain or branched alkyl group is more preferably an alkyl group having 1 to 16 carbon atoms, and particularly preferably a methyl group, an ethyl group, an n-butyl group, an n-hexadecyl group or a 2-ethylhexyl group.

As R₁Examples of the aryl group in (1) include phenyl and naphthyl. The aryl group may be substituted with 1 to 3 substituents, for example: an alkyl group having 1 to 6 carbon atoms such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an n-hexyl group, a cyclopropyl group, a cyclobutyl group, a cyclohexyl group and the like, an alkoxy group having 1 to 6 carbon atoms such as a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy group, an isobutoxy group, a tert-butoxy group and the like, a halogen atom such as a hydroxyl group, an amino group, a nitro group, a cyano group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like, a carboxyl group and the like.

As R₁The aralkyl group shown in (1) is preferably composed of an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 20 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, and cyclohexyl, and examples of the aryl group having 6 to 20 carbon atoms include phenyl and naphthyl. Among the aralkyl groups, a benzyl group or a phenethyl group is preferable, and a benzyl group is particularly preferable. The aryl group of the aralkyl group may be substituted with 1 to 3 substituents as follows: the above-mentioned carbon number of 1 to 6An alkoxy group having 1 to 6 carbon atoms such as an alkyl group, a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy group, an isobutoxy group, or a tert-butoxy group, a halogen atom such as a hydroxyl group, an amino group, a nitro group, a cyano group, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, a carboxyl group, or the like.

In the preventive and/or therapeutic agent for flavivirus infection according to the embodiment of the present invention, at least one selected from 5-ALA, an ester thereof, or a salt thereof may be used as the active ingredient, and the active ingredient may be used alone or in combination of two or more. For example, the active ingredient used in the present embodiment may be any of 5-ALA, a 5-ALA ester, a 5-ALA salt, or a salt of a 5-ALA ester. Further, for example, a combination of salts of 5-ALA and 5-ALA ester may be used. At least one selected from the group consisting of 5-ALA, an ester thereof, and a salt thereof used in the present embodiment may be a purified substance, a roughly purified substance, or a synthesized mixture. In the present embodiment, it is preferable to use 5-ALA salt as the active ingredient, and it is more preferable to use 5-ALA hydrochloride and/or 5-ALA phosphate as the active ingredient. At least one selected from 5-ALA or an ester thereof, or a salt thereof for use in the present invention can be produced by a known method.

In the present invention, "flavivirus" refers to a virus classified into the genus flavivirus of the family flaviviridae. Examples of the flavivirus include dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, Murray Valley encephalitis virus, St.Louis encephalitis virus, Omsk hemorrhagic fever virus, tick-borne encephalitis virus, and the like. In one embodiment of the present invention, the flavivirus to be prevented and/or treated is a virus classified into the genus flavivirus of the family flaviviridae, but the flavivirus to be prevented and/or treated is preferably dengue virus, zika virus, japanese encephalitis virus, tick-borne encephalitis virus, west nile virus, or yellow fever virus, and more preferably dengue virus, zika virus, or japanese encephalitis virus.

In the present invention, "flavivirus infection" refers to a disease in which a subject such as a human or an animal other than a human is infected with flavivirus. Examples of the flavivirus infection diseases for humans include dengue fever, dengue hemorrhagic fever, and dengue shock syndrome caused by infection with dengue virus, Zika virus disease and congenital Zika virus infection caused by infection with Zika virus, Japanese encephalitis caused by infection with Japanese encephalitis virus, West Nile fever and West Nile encephalitis caused by infection with West Nile virus, yellow fever caused by infection with yellow fever virus, Murray Valley encephalitis caused by infection with Murray Valley encephalitis virus, St.Louis encephalitis caused by infection with St.Louis encephalitis virus, St.Omsk hemorrhagic fever caused by infection with St.Omsk hemorrhagic fever virus, and tick-borne encephalitis caused by infection with tick-borne encephalitis virus. Examples of the flavivirus infections of non-human animals include, but are not limited to, encephalitis caused by horse infection with japanese encephalitis virus, abortion caused by infection with pregnant pig, encephalitis caused by horse infection with west nile virus, abortion caused by infection with pregnant sheep, and the like.

