JP2021130634A - Human norovirus inactivator - Google Patents

Abstract

To provide a new human norovirus inactivator that has high human norovirus inactivation effect and also has excellent handleability.SOLUTION: A human norovirus inactivator contains (a) alkanolamine as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a human norovirus inactivating agent, a human norovirus inactivating method, and a method for treating an object.

Norovirus is an RNA virus that does not have an envelope (membrane structure) classified into the Caliciviridae and Norovirus genus, has strong resistance to acidity (gastric acid), and has a small amount (about 10 to 100). It is known to be infected with.
At present, there are no vaccines or therapeutic agents for norovirus, so it is important to prevent infection by removing the virus or inactivating the virus by cleaning and disinfecting cooking utensils, clothes, fingers, etc. to which the virus can adhere. be.

Since norovirus has strong physicochemical resistance, chlorine-based disinfectants (sodium hypochlorite, etc.), iodine agents (povidone iodine, etc.), aldehyde agents (glutaral, etc.), peracetic acid preparations, etc. can be used to inactivate norovirus. Is used. However, chlorine-based disinfectants have the effect of bleaching the color pattern of fibers, and there is a problem that their use in textile products and clothing is restricted. In addition, disinfectants containing ethanol and cationic surfactants, which are effective against many bacteria, may not be sufficiently effective against norovirus in general usage. Therefore, a safer and more efficient norovirus inactivating technique is required.

In Patent Documents 1 and 2, a predetermined aqueous disinfectant containing a quaternary ammonium salt-type surfactant and an alkaline agent is impregnated into a base sheet containing a predetermined fiber, and is virus-removing or viral. A cleaning sheet having an inactivating effect is disclosed.
Further, in Patent Document 3, 1,4-bis (3,3'-(1-decylpyridinium) methyloxy) butanedibromide is used as a virus inactivating component, and as an efficacy enhancer of the virus inactivating component. The 1,4-bis (3,3'-(1-decylpyridinium) methyloxy) butane dibromide is formed by dissolving one or more selected from carbonate, bicarbonate, and alkanolamine in water. A virus-inactivating composition having a blending amount of 0.05% by mass or more and 1.0% by mass or less is disclosed.
Further, Patent Document 4 contains (A) ethanol in an amount of 50 to 70% by weight, and (B) an organic acid and / or an organic acid salt in a total amount of 0.05 to 4.50% by weight of ethanolamines. , The pH is 6 to 12, and the ethanolamines are at least one selected from the group consisting of monoethanolamine, diethanolamine, triethanolamine, isopropanolamine, and morpholin, disclosed as a disinfectant for norovirus. Has been done.
Further, Patent Document 5 contains (A) a quaternary ammonium salt which is at least one of dioctyldimethylammonium chloride and alkyldimethylbenzylammonium chloride, (B) an alkaline agent, and (C) a chelating agent in a predetermined ratio. However, a liquid sterilizing detergent composition for laundry, in which the pH of a 0.1% by mass aqueous solution at 25 ° C. is in the range of 10.0 to 12.0 and has inactivation against non-enveloped viruses, is disclosed.

By the way, conventionally, norovirus that infects humans (hereinafter referred to as human norovirus) has not been established in a laboratory culture system. Therefore, in order to evaluate the inactivating effect of norovirus, the same alternative virus of the Caliciviridae family is used. Feline calicivirus and murine norovirus were used. However, feline calicivirus is not a diarrhea infection but a virus that causes respiratory infections and is vulnerable to acids and alkalis, and many differences from human norovirus have been reported. In addition, it has been reported that mouse norovirus is remarkably vulnerable to alcohol as compared with other enveloped viruses such as human norovirus (Non-Patent Document 1). In fact, Non-Patent Document 2 reports that in the recently established human norovirus culture system, alcohol, which has been thought to be effective in some alternative viruses, is insufficient for inactivating human norovirus. Was done. That is, it has become clear in recent years whether an agent whose effect has been confirmed as an alternative virus can actually inactivate human norovirus. Therefore, there is a strong demand for more accurate inactivating techniques for human norovirus.

Cromeans Theresa et al., Appl. Environ. Microbiol. 80.18 (2014): 5743-5751. Costantini, Veronica, et al. "Human norovirus replicationin human intestinal enteroids as model to evaluate virus inactivation." Emerging infectious diseases 24.8 (2018): 1453.

Japanese Unexamined Patent Publication No. 2019-11342 JP-A-2018-27916 JP-A-2018-127401 JP-A-2016-210807 Japanese Unexamined Patent Publication No. 2016-60787

The present invention provides a new human norovirus inactivating agent having a high inactivating effect on human norovirus and excellent handleability.

The present invention relates to (a) a human norovirus inactivating agent containing an alkanolamine as an active ingredient.

The present invention also relates to a method for inactivating human norovirus in which a human norovirus inactivating agent containing alkanolamine as an active ingredient is brought into contact with an object in which human norovirus is present and left for a predetermined time.

The present invention also relates to a method for treating an object, in which a human norovirus inactivating agent containing alkanolamine as an active ingredient is attached to the object to form a portion of the object that inactivates human norovirus.

Hereinafter, (a) alkanolamine will be described as (a) component.

According to the present invention, a new human norovirus inactivating agent and a human norovirus inactivating method, which have a high inactivating effect on human norovirus and are excellent in handleability, are provided.

The inactivating agent of the present invention contains the component (a) alkanolamine as an active ingredient. The component (a) has conventionally been used in various preparations for the purpose of pH adjustment and the like. Further, Patent Document 3 proposes to use the component (a) as a drag enhancer for a specific virus inactivating component. However, it has not been known in the art that the component (a) itself has an inactivating effect on human norovirus. In the present invention, the effect is actually confirmed using human norovirus as in the examples described later, and the inactivating effect of the component (a) on human norovirus is shown more accurately than the conventional technique. There is.

Examples of the alkanolamine include alkanolamines having 1 or more and 9 or less carbon atoms.

As the alkanolamine, monoalkanolamine is preferable. Further, from the viewpoint of the inactivating effect of human norovirus, monoalkanolamine having 1 to 6 carbon atoms and monoethanolamine having 2 carbon atoms are preferable.
Alkanolamines may be used alone or in combination of two or more.

The inactivating agent of the present invention contains the component (a) in an amount effective for inactivating human norovirus. Here, the effective amount of the present invention means, for example, that the amount of human norovirus in the drug-treated group 7 days after the start of the test is reduced by 1 Log or more as compared with the amount of human norovirus in the drug-untreated group by the method of Examples described later, preferably. It is an amount that is reduced by 2 Logs or more, and in particular, is inactivated to the detection limit. For example, the inactivating agent of the present invention contains the component (a) in an amount of 0.5% by mass or more, further 0.8% by mass or more, further 1.1% by mass or more, and further 1.5% by mass from the viewpoint of the inactivating effect. % Or more, and 10% by mass or less, and further 5% by mass or less from the viewpoint of odor and irritation. The content of the component (a) is calculated as an amine.

The pH of the inactivating agent can be adjusted with the type and amount of the component (a) and, if necessary, an alkaline compound other than the component (a). Examples of the alkaline compound include inorganic hydroxides such as sodium hydroxide, potassium hydroxide, magnesium hydroxide and calcium hydroxide, inorganic carbonates such as sodium carbonate and potassium carbonate, and inorganic silicates such as sodium silicate. Can be mentioned.

The inactivating agent of the present invention can contain water. The inactivating agent of the present invention may be an inactivating agent containing the component (a) and water.

When the inactivating agent of the present invention contains water, the pH at 25 ° C. is preferably 10 or more, more preferably 10.5 or more, and preferably 14 or less, more preferably 12 or less. The pH can be measured by the glass electrode method.

The inactivating agent of the present invention exhibits an excellent human norovirus inactivating effect even if the content of (b) a cationic bactericidal agent [hereinafter referred to as (b) component] is small. The inactivating agent of the present invention may contain the component (b) in an amount of less than 0.1% by mass, further less than 0.05% by mass, further less than 0.01% by mass, and further 0% by mass.

Examples of the component (b) include a cationic bactericide selected from a quaternary ammonium salt type bactericide and a pyridinium salt type bactericide. That is, in the present invention, the content of the cationic fungicide selected from the quaternary ammonium salt type fungicide and the pyridinium salt type fungicide may be in the above range, and the content of the cationic fungicide is not contained. May be. As the quaternary ammonium salt type bactericide, one or two of the groups bonded to the quaternary nitrogen are alkyl groups having 8 or more carbon atoms and 14 or less carbon atoms, and the rest are alkyl groups having 1 or more carbon atoms and 3 or less carbon atoms. Examples thereof include a quaternary ammonium salt having a hydroxyalkyl group or a benzyl group having 1 to 3 carbon atoms. Examples of the pyridinium salt type disinfectant include cetylpyridinium salt and 1,4-bis (3,3'-(1-decylpyridinium) methyloxy) butandi salt. The counter anion of each salt is a halide ion, preferably a chloride ion or a bromide ion.

Generally, ethanol is known as an inactivating agent for norovirus. For example, Non-Patent Document 2 describes one containing 50 to 70% of ethanol, but human norovirus has not been completely inactivated. In addition, ethanol is highly volatile, has a pungent odor, and has a flash point, making it extremely difficult to handle. The present invention can exhibit an excellent human norovirus inactivating effect even if the content of (c) ethanol [hereinafter referred to as the component (c)] is small or not. The inactivating agent of the present invention may contain the component (c) in an amount of less than 10% by mass, further 5% by mass or less, and further 0% by mass.

The inactivating agent of the present invention can be applied to a cleaning agent. For that purpose, the inactivating agents of the present invention include (d) (d1) surfactants [however, excluding component (b)] [hereinafter referred to as component (d1)] and (d2) organic solvent [however, component (c)). It is possible to contain one or more components [hereinafter referred to as (d) component] selected from [excluding] [hereinafter referred to as (d2) component].
Since many of the objects that inactivate human norovirus, such as vomitus, coexist with a large amount of contaminants, it is preferable to contain the component (d) from the viewpoint of removing them.

Examples of the surfactant of the component (d1) include a nonionic surfactant, an amphoteric surfactant, an anionic surfactant and the like.
As a nonionic surfactant, an alkyl alkoxylate type surfactant having an alkyl group having 10 or more and 18 or less carbon atoms (alkoxylate is one or more selected from ethoxylate or propoxylate, and the average number of added moles is 3 to 3 to 20), preferably selected from alkyl glycoside-type surfactants having an alkyl group having 10 or more and 18 or less carbon atoms. As amphoteric surfactants, amine oxide type surfactants having one alkyl group having 10 or more and 18 or less carbon atoms and two alkyl groups or hydroxyalkyl groups having 1 or more and 3 or less carbon atoms, carbobetaine type surfactants, Alternatively, a sulfobetaine type surfactant is preferable. Examples of anionic surfactants include fatty acid salts having 8 to 18 carbon atoms, alkyl sulfates having an alkyl group having 8 to 18 carbon atoms, alkyl ether sulfates having an alkyl group having 8 to 18 carbon atoms, and carbon atoms. Those selected from alkylarylsulfonic acids having an alkyl group of 8 or more and 18 or less are preferable.

When the component (d1) is contained, the content thereof is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, and preferably 10% by mass or less in the inactivating agent of the present invention. More preferably, it is 5% by mass or less.

Examples of the organic solvent of the component (d2) include monovalent or polyhydric alcohol solvents and glycol ether solvents. As the monohydric or polyhydric alcohol solvent, propanol, butanol, glycerin, ethylene glycol, propylene glycol and butylene glycol are preferable. Examples of the glycol ether solvent include a glycol ether solvent having an alkyl group having 1 or more carbon atoms and 6 or less carbon atoms and having an addition molar number of ethylene oxide or propylene oxide of 1 or more and 3 or less, for example, diethylene glycol monobutyl ether and dipropylene glycol. Examples thereof include monobutyl ether.

When the component (d2) is contained, the content thereof is preferably 1% by mass or more, more preferably 2% by mass or more, and preferably 15% by mass or less, more preferably 8 in the inactivating agent of the present invention. It is less than mass%.

In addition, the inactivating agent of the present invention has optional components such as a pH adjuster, a pigment, a fragrance, a chelating agent, a dispersant, a stabilizer, and a preservative (excluding the components (a) to (d)). Can be contained.
The inactivating agent of the present invention can be used as an optional component, for example.
(1) Citric acid, ethylenediamine tetraacetic acid, succinic acid, fumaric acid, methyliminodiacetic acid, iminodisuccinic acid, ethylenediaminedisuccinic acid, taurinediacetic acid, hydroxyethyliminodiacetic acid, β-alaninediacetic acid, hydroxyiminodisuccinic acid, methylglycindi Polyvalent carboxylic acids such as acetic acid, diacetic acid glutamate, asparagine diacetic acid, serine diacetic acid, and salts thereof (examples of salts include alkali metal salts and alkaline earth metal salts, preferably sodium salts and potassium salts).
(2) Inorganic acids such as phosphoric acid, boric acid, carbonic acid, silicic acid and salts thereof, and salts thereof (examples of salts include alkali metal salts and alkaline earth metal salts, and sodium salts and potassium salts are preferable. )
(3) A non-carboxylic acid-based nitrogen-containing compound such as tris (hydroxymethyl) aminomethane or diethylbarbituric acid can be contained. The inactivating agent of the present invention preferably contains a component selected from the polyvalent carboxylic acid of (1) and a salt thereof, and more preferably contains citric acid or a salt thereof.

In the present invention, the polyvalent carboxylic acid and salts thereof are preferably 0.01% by mass or more, more preferably 0.03% by mass or more, further preferably 0.08% by mass or more, and preferably 1% by mass. Hereinafter, the content is more preferably 0.8% by mass or less, still more preferably 0.5% by mass or less. Further, in the present invention, (a) / (polyvalent carboxylic acid and salts thereof), which is a mass ratio of the content of the component (a) to the content of the polyvalent carboxylic acid and its salt, is preferable. It is 1 or more, more preferably 2 or more, still more preferably 3 or more, still more preferably 6 or more, and preferably 300 or less, more preferably 100 or less, still more preferably 37.5 or less.

The inactivating agent of the present invention exhibits an excellent inactivating effect on human norovirus. The human norovirus may belong to the Caliciviridae, Norovirus genus, Norwalk virus species according to the classification of the International Committee on Taxonomy of Viruses.

The inactivating agent of the present invention may be blended with a disinfectant, a cleaning agent, a deodorant and the like. For example, the inactivating agent of the present invention can be blended with a cleaning agent for a hard surface to obtain a cleaning agent composition for a hard surface having an inactivating effect on human norovirus. A contact-type human norovirus inactivating agent used in contact with an object is a preferred embodiment of the present invention.

The present invention provides a method for inactivating human norovirus, which applies an effective amount of alkanolamine to human norovirus. The effective amount may be an amount at which inactivation of human norovirus is recognized by application, and may be, for example, an amount that reduces human norovirus as compared with the time of contact in the evaluation method of Examples described later. Specifically, the present invention provides a method for inactivating human norovirus in which a human norovirus inactivating agent containing the component (a) as an active ingredient is brought into contact with an object in which human norovirus is present and left for a predetermined time.

The object may be selected from articles, emissions from the human body, and the like.
Articles include, for example, (1) textile products such as clothing, gloves, hats, bedding, and curtains, (2) toilets, washbasins, bathtubs, tables, chairs, tableware, refrigerators, bags, electronic devices, doorknobs, switches, etc. Examples include hard articles, (3) structures such as floors, walls, ceilings, pillars, and windows.
In addition, examples of excrement from the human body include excrement and vomiting of a person infected with human norovirus.

The inactivating agent of the present invention can be brought into contact with an object by a method such as coating, dipping, or spraying. For example, when the inactivating agent of the present invention is sprayed and brought into contact with an object, the inactivating agent of the present invention can be contained and used in a general-purpose container such as a trigger spray or a dispenser.
Further, when the inactivating agent of the present invention is applied to an object, for example, the human norovirus inactivating the object can be inactivated by wiping with a base sheet carrying the inactivating agent of the present invention. Examples of the material of the base material sheet include recycled fibers, synthetic fibers, plant fibers, non-woven fabrics made of mixed fibers thereof, woven fabrics, and net cloths.

In addition, for example, a vinyl sprayed with the inactivating agent of the present invention when disposing of clothing, disposable masