WO2018230674A1

WO2018230674A1 - Antiviral or virus-inactivating agent

Info

Publication number: WO2018230674A1
Application number: PCT/JP2018/022821
Authority: WO
Inventors: 小山　一; 池田　敬子
Original assignee: 公立大学法人和歌山県立医科大学
Priority date: 2017-06-16
Filing date: 2018-06-15
Publication date: 2018-12-20
Also published as: JP2020128342A

Abstract

The purpose of the present invention is to provide an antiviral agent which has a higher safety to living organisms and is appropriately applicable not only to articles but also to living organisms. An antiviral agent which comprises at least one member selected from the group consisting of a compound represented by general formula (1) such as N-acetyl tryptophan, a salt thereof and a solvate of the same.

Description

Antiviral or virus inactivating agent

The present invention relates to an antiviral or virus inactivating agent or the like.

Many viral diseases such as influenza, measles and chickenpox are easily transmitted by viruses attached to familiar items, droplet infection, and droplet nuclear infection. For this reason, virus infection countermeasures are a major issue in modern society. There are two aspects of virus infection control: treatment and prevention. The importance of education does not wait for prevention, but isolation and disinfection are fundamental in terms of technique.

Disinfectant is indispensable for disinfection, but chlorine-based disinfectant (Patent Document 1) that is generally used has strong tissue damage and its usage is limited. In order to perform effective disinfection in a safe manner for users in old-age facilities and infant facilities, an effective disinfectant that is light in tissue damage is desired.

In general, disinfectants are limited in their application to the human body due to their tissue damage and cannot be used as therapeutic agents. However, if an inactivating agent with little tissue damage can be developed, it can be considered not only a safe disinfectant but also a prophylactic / therapeutic agent for viral infection on the body surface.

JP 2009-148519 A

An object of the present invention is to provide an antiviral agent having higher safety for living bodies and suitable not only for application to articles but also for living bodies. Preferably, the present invention provides an antiviral agent that has higher safety for living bodies, is suitable not only for application to articles but also for living bodies, and has a higher antiviral or virus inactivating action. Is an issue.

As a result of earnestly, systematically and exhaustively researched in view of the above problems, the present inventor is able to solve the above problems by using a compound represented by the general formula (1) such as N acetyltryptophan. I found it. The present inventor has completed the present invention by further research based on this finding.

That is, the present invention includes the following embodiments:
Item 1. General formula (1):

[Wherein R ¹ represents an alkyl group having 1 to 6 carbon atoms. R ² represents an alkylene group having 1 to 6 carbon atoms. R ³ represents a group derived from a nitrogen-containing heterocycle. ]
An antiviral or viral inactivating agent comprising at least one selected from the group consisting of a compound represented by the formula: salt thereof, and solvate thereof.

Item 2. The compound is represented by the general formula (1A):

[Wherein, R ¹ and R ² are the same as defined above. ]
Item 2. The antiviral or virus inactivating agent according to Item 1, which is a compound represented by:

Item 3. The compound is represented by the general formula (1AA):

[Wherein, R ¹ is the same as defined above. ]
Item 3. The antiviral or virus inactivating agent according to

Item

1 or 2, which is a compound represented by:

Item 4. Item 4. The antiviral or virus inactivating agent according to any one of Items 1 to 3, wherein the alkyl group represented by R ¹ has 1 to 3 carbon atoms.

Item 5. Item 5. The antiviral or virus inactivating agent according to any one of Items 1 to 4, which is used for application to a living body.

Item 6. Item 6. The antiviral or virus inactivating agent according to Item 5, which is a pharmaceutical, cosmetic, disinfectant or detergent.

Item 7. Item 5. The antiviral or virus inactivating agent according to any one of Items 1 to 4, which is used for application to articles.

Item 8. Item 8. The antiviral or virus inactivating agent according to Item 7, which is a disinfectant or a cleaning agent.

Item 9. General formula (1):

[Wherein R ¹ represents an alkyl group having 1 to 6 carbon atoms. R ² represents an alkylene group having 1 to 6 carbon atoms. R ³ represents a group derived from a nitrogen-containing heterocycle. ]
A method for producing a whole vaccine, comprising treating a virus with at least one selected from the group consisting of a compound represented by the formula: salt thereof, and solvate thereof.