In the present invention, "prevention of flavivirus infection" refers to, for example, inhibition of the onset of flavivirus infection, i.e., complete inhibition of the onset or reduction of the incidence, but is not limited thereto. The "treatment of flavivirus infection" includes, for example, prevention, alleviation, and complete cure of severe flavivirus infection, but is not limited thereto. The "prevention and/or treatment" in the present invention means any of the following (1) to (3): (1) prevention, (2) treatment, (3) both prevention and treatment.

In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may be any of administration routes, dosage forms and compositions as long as it can prevent and/or treat infection administered to a subject. For example, in one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection includes oral administration, intravenous administration, intranasal administration, transdermal administration, inhalation administration, administration using suppository, and the like, but is not limited thereto, and may be administered in various routes. Preferably, the route of administration of the preventive and/or therapeutic agent for flavivirus infection is oral administration. In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may be in a form suitable for the administration route, for example, in the form of tablets, powders, capsules, elixirs, suspensions, emulsions, solutions, syrups, ointments, suppositories, patches, and the like. In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may be in the form of a food. In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may be in the form of a feed to animals other than humans.

In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection contains an iron compound. The iron compound is not particularly limited, and examples of the iron compound include organic salts and inorganic salts of iron, and complexes of iron with high-molecular compounds such as proteins. Examples of the organic salt of iron include citrates such as ferrous citrate, ferric sodium citrate, ferrous sodium citrate and ferric ammonium citrate, hydroxycarboxylic acid salts such as ferric malate, ferric sodium citrate succinate, ferric lactate, ferric tartrate and ferric glycolate, ferrous succinate, ferric acetate, ferric oxalate, ferric dextran, ferric gluconate, ferric sodium ethylenediamine tetraacetate, ferric potassium ethylenediamine tetraacetate, ferric ammonium ethylenediamine tetraacetate, ferric sodium diethylenetriamine pentaacetate, ferric potassium diethylenetriamine pentaacetate, ferric ammonium diethylenetriamine pentaacetate and ferric glycerophosphate. Examples of the inorganic salt of iron include iron oxide, iron chloride, iron nitrate, iron sulfate, ammonium iron sulfate, ferrous pyrophosphate, and ferric pyrophosphate. The iron compound may be iron-binding proteins such as lactoferrin iron and transferrin iron, and heme iron. The iron compound may be 1 kind, or a plurality of kinds may be used in combination. The iron compound is preferably sodium ferrous citrate.

In one embodiment of the present invention, the amount of the iron compound contained in the preventive and/or therapeutic agent for flavivirus infection is 1:0.05 to 1:5, preferably 1:0.1 to 1:1, in terms of the total molar amount of 5-aminolevulinic acid (5-ALA), an ester thereof, or a salt thereof, relative to the molar amount of the iron atom in the iron compound.

In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may contain various pharmaceutically or food hygienically acceptable carriers and additives, or various carriers and additives acceptable as a feed for non-human animals, and the like, as required. Examples of the various carriers and additives include excipients, lubricants, stabilizers, dispersants, binders, diluents, flavors, sweeteners, flavoring agents, and coloring agents. In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection may be a composition for preventing and/or treating flavivirus infection containing these essential components.

Another embodiment of the present invention is a method for preventing and/or treating a flavivirus infection, comprising: administering to a subject a preventive and/or therapeutic agent for flavivirus infection comprising at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof. In addition, another embodiment of the present invention is the use of at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, in a subject for the prevention and/or treatment of a flavivirus infection.

In the method for preventing and/or treating flavivirus infection according to the embodiment of the present invention, the target of the prevention and/or treatment of flavivirus infection is not particularly limited, and examples thereof include humans, non-human mammals, and birds.

In the method for preventing and/or treating flavivirus infection of the present invention, the amount of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof to be administered may be appropriately determined depending on the type of virus, the degree of symptoms, and the like. The frequency and duration of administration are not particularly limited.

In one embodiment of the present invention, the preventive and/or therapeutic agent for flavivirus infection is a preventive and/or therapeutic agent for flavivirus infection containing at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, and not requiring light irradiation. In another embodiment, the preventive and/or therapeutic agent for flavivirus infection is a preventive and/or therapeutic agent for flavivirus infection containing at least one selected from 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, and not subjected to light irradiation. In addition, in one embodiment of the present invention, the method for preventing and/or treating a flavivirus infection is a method for preventing and/or treating a flavivirus infection that includes the step of administering to a subject a flavivirus infection preventing and/or treating agent containing at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, and that does not require light irradiation. In another embodiment, the method for preventing and/or treating a flavivirus infection is a method for preventing and/or treating a flavivirus infection that includes the step of administering to a subject a preventive and/or therapeutic agent for a flavivirus infection that includes at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, without light irradiation. The "light irradiation" in these embodiments includes, for example, light irradiation used for photodynamic therapy (PDT) utilizing photosensitivity of protoporphyrin (protoporphyrin IX) accumulated in cells.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the scope of the examples.

Examples

The inhibitory effect on the replication of dengue virus (DENV) or Japanese Encephalitis Virus (JEV) when cells are treated with an agent such as 5-ALA before, simultaneously with, or after infection with flavivirus is confirmed according to the following procedure.

(1) Vero cells were seeded in 24-well plates at 37 ℃ with 5% CO on the Day before infection (Day0)₂And (5) culturing. The culture medium was MEM + 10% fetal bovine serum +10mM non-essential amino acids +1000U/ml penicillin + 100. mu.g/ml streptomycin. In experiments with pre-infection treatment with agents, the study subject agent was added to the culture at this point. In all experiments, 5-aminolevulinic acid hydrochloride was used as 5-ALA, Sodium Ferrous Citrate (SFC) as the iron-containing compound, 300 μ M ribavirin or various concentrations of lucidone were used as the positive control, and water was used as the negative control.

(2) On the Day of infection (Day1), the culture medium of the prepared plate was removed, and cells were inoculated with dengue virus type 2 New guineac strain (DENV2 NGC) or japanese encephalitis virus zhongshan strain (JEV Nakayama) at MOI (multiplicity of infection) 2 and MOI (MOI) 5, respectively (the amount of inoculum was 200 μ l). After allowing the virus to adsorb to the cells for 1 hour, phosphorus was addedThe cells were washed with acid buffered saline (PBS) and the inoculum was completely removed. 1ml of maintenance medium (MEM + 1% fetal bovine serum +10mM non-essential amino acids +1000U/ml penicillin + 100. mu.g/ml streptomycin +1mM HEPES) was added to the plate, and the mixture was incubated at 37 ℃ with 5% CO₂Cells were cultured under the conditions described above. In an experiment in which a drug is added simultaneously with infection, the drug to be studied is added to the maintenance culture solution at this time.

(3) After 24 hours (Day2), the culture medium was collected from the plate, and 1ml of the culture medium for maintenance was added to the plate again at 37 ℃ with 5% CO₂Cells were cultured under the conditions described above. In experiments in which a drug is added after infection, the drug to be studied is added at a predetermined time after infection. The recovered culture broth was centrifuged at 3000rpm for 5 minutes, and the supernatant was cryopreserved.

(4) The culture medium was recovered and added every 24 hours thereafter. Note that 2 wells were used for all conditions in the infection experiment.

The infectious titer of the virus was determined according to the following procedure.

(1) On the day before the experiment, Vero cells were seeded in 24-well culture plates at 37 ℃ with 5% CO₂And (5) culturing.

(2) The titer-measuring sample was thawed, and a 10-fold stepwise dilution series was prepared using a maintenance culture medium.

(3) The culture solution of the prepared plate was removed, and the dilution series prepared in step (2) was inoculated at 200. mu.l each. After 1 hour of adsorption, the inoculum was removed from the plate, 1ml of 1% methylcellulose (MEM + 1% methylcellulose + 1% fetal bovine serum) was added to the plate, and the plate was incubated at 37 ℃ with 5% CO₂Let stand still (14 days for DENV and 10 days for JEV).

(4) After removing the methylcellulose solution from the plate and washing with PBS, 1ml of crystal violet (0.05% crystal violet 20% methanol) was added to the plate and allowed to stand at room temperature for 10 minutes. The crystal violet solution was removed from the plate, washed with water and the number of plaques after drying was counted. The virus titer in the culture broth was calculated from the number of plaques.

MTT assay

(1) On the day before the experiment, Vero cells were seeded in 96-well culture plates at 37 ℃ C. 5% CO₂And (5) culturing.

(2) A2-fold stepwise dilution series (. about.0.8 mM) of 5-ALA was prepared using a maintenance medium (MEM + 1% fetal bovine serum +10mM non-essential amino acids +1000U/ml penicillin + 100. mu.g/ml streptomycin +1mM HEPES). Water was used as a negative control. A2-fold dilution series (160. mu.M) was prepared in the same manner for Ganoderma lucidum ketone.

(3) Removing the culture medium from the prepared plate, and adding the 5-ALA solution or the Ganoderma lucidum ketone solution prepared in (2) to the plate. 100 μ l per 1 well, 3 wells per 1 condition were used.

(4) The old culture medium was removed from the plate every 24 hours, and a new 5-ALA solution or a Ganoderma lucidum ketone solution was added to the plate for culture.

(5) After culturing for 2 days, 4 days or 7 days with 5-ALA solution or 7 days with Ganoderma lucidum ketone solution, the cells were washed with PBS 2 times, added with 100. mu.l of MTT solution, and cultured at 37 ℃ with 5% CO₂The cells were incubated for 3 hours. As the MTT solution, a solution prepared by dissolving thiazole blue tetrazolium bromide in an equivalent amount of a mixture (10mg/ml) of 2 XMEM and sterile water and subjecting the solution to filtration sterilization was used (in real time).

(6) The culture medium was removed from the plate, and 100. mu.l of isopropanol was added to the plate, followed by shaking at room temperature for about 5 minutes.

(7) The absorbance at 570nm was measured, and the percentage of absorbance with respect to the absorbance in the 0 mM-treated well was calculated.

Fig. 21 illustrates the timing of drug treatment, virus infection treatment, and the like in each of the following examples.

Example 1

The inhibitory effect of dengue virus replication in pre-infection drug treatment was studied.

The agents used were 5-ALA (1mM), 5-ALA (1mM) + SFC (0.25mM) (i.e.5-ALA in combination with SFC), SFC (0.25mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). The titer of the progeny virus released from the cells treated with the agent was evaluated and is shown in log (PFU/ml) (PFU: plaque forming unit) in FIG. 1. As shown in FIG. 1, 5-ALA (1mM) and 5-ALA (1mM) + SFC (0.25mM) showed no inhibitory effect on dengue virus replication in the case of administration prior to infection.

Example 2

The inhibitory effect of dengue virus replication in drug treatment concurrent with infection was studied.

The agents used were 5-ALA (1mM), 5-ALA (1mM) + SFC (0.25mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 1 Day concurrent with infection (Day 1). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) (PFU: plaque forming unit) in FIG. 2. As shown in FIG. 2, 5-ALA (1mM) and 5-ALA (1mM) + SFC (0.25mM) showed inhibitory effects on dengue virus replication in the case of simultaneous treatment with infection. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

Example 3

The inhibitory effect of dengue virus replication in drug treatment after infection was studied.

The agents used were 5-ALA (1mM), 5-ALA (1mM) + SFC (0.25mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 1 Day from 1 Day after infection (Day 2). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) (PFU: plaque forming unit) in FIG. 3. As shown in FIG. 3, 5-ALA (1mM) and 5-ALA (1mM) + SFC (0.25mM) showed inhibitory effects on dengue virus replication in the case of post-infection treatment. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

Example 4

The dose-dependent inhibitory effect of dengue virus replication in drug treatment after infection was studied.

The agents used were 5-ALA (1mM), 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 1 Day from 1 Day after infection (Day 2). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 4. As shown in FIG. 4, 5-ALA showed the inhibitory effect of dengue virus replication dose-dependently at a concentration of 0.05mM to 1mM in the case of post-infection treatment. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

Example 5

The inhibitory effect of japanese encephalitis virus replication in drug treatment after infection was investigated.

The agents used were 5-ALA (1mM), 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 1 Day from 1 Day after infection (Day 2). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 5. As shown in FIG. 5, 5-ALA in the case of post-infection treatment showed inhibitory effect of Japanese encephalitis virus replication in a dose-dependent manner at a concentration of 0.05mM to 1 mM. By removing the agent from the culture, the inhibitory effect disappears with the passage of time, but the inhibitory effect is more durable than in the case of the dengue virus of example 4.

Example 6

The inhibitory effect of dengue virus replication in drug treatment 7 days after infection was studied.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 6. As shown in FIG. 6, treatment with 5-ALA (0.2mM) until Day7 showed an increased inhibitory effect on dengue virus replication with the passage of the treatment period. Treatment with 5-ALA (0.2mM) resulted in a stronger inhibitory effect on dengue virus replication than did ribavirin (300. mu.M). In Day8, the inhibitory effect on dengue virus replication was reduced by treatment with 5-ALA (0.2 mM). One of the causes of the reduction of the inhibitory effect is the appearance of 5-ALA-resistant viruses. The reduction in inhibitory effect caused by continued treatment with the agent was also found in ribavirin treatment.

Example 7

The inhibitory effect of japanese encephalitis virus replication in drug treatment 7 days after infection was investigated.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 7. As shown in FIG. 7, treatment with 5-ALA (0.2mM and 0.05mM) showed inhibitory effects on Japanese encephalitis virus replication. Treatment with 5-ALA (0.2mM) resulted in a stronger inhibitory effect on Japanese encephalitis virus replication than with ribavirin (300. mu.M). It was found that the inhibition effect of the replication of Japanese encephalitis virus decreased by the continuous treatment with the drug, and one of the causes is thought to be the occurrence of drug-resistant Japanese encephalitis virus. Based on the results of FIG. 7, it is believed that 5-ALA delayed the appearance of resistant virus compared to ribavirin.

Example 8

The inhibitory effect of dengue virus replication in the 4 days post-infection drug treatment was studied.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 4 days from 1 Day after infection (from Day2 to the end of Day 6). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 8. As shown in FIG. 8, treatment with 5-ALA (0.2mM) showed inhibitory effect of dengue virus replication. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

Example 9

The inhibitory effect of dengue virus replication in the drug treatment 2 days after infection was studied.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 2 days from 1 Day after infection (from Day2 to the end of Day 4). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 9. As shown in FIG. 9, treatment with 5-ALA (0.2mM) showed inhibitory effect of dengue virus replication. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time. From the results of FIGS. 6, 8 and 9, it is understood that the 5-ALA treatments for 4 days and 2 days showed a lower inhibitory effect on dengue virus replication than the 5-ALA treatment for 7 days.

Example 10

The inhibitory effect of japanese encephalitis virus replication in the drug treatment 4 days after infection was investigated.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 4 days from 1 Day after infection (from Day2 to the end of Day 6). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 10. As shown in FIG. 10, treatment with 5-ALA (0.2mM) showed inhibitory effect on Japanese encephalitis virus replication. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

Example 11

The inhibitory effect of japanese encephalitis virus replication in drug treatment 2 days after infection was investigated.

The agents used were 5-ALA (0.2mM), 5-ALA (0.05mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure). Treatment with the agent was 2 days from 1 Day after infection (from Day2 to the end of Day 4). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 11. As shown in FIG. 11, treatment with 5-ALA (0.2mM) showed inhibitory effect on Japanese encephalitis virus replication. By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time. From the results of FIGS. 7, 10 and 11, it is clear that 5-ALA treatment for 4 days and 2 days showed a lower inhibitory effect on Japanese encephalitis virus replication than 5-ALA treatment for 7 days.

Example 12

Cytotoxicity was investigated on non-infected cells treated with 5-ALA solution for 2 days according to the MTT assay described above. The results of cytotoxicity of 2-day treatment with 5-ALA solution are shown in FIG. 12.

Example 13

Cytotoxicity was investigated for 4 days of treatment with 5-ALA solution on non-infected cells according to the MTT assay described above. The results of cytotoxicity of 4 days of treatment with 5-ALA solution are shown in FIG. 13.

Example 14

Cytotoxicity was investigated on non-infected cells treated with 5-ALA solution for 7 days according to the MTT assay described above. The results of cytotoxicity of the treatment with 5-ALA solution for 7 days are shown in FIG. 14.

"Cont" in FIGS. 12, 13 and 14 is a negative control. For non-infected cells, the cytotoxicity of 5-ALA was confirmed dose-dependently. CC50 (concentration of 50% of cytotoxic agents in cells) in 7 days of treatment with 5-ALA exceeded 0.8 mM. The concentration of 0.8mM was higher than that of 0.2mM of 5-ALA in examples 6 and 7, which confirmed the inhibitory effect on viral replication. It is thus presumed that 5-ALA can prevent and/or treat flavivirus infection while suppressing side effects due to cytotoxicity.

Example 15

Cytotoxicity was investigated at 7 days of treatment with columbin according to the above MTT assay.

The results of cytotoxicity of non-infected cells treated with ganoderma lucidum ketone for 7 days are shown in fig. 15. "Cont" in FIG. 15 is a negative control. The cytotoxicity of the ganoderma lucidum ketone is confirmed in a dose-dependent manner. CC50 (concentration of 50% of cytotoxic agents in cells) was over 60 μ M in 7 day treatments with lucidone. For cells not infected with flavivirus, lucidone showed no cytotoxicity at a concentration of 40 μ M. Thus, in the following examples, the concentration at which anti-flavivirus activity of lucidone was studied was set to 40. mu.M.

Example 16

The inhibitory effect of dengue virus replication of 5-ALA in the drug treatment 7 days after infection was compared with 40. mu.M lucidone.

The used agents were 5-ALA (0.2mM), Ganoderma lucidum (40. mu.M) and water (Cont in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown in log (PFU/ml) in FIG. 16. As shown in FIG. 16, 40. mu.M of lucidone showed a stronger inhibitory effect against dengue virus replication than 0.2mM of 5-ALA.

Example 17

The inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the drug treatment 7 days after infection was compared with 40. mu.M columbine.

The used agents were 5-ALA (0.2mM), Ganoderma lucidum (40. mu.M) and water (Cont in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown as log (PFU/ml) in FIG. 17. As shown in figure 17 shows that 40 u M red ZhiKetone showed than 0.2mM 5-ALA strong inhibition of Japanese encephalitis virus replication effect.

Example 18

The cytotoxicity of Ganoderma lucidum ketone and 5-ALA on cells infected with flavivirus was studied.

In the experiments of example 16 and example 17, the state of cells when cells infected with dengue virus type 2 New Guinea C strain (DENV2 NGC) or Japanese encephalitis virus Zhongshan strain (JEV Nakayama) were treated with a drug for 2 days was observed with an optical microscope. Fig. 18 shows a microphotograph showing the state of each cell. In FIG. 18, NGC represents dengue virus type 2 New Guinea C strain (DENV2 NGC) and Nakayama represents Japanese encephalitis virus Zhongshan strain (JEV Nakayama). Cells infected with dengue virus and cells infected with Japanese encephalitis virus treated with lucidone both showed significant cell detachment. This indicates that 40. mu.M of lucidone is cytotoxic to cells infected with dengue virus and cells infected with Japanese encephalitis virus. In contrast, 0.2mM 5-ALA showed no detachment of cells infected with dengue virus and cells infected with Japanese encephalitis virus. That is, 0.2mM of 5-ALA was not considered cytotoxic to these infected cells.

It is thus presumed that the anti-flavivirus activity of 40. mu.M of lucidone shown in example 16 (i.e., FIG. 16) and example 17 (i.e., FIG. 17) reflects the cytotoxicity of lucidone to infected cells. That is, the chiroprione kills infected cells themselves which become hosts of dengue virus and japanese encephalitis virus, and the number of host cells for virus propagation in the experimental system is reduced. As a result, it is considered that the amount of virus present in the culture medium was reduced in the presence of Ganoderma lucidum ketone. In other words, it can be said that the results of examples 16 and 17 are not the results of correctly comparing the anti-flavivirus activity other than the cytotoxicity of Ganoderma lucidum ketone with the anti-flavivirus activity of 5-ALA. The anti-flavivirus activity of Ganoderma lucidum ketone and 5-ALA should be compared at a concentration where each agent is not cytotoxic. Thus, in examples 19 and 20 below, comparison of anti-flavivirus activity of columbione and 5-ALA was performed at concentrations that did not produce cytotoxicity.

Example 19

The inhibitory effect of dengue virus replication of 5-ALA in the drug treatment 7 days after infection was compared with 10. mu.M and 2.5. mu.M of lucidone.

The used agents are 5-ALA (0.2mM), Ganoderma lucidum ketone (10 μ M), Ganoderma lucidum ketone (2.5 μ M) and water (NC in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown as log (PFU/ml) in FIG. 19. As shown in FIG. 19, 5-ALA at 0.2mM showed a stronger inhibitory effect on dengue virus replication than 10. mu.M and 2.5. mu.M could be obtained.

Example 20

The inhibitory effect of 5-ALA on Japanese encephalitis virus replication in the drug treatment 7 days after infection was compared with 10. mu.M and 2.5. mu.M lucidone.

The used agents are 5-ALA (0.2mM), Ganoderma lucidum ketone (10 μ M), Ganoderma lucidum ketone (2.5 μ M) and water (NC in the figure). The treatment with the agent was 7 days from 1 Day after infection (from Day2 to the end of Day 8). The titer of progeny virus released from the treated cells was evaluated and is shown as log (PFU/ml) in FIG. 20. As shown in FIG. 20, 0.2mM of 5-ALA showed a stronger inhibitory effect on Japanese encephalitis virus replication than 10. mu.M and 2.5. mu.M of Ganoderma lucidum.

According to the results of the examples comparing chidonone with 5-ALA, the difference in concentration (i.e., treatment window) showing cytotoxicity and efficacy was as follows.

Anti-flavivirus activity of 5-ALA was confirmed above 0.2mM with cytotoxicity of at least 0.8mM (cytotoxicity of 5-ALA above 0.8mM was not investigated). Thus, the therapeutic window for 5-ALA is 0.2mM < 5-ALA < 0.8mM or more.

On the other hand, the anti-flavivirus activity of ganoderma lucidum ketone was confirmed at 40 μ M, and cytotoxicity was also confirmed at 40 μ M, thus there was no therapeutic window.

Therefore, 5-ALA is known to be useful as a preventive and/or therapeutic agent for flavivirus infections, compared to Ganoderma lucidum. As one of the mechanisms of action of anti-dengue virus activity of lucidone, it is known that HO-1 increases in the cell. Thus, even though it is known that the antiviral action of 5-ALA is increased by intracellular HO-1, it is clear that the usefulness of 5-ALA as a preventive and/or therapeutic agent for flavivirus infections is not predicted only by the increase in HO-1. It can be said that the advantageous effect of 5-ALA is an unexpected effect far exceeding the effect shown by Ganoderma lucidum ketone brought about by only the increase of HO-1.

Example 21

In the same manner as in example 6, the inhibitory effect on replication of each virus in drug treatment 7 days after infection was confirmed for 4 types of dengue virus, i.e., dengue virus type 1 Homoped strain (DENV1), dengue virus type 3 CH53489 strain (DENV3), dengue virus type 4 TVP360 strain (DENV4), and Zika virus PRVABC59 strain (ZKV). The inoculation on the Day of infection (Day1) was performed with MOI 0.2, MOI 0.01, MOI 0.05 on DENV1, DENV3, DENV4, ZKV, respectively. In addition, in the step (3) of measuring the infectious titer of the virus described above, the plates were incubated at 37 ℃ and 5% CO₂The period of lower standing was 10 days for DENV1, DENV3, and DENV4, and 6 days for ZKV. The drugs used were 5-ALA (0.2mM), 5-ALA (0.04mM), ribavirin (300. mu.M, Rib in the figure) and water (NC in the figure).

Example 21-1

Study of the inhibitory Effect of dengue Virus type 1 replication in drug treatment 7 days post infection

As shown in FIG. 22, treatment with 5-ALA (0.2mM) until Day9 showed an inhibitory effect of dengue virus type 1. Treatment with 5-ALA (0.2mM) resulted in a stronger inhibitory effect on dengue virus replication than did ribavirin (300. mu.M). By removing the agent from the culture medium, the inhibitory effect disappears with the passage of time.

(example 21-2)

Study of the inhibitory Effect of dengue Virus type 3 replication in drug treatment 7 days post infection

As shown in FIG. 23, treatment with 5-ALA (0.2mM) until Day7 showed an inhibitory effect of dengue virus type 3. Treatment with 5-ALA (0.2mM) resulted in a stronger inhibitory effect on dengue virus replication than did ribavirin (300. mu.M). In Day8, the inhibitory effect on dengue virus replication was reduced by treatment with 5-ALA (0.2 mM). One of the causes of the reduction of the inhibitory effect is the appearance of 5-ALA-resistant viruses. The reduction in inhibitory effect caused by continued treatment with the agent was also found in ribavirin treatment starting from Day 7.

(examples 21 to 3)

Study of the inhibitory Effect of dengue Virus type 4 replication in drug treatment 7 days post infection

As shown in FIG. 24, treatment with 5-ALA (0.2mM) until Day7 showed an inhibitory effect of dengue virus type 4. The effect of ribavirin (300. mu.M) was greater than the inhibitory effect of dengue virus replication by treatment with 5-ALA (0.2 mM). In Day8, the inhibitory effect on dengue virus replication was reduced by treatment with 5-ALA (0.2 mM). One of the causes of the reduction of the inhibitory effect is the appearance of 5-ALA-resistant viruses. The reduction in inhibitory effect caused by continued treatment with the agent was also found in ribavirin treatment.

(examples 21 to 4)

Study of inhibitory Effect of Zika Virus replication in drug treatment 7 days after infection

As shown in FIG. 25, the treatment with 5-ALA (0.2mM) showed inhibitory effects against Zika virus up to Day 8. The inhibition effect of Zika virus replication by treatment with 5-ALA (0.2mM) was stronger among Day5, 6 and 8 than that of ribavirin (300. mu.M). After Day9, the inhibitory effect on dengue virus replication was reduced by treatment with 5-ALA (0.2 mM). One of the causes of the reduction of the inhibitory effect is the appearance of 5-ALA-resistant viruses. The reduction in inhibitory effect caused by continued treatment with the agent was also found in ribavirin treatment.

Industrial applicability

The agent for preventing and/or treating flavivirus infection containing at least one selected from the group consisting of 5-aminolevulinic acid (5-ALA) or an ester thereof, or a salt thereof, which is an embodiment of the present invention, and the method for preventing and/or treating flavivirus infection, which is an embodiment of the present invention, are useful for the prevention and/or treatment of flavivirus infection.