Item 10. General formula (1):

[Wherein R ¹ represents an alkyl group having 1 to 6 carbon atoms. R ² represents an alkylene group having 1 to 6 carbon atoms. R ³ represents a group derived from a nitrogen-containing heterocycle. ]
A whole vaccine comprising at least one selected from the group consisting of a compound represented by the formula: salts thereof, and solvates thereof; and an inactivated virus.

According to the present invention, it is possible to provide an antiviral or virus inactivating agent that has higher safety for living bodies and is suitable not only for application to articles but also for living bodies. Moreover, the antiviral or virus inactivating agent of the present invention can exert an antiviral or viral inactivating action not only on enveloped viruses but also on non-enveloped viruses. Furthermore, since the active ingredient of the antiviral or virus inactivating agent of the present invention has higher safety against living bodies, it can be suitably used for the production of a whole vaccine consisting of whole virus particles.

The result of Test Example 1 is shown. Virus mixed with various concentrations of N-acetyltryptophan aqueous solution, herpes simplex virus type 1 (hereinafter referred to as HSV-1) and influenza virus at 30 ° C for 5 minutes, feline calicivirus (hereinafter referred to as FCV) and human rhinovirus ( In the following, HRV) shows the relative value of the residual virus infectivity when incubated at 37 ° C. for 20 minutes. The value when kept warm in a solution containing no N-acetyltryptophan is 1. For HRV only, use 10 mM citrate buffer (pH 6.0). ○: HSV-1 F strain; △: Influenza virus A0PR8 strain; □: FCV; ◇: HRV The result of Test Example 2 is shown. Each type and subtype of influenza virus is mixed in various concentrations of N-acetyltryptophan aqueous solution, and the relative values of the residual virus infectivity when incubated at 30 ° C. for 10 minutes are shown. The value when kept warm in a solution containing no N-acetyltryptophan is 1. ○: Influenza A virus A0PR8 strain (H1N1 subtype); △: Influenza A virus A / Aichi / 68 strain (H3N2 subtype); □: Influenza B virus B / Tokyo strain The result of Test Example 3 is shown. HSV-1 F strain (○, ●) or influenza virus A0PR8 strain (△, ▲) was mixed with various concentrations of N-acetyltryptophan aqueous solution, and the relative value of the residual virus infectivity when incubated at 30 ° C for 5 minutes was calculated. Show. In order to achieve a final concentration of 0.25% in the reaction solution, a concentrated bovine serum albumin (BSA) solution (10%) was added to the virus solution in advance (●, と ▲) and a solvent ( Two types of virus solutions were used, one with phosphate buffered saline (PBS) added (○, △). The value when kept warm in a solution containing no N-acetyltryptophan was taken as 1. The result at the time of using influenza virus in the test example 4 is shown. Shows the relative value of residual virus infectivity when influenza virus A0PR8 strain is added to a reaction solution in which N-acetyltryptophan at the indicated concentration is dissolved in 10 mM citrate buffer adjusted to various pH and incubated at 30 ° C for 5 minutes. . ○: pH 7.0; △: pH 6.0; □: pH 5.8; ●: pH 5.5; ▲: pH 5.2; ■: pH 5.0. The value when kept in a phosphate buffered saline solution (hereinafter referred to as PBS) not containing N-acetyltryptophan was set to 1. The result at the time of using a feline calicivirus in the test example 4 is shown. The relative value of the residual virus infectivity when feline calicivirus was added to a reaction solution in which N-acetyltryptophan at the indicated concentration was dissolved in 10 mM citrate buffer adjusted to various pH and incubated at 37 ° C. for 20 minutes is shown. ○: pH 7.0; △: pH 6.0; □: pH 5.8; ●: pH 5.5; ▲: pH 5.2; ■: pH 5.0. The value when kept warm in PBS without N-acetyltryptophan was taken as 1. The result of Test Example 5 is shown. The relative value of the residual virus infectivity when HSV-1 is added to a reaction solution prepared by dissolving N acetyltryptophan in 5.0 mM in various buffer solutions adjusted to the indicated pH and kept at 30 ° C. for 5 minutes is shown. ◯: 10 mM mM phosphate buffer; Δ: 10 mM mM acetate buffer. The value when kept warm in PBS without N-acetyltryptophan was taken as 1. The result of Test Example 6 is shown. The relative values of the residual virus infectivity when the influenza virus A0PR8 strain was added to 10 mM citrate buffer (pH 5.0) containing the indicated concentration of N-acetyltryptophan and incubated at various temperatures for 5 minutes are shown. ○: 0 ° C; △: 20 ° C; □: 25 ° C; ◇: 30 ° C; ▲: 34 ° C; ■: 37 ° C. The residual infectivity titer was 1 when incubated at 37 ° C for 5 minutes in PBS containing no N-acetyltryptophan. The result at the time of using the herpes simplex virus in the test example 7 is shown. After infecting Vero cells with herpes simplex virus type 1 F at 10 infectious units per cell, the relative value of virus yield after overnight incubation of these virus-infected cells in a culture medium containing various concentrations of N-acetyltryptophan Show. ○: N-acetyltryptophan-containing culture solution; Δ: control culture solution (containing methanol as solvent). The virus yield value in a culture medium containing no N-acetyltryptophan was taken as 1. The result at the time of using influenza virus in the test example 7 is shown. Shows the relative value of virus yield after infecting MDCK cells with 4 infectious influenza virus A / Aichi strains per cell and then cultivating the virus-infected cells overnight in a medium containing the indicated concentration of N-acetyltryptophan. . ○: N-acetyltryptophan-containing culture solution; Δ: control culture solution (containing methanol as solvent). The virus yield value in a culture medium containing no N-acetyltryptophan was taken as 1.

In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

1． Antiviral or virus inactivating agent The present invention, as one aspect, contains at least one selected from the group consisting of a compound represented by the general formula (1), a salt thereof, and a solvate thereof, The present invention relates to a virus or a virus inactivating agent (in this specification, sometimes referred to as “agent of the present invention”). This will be described below.

1-1. The compound represented by the general formula (1) has the following general formula (1).

R ¹ represents an alkyl group having 1 to 6 carbon atoms. The alkyl group represented by R ¹ includes any of linear or branched (preferably linear). The number of carbon atoms of the alkyl group is preferably 1 to 4, more preferably 1 to 3, further preferably 1 to 2, and still more preferably 1. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n -Hexyl group, 3-methylpentyl group and the like.

R ² represents an alkylene group having 1 to 6 carbon atoms. The alkylene group represented by R ² includes any of linear or branched (preferably linear). The number of carbon atoms of the alkylene group is preferably 1 to 4, more preferably 1 to 3, further preferably 1 to 2, and still more preferably 1. Specific examples of the alkylene group include methylene group, ethylene group, n-propylene group, isopropylene group, n-butylene group, isobutylene group, n-hexylene group and the like.

R ³ represents a group derived from a nitrogen-containing heterocycle (a group obtained by removing one hydrogen atom (preferably a hydrogen atom bonded to a carbon atom) from the nitrogen-containing heterocycle). The group derived from the nitrogen-containing heterocycle represented by R ³ is not particularly limited, and examples thereof include heteroaryl groups having 3 to 20, preferably 3 to 12, and more preferably 7 to 11 ring members. . The number of nitrogen atoms contained in the group derived from the nitrogen-containing heterocycle is, for example, 1 to 3, preferably 1 to 2, and more preferably 1. The group derived from the nitrogen-containing heterocycle is preferably a group having —NH— in the ring. The group derived from the nitrogen-containing heterocycle is preferably a group that does not contain a heteroatom other than a nitrogen atom. The group derived from the nitrogen-containing heterocycle is, for example, monocyclic or polycyclic (bicyclic, tricyclic, etc.), and preferably bicyclic. The group derived from the nitrogen-containing heterocycle is preferably a nitrogen-containing heteroaryl group. Specific examples of the group derived from the nitrogen-containing heterocyclic ring include pyrrolyl group, pyridyl group, pyrrolidyl group, piperidyl group, imidazolyl group, pyrazolyl group, pyrazyl group, pyrimidyl group, pyridazyl group, piperazyl group, triazinyl group, oxazolyl group, Examples include isoxazolyl group, morpholyl group, thiazolyl group, isothiazolyl group, indolyl group, quinolyl group, isoquinolyl group, benzimidazolyl group, quinazolyl group, phthalazyl group, purinyl group, and pteridyl group. Among these, an indolyl group is preferable, and a 3-indolyl group is more preferable.

The compound represented by the general formula (1) is preferably the general formula (1A):

[Wherein, R ¹ and R ² are the same as defined above. ]
And more preferably a compound represented by the general formula (1AA)

[Wherein, R ¹ is the same as defined above. ]
The compound represented by these is mentioned.

The salt of the compound represented by the general formula (1) is not particularly limited, and any appropriate acceptable salt can be adopted depending on the application. As the salt, either an acidic salt or a basic salt can be employed. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid Organic salts such as salts, citrates, methanesulfonates, paratoluenesulfonates, and the like. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; calcium salts, magnesium Alkaline earth metal salts such as salts; salts with ammonia; morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono (hydroxyalkyl) amine, di (hydroxyalkyl) amine, tri (hydroxyalkyl) Examples thereof include salts with organic amines such as amines.

The compound represented by the general formula (1) or a salt thereof may be a solvate. As the solvent, an appropriate suitable solvent can be adopted depending on the application. Examples of the solvent include water, ethanol, glycerol, acetic acid and the like.

The compound represented by the general formula (1) can be synthesized by various methods, for example, according to or according to a known synthesis method. In addition, a commercially available product can be used as it is or appropriately modified as the compound represented by the general formula (1).

1-2. Uses Although the target virus of the agent of the present invention is not particularly limited, for example, influenza virus (for example, type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus , Mumps virus, arbovirus, RS virus, SARS virus, hepatitis virus (eg, hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, nipavirus, lisa virus, etc. Envelope virus (virus with envelope); adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackie virus, human parvovirus, encephalomyocarditis virus, polio Luz, non-enveloped viruses such as rhinovirus (virus non-enveloped), and the like. Among these, respiratory infection virus that spreads through droplets (droplets) and contact infection viruses that spread through contact with secretory fluid (mucus), and more preferably influenza viruses, herpes viruses, Examples include rhinovirus.

The agent of the present invention can be widely used in various fields requiring antiviral properties. The agent of the present invention can be used in various fields such as industry, washing, medical care, food, daily necessities and the like. The agent of the present invention is classified into a use applied to a living body and a use applied to an article described later.

1-2-1. Uses applied to living bodies Examples of uses applied to living bodies include pharmaceuticals, cosmetics, disinfectants, and cleaning agents. The application target in this case is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats and deer.

By applying the agent of the present invention to a living body, an antiviral effect and / or a virus inactivating effect can be exhibited at a site where the active ingredient comes into contact.

The form of the agent of the present invention is not particularly limited, and can take a form normally used in each application depending on the application of the agent of the present invention.

As a form, when the use is a medicine, for example, a patch (plaster, plaster, etc., tape (reservoir type, matrix type, etc.), cataplasm, patch, microneedle, etc.), ointment, external liquid (Liniments, lotions, etc.), sprays (external aerosols, pump sprays, etc.), creams, gels, eye drops, eye ointments, nasal drops, suppositories, rectal semisolids, enemas Formulation forms suitable for parenteral intake such as external preparation forms; tablets (including oral disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), pills, granules, Examples of dosage forms suitable for oral consumption (oral dosage forms) such as fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly And Use formulation are preferably exemplified.

As a form, when a use is cosmetics, a liquid agent, a gel agent, a cream agent, an ointment, a stick agent etc. are mentioned, for example.

As the form, when the use is a disinfectant or a cleaning agent, for example, liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate agent, capsule) , Film agents, kneaded materials, molten solids, waxy solids, elastic solids, and the like). For example, when applied to the oral cavity, more specifically, dentifrices (toothpaste, liquid dentifrice, liquid dentifrice, powder dentifrice, etc.), mouthwashes, coating agents, patches, mouth fresheners, food ( For example, chewing gum, tablet candy, candy, gummi, film, troche, etc.). Moreover, if it is a case where it applies to a nasal cavity, a spray type nasal drop etc. will be mentioned more specifically. In the case of application to the skin, soap, body soap, shampoo, rinse, spray agent and the like can be mentioned.

The agent of the present invention may further contain other components as necessary. The other components are not particularly limited as long as they are components that can be blended in, for example, pharmaceuticals, cosmetics, disinfectants, detergents, and the like. For example, bases, carriers, solvents, dispersants, emulsifiers, buffers, Stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents and the like can be mentioned.

The buffering agent is not particularly limited, and an appropriate suitable one can be adopted depending on the application. Preferred examples include phosphate buffer and acetate buffer.

The agent of the present invention is preferably used so that the pH becomes acidic when applied to a living body. From this viewpoint, the pH of the agent of the present invention is preferably, for example, less than 7, preferably 6 or less, more preferably 5.5 or less, and further preferably 5 or less. The lower limit of pH is not particularly limited, and is 1, 2, 3, or 4, for example.

The content of the active ingredient of the agent of the present invention depends on the type, use, usage mode, application target, application target state, and the like of the active ingredient, and is not limited to, for example, 0.000001 to 100% by weight, Preferably, it can be 0.01 to 50% by weight.

The amount of application (for example, administration, ingestion, inoculation, etc.) of the agent of the present invention is not particularly limited as long as it is an effective amount that exhibits a desired effect, and is generally 0.1 to 1 per day as the weight of the active ingredient. 1000 mg / kg body weight. The above dose is preferably administered once a day or divided into 2 to 3 times a day, and can be appropriately increased or decreased depending on age, disease state, and symptoms.

1-2-2. Uses applied to articles Examples of uses applied to articles include disinfectants and cleaning agents. The application object in this case is not particularly limited, and includes industrial products and raw materials used in various fields.

Specific examples of goods include OA equipment, home appliances, air conditioning equipment, vacuum cleaners, desks, chairs, sofas, benches, windows, straps, handles, seats, automatic ticket gates, automatic ticket vending machines, vending machines, doors, fences , Handrails, tableware, cooking utensils, packaging films, packaging bags, bottles, bottles, packaging packs, sinks, toilet bowls, stationery, books, shelves, toothbrushes, mirrors, air conditioning filters, masks, coats, jackets, pants, skirts, shirts, Knit shirt, blouse, sweater, cardigan, nightwear, underwear, underwear, diaper, supporter, socks, tights, stockings, hat, scarf, scarf, collar, stall, gloves, clothes lining, clothes lining, clothes Filling, work clothes, uniforms, school uniforms, curtains, net doors, futon mats, futon cotton, duvet covers, pillowcases, sheets, mats, cars Tsu door, towel, handkerchief, wall cloth, band-aids, bandages, and the like.

By applying the agent of the present invention to an article, an antiviral effect and / or a virus inactivating effect can be exhibited at a site where the active ingredient comes into contact. In addition, the site | part which an active ingredient contacts is not restricted to the site | part on an article | item, When the article | item is applied to the biological body etc., the site | part in a biological body is also included.

The dosage form of the agent of the present invention is not particularly limited, and can be appropriately selected according to its use. Examples of the dosage form include liquids such as liquids, emulsions, suspensions, dispersants, and aerosols; solid or semi-solids such as wettable powders, powders, granules, fine granules, and flowables.

The agent of the present invention may further contain other components as necessary. The other components are not particularly limited as long as they are components that can be blended in, for example, an article cleaning agent, disinfectant, etc. For example, a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer. , Excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents and the like.

The content of the active ingredient of the agent of the present invention depends on the type, use, usage mode, application target, application target state, and the like of the active ingredient, and is not limited to, for example, 0.000001 to 100% by weight, Preferably, it can be 0.001 to 50% by weight.

2． Whole vaccine The present invention, as one aspect, comprises at least one selected from the group consisting of a compound represented by the general formula (1), a salt thereof, and a solvate thereof, and an inactivated virus. The present invention relates to a vaccine (sometimes referred to herein as “the vaccine of the present invention”) and a method for producing the same.

The compound represented by the general formula (1), a salt thereof, and a solvate thereof are the same as those in the agent of the present invention.

The vaccine of the present invention can be produced by treating a virus with at least one selected from the group consisting of a compound represented by the general formula (1), a salt thereof, and a solvate thereof. it can. By this step, the virus is inactivated and becomes an active ingredient of the vaccine.

Although it does not restrict | limit especially as a virus, For example, influenza viruses (for example, A type, B type, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, herpes simplex virus, mumps virus, arbovirus, Envelope viruses such as RS virus, SARS virus, hepatitis virus (eg, hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, AIDS virus, rabies virus, hantavirus, dengue virus, nipah virus, lisa virus, etc. ); Adenovirus, Norovirus, Rotavirus, Human papillomavirus, Poliovirus, Enterovirus, Coxsackie virus, Human parvovirus, Encephalomyocarditis virus, Poliovirus, Rhinoui Non-enveloped viruses such as Rus (viruses without an envelope). Among these, Preferably, envelope virus is mentioned, More preferably, influenza virus, herpes virus, rhinovirus etc. are mentioned.

In the treatment of viruses, the agent of the present invention can also be used.

The mode of virus treatment is not particularly limited as long as the virus is inactivated. The well-known aspect which can be employ | adopted as a whole vaccine manufacturing method is employable.

In the treatment of viruses, other antiviral agents and / or virus inactivating agents can be used in combination as appropriate.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Materials and Methods The materials and methods of each experiment conducted in the following test examples are as shown below unless otherwise limited.

Materials and methods As viral envelope virus, influenza virus A / Aichi / 68 (H3N2) strain, A / PR8 / 34 (H1N1 ) strain, B / Tokyo strain, and herpes simplex virus type 1 strain F (hereinafter, HSV-1 and approximately) the Using. Feline calicivirus (hereinafter abbreviated as FCV) and human rhinovirus type 1 (hereinafter abbreviated as HRV) were used as non-enveloped viruses.

Influenza virus (Orthomyxoviridae) was originally a waterfowl digestive infection virus (transmitted by faecal infection), but in humans it was a respiratory infection virus transmitted by droplet infection and contact infection, and herpes simplex virus (Herpesviridae) is a body surface mucosal infection virus transmitted by contact infection. Human rhinovirus is also a Picornaviridae, but it is respiratory-infected and is the largest causative virus for human cold syndrome. Feline calicivirus (Caliciviridae) has almost the same viral particle structure as poliovirus. In cats, it is not a digestive organ infection but a respiratory infection virus, but it is also treated by the Ministry of Health, Labor and Welfare as a norovirus substitute virus because it is the Caliciviridae family like norovirus.

Materials and methods MDCK cells derived from cell canine kidney was used for the infectivity of measurement and growth experiments of the influenza virus. Vero cells derived from African green monkey kidney were used for measurement of HSV-1 infectious titer, and cat-derived CRFK cells or human-derived HeLa cells were used to measure infectious titers of FCV and HRV, respectively. Eagle minimum essential medium (MEM) containing 5% fetal bovine serum (FBS) was used for cell culture.

Materials and methods Quantification of virus infectivity titer The virus infectivity titer was determined by the plaque method except for HRV (HRV was determined by the 50% tissue culture infectious dose method). Dilute each virus sample 10-fold with Dulbecco's phosphate buffered saline (PBS) and inoculate 0.5 ml of this into monolayer culture cells grown to saturation in a 50 mm dish and slowly incubate for 1 hour at room temperature. Viral adsorption was performed by mechanical shaking. In order to suppress non-specific inactivation of the virus in the diluent, 0.1% bovine serum albumin (BSA) is used in the diluent for influenza virus, and 0.5% fetal calf serum (in the diluent for HSV-1 or FCV). FBS) was added.

After adsorbing the virus, the unadsorbed virus was removed by suction, and then the MDCK cells infected with influenza virus were HSV-1 or FCV in MEM containing 0.6% agar (Difco purified agar) and acetylated trypsin (6μg / ml). Vero cells infected with A. were cultured in MEM containing 0.5% FBS and 0.6% methylcellulose, respectively. Influenza virus, HSV-1, and FCV were cultured at 37 ° C, the former being cultured for 2 days and the latter being cultured for 2-3 days. After culturing, dishes containing infected cells were fixed and stained with a solution containing 10% formalin and 0.5% (w / v) crystal violet, and plaques were counted visually after washing and air drying.

Materials and methods Measurement of antiviral effects The effect of each sample on virus growth was performed as follows. Vero cells (when investigating the antiviral effect on HSV-1 or PV-1) or MDCK cells (when investigating the effect on influenza virus) in a 6-well dish (diameter 33 mm) until the entire bottom of the well is covered Monolayer culture was performed. Each virus was added to 4 to 10 cells per cell (PFU; infectious unit), and virus adsorption was performed on a rocker platform at room temperature for 60 minutes. After virus adsorption is completed, add 1.0 ml of MEM containing 0.1% BSA to each virus-infected cell in each well of a 6-well dish as a culture solution, and change the amount of sample solution added to each well to obtain various sample concentrations. After being added to the culture solution, the cells were cultured for the time necessary for complete growth of each virus (16 to 28 hours for HSV-1; 12 to 18 hours for influenza virus).

For the quantification of the progeny virus produced, a part of the culture supernatant was taken in the case of influenza virus, and the amount of infectious virus released therein was measured by the plaque method. In the case of HSV-1, the infected cells are frozen and thawed twice with the culture solution at -80 ° C, the infected cells are crushed under mild conditions, and the intracellular virus is also released outside the cell. Each infectious virus was quantified by the plaque method as the amount of total progeny virus.

The degree of inhibition of virus growth was produced in the culture solution containing the amount of sample solution at each concentration when the amount of infectious progeny virus produced when the sample was not added to the culture solution of infected cells was 1. It was expressed as a relative ratio of the amount of progeny virus.

Materials and methods Measurement of virus inactivation effect (virucidal effect) Each sample solution was added to an Assist tube and ice-cooled, and the virus solution was added to the sample so that the amount was 1/19 of the sample. The well-mixed sample-virus mixture is allowed to stand at 30 ° C for 30 minutes, then immediately diluted 10-fold with cold virus dilution (PBS with serum or BSA added depending on the test virus species), and infection in each dilution Sex virus amount was measured by plaque method.

The degree of inactivation is the amount of residual infectious virus when kept in the sample solution at each concentration, and the amount of residual infectious virus in the sample kept warm using virus dilution instead of sample solution is 1. Expressed as a relative ratio.

Test Example 1. Inactivation of various viruses by N-acetyltryptophan Residual virus infection when the virus is mixed in an aqueous solution or citrate buffer containing various concentrations of N-acetyltryptophan (hereinafter abbreviated as N-AcTrp) and kept at a predetermined temperature The titer (infectious viral load) was examined. The results are shown in FIG.

As shown in FIG. 1, a significant decrease was observed depending on the concentration of N-AcTrp compared to the value when incubated in a control solution containing no N-AcTrp. The degree of virus inactivation was prominent in enveloped virus, highest in influenza virus, and inactivated to 3 mM or below the detection limit (10 ^-5 ). Although it was necessary to increase the incubation temperature and incubation time, the non-enveloped viruses HRV and FCV were also inactivated by N-AcTrp.

Test Example 2. Inactivation of influenza virus N-AcTrp showed a strong inactivation action against influenza virus, and inactivated non-enveloped viruses, which are generally known to be difficult to disinfect, such as HRV and FCV, which are respiratory infections. Therefore, we examined the inactivating effect of N-AcTrp on other types and subtypes of influenza viruses that are of great social interest as respiratory infection viruses. The results are shown in FIG.

As shown in Figure 2, all influenza virus species (two subtypes of influenza A virus, (H1N1 subtype and H3N2 subtype)) that are known to infect humans as the causative virus of seasonal influenza, In addition, a remarkable inactivation effect was observed in influenza virus B).

Test Example 3. Virus inactivation in the presence of protein It is known that the inactivation ability of many virus inactivating active substances is clearly reduced by the coexistence of proteins even in a very small amount. Thus, we investigated how much protein N-AcTrp suffers from protein interference. Highly sensitive influenza virus and HSV-1 were used as test viruses. The results are shown in FIG.

As shown in FIG. 3, in any virus, inactivation by N-AcTrp is greatly inhibited in the presence of 0.25% bovine serum albumin (BSA; bovine serum albumin) compared to the case without protein. As shown in HSV-1, when N-AcTrp concentration is increased, it is inactivated to below the detection limit even in the presence of BSA. Considering BSA concentration, the degree of inhibition received from BSA is light, and N-AcTrp is compared It is characterized by being less susceptible to protein interference.

Similar results were obtained when human immunoglobulin protein was used instead of BSA.

Test Example 4. Effect of pH on virus inactivation of N-AcTrp Regarding the effect of pH on virus inactivation by N-AcTrp, the most sensitive influenza virus (envelope virus) and the least sensitive FCV (non-enveloped virus) It investigated using. FIG. 4 shows the results with influenza virus, and FIG. 5 shows the results with FCV.

As shown in FIGS. 4 and 5, in any case, although significant inactivation was observed at neutral pH, the inactivation action was weak, and stronger inactivation was observed as the pH became acidic.

Test Example 5. Effect of buffer composition on virus inactivation of N-AcTrp I noticed that there was a difference in the degree of inactivation even when using the same concentration of N-AcTrp. In order to investigate the cause, virus inactivation by N-AcTrp The influence of the buffer material on the effect was also investigated. HSV-1 (envelope virus) was used as the virus. The results are shown in FIG.

As shown in FIG. 6, as estimated, the virus inactivating action of N-AcTrp was greatly affected by the type of organic acid used for preparing the buffer solution. The virus inactivation activity was stronger when using a phosphate buffer than when using an acetate buffer.

Test Example 6. Effect of incubation temperature on virus inactivation of N-AcTrp The effect of incubation temperature on inactivation of influenza virus by N-AcTrp in 10 mM citrate buffer (pH 5.0) was investigated. The results are shown in FIG.

As shown in FIG. 7, virus inactivation by acidic pH is also affected by temperature (when the N-AcTrp concentration is 0 mM), but virus inactivation by N-AcTrp was clearly seen. The virus inactivation effect of N-AcTrp occurs even at ice temperature (0 ° C), but the inactivation effect was limited below 25 ° C. Beyond 30 ° C, it became more prominent as the heat retention temperature increased.

Test Example 7. Inhibition of virus growth by N-AcTrp (antiviral effect)
The inhibitory effect of virus growth by N-AcTrp was examined. The results when HSV-1 is used are shown in FIG. 8, and the results when influenza virus is used are shown in FIG.

As shown in FIGS. 8 and 9, the virus yield after one-stage growth decreased with increasing concentration of N-AcTrp, and N-AcTrp not only inactivated the virus, but also HSV-1 And virus growth in influenza virus-infected cells were significantly inhibited.

Claims

General formula (1):

[Wherein R 1 represents an alkyl group having 1 to 6 carbon atoms. R 2 represents an alkylene group having 1 to 6 carbon atoms. R 3 represents a group derived from a nitrogen-containing heterocycle. ]
An antiviral or viral inactivating agent comprising at least one selected from the group consisting of a compound represented by the formula: salt thereof, and solvate thereof.
The compound has the general formula (1A):

[Wherein, R 1 and R 2 are the same as defined above. ]
The antiviral or virus inactivating agent according to claim 1, which is a compound represented by:
The compound has the general formula (1AA):

[Wherein, R 1 is the same as defined above. ]
The antiviral or virus inactivating agent according to claim 1 or 2, which is a compound represented by the formula:
The antiviral or viral inactivating agent according to any one of claims 1 to 3, wherein the alkyl group represented by R 1 has 1 to 3 carbon atoms.
The antiviral or viral inactivating agent according to any one of claims 1 to 4, which is used for application to a living body.
The antiviral or viral inactivating agent according to claim 5, which is a medicine, cosmetics, disinfectant or detergent.
The antiviral or virus inactivating agent according to any one of claims 1 to 4, which is used for application to an article.
The antiviral or virus inactivating agent according to claim 7, which is a disinfectant or a cleaning agent.
General formula (1):

[Wherein R 1 represents an alkyl group having 1 to 6 carbon atoms. R 2 represents an alkylene group having 1 to 6 carbon atoms. R 3 represents a group derived from a nitrogen-containing heterocycle. ]
A method for producing a whole vaccine, comprising treating a virus with at least one selected from the group consisting of a compound represented by the formula: salt thereof, and solvate thereof.
General formula (1):

[Wherein R 1 represents an alkyl group having 1 to 6 carbon atoms. R 2 represents an alkylene group having 1 to 6 carbon atoms. R 3 represents a group derived from a nitrogen-containing heterocycle. ]
A whole vaccine comprising at least one selected from the group consisting of a compound represented by the formula: salts thereof, and solvates thereof; and an inactivated virus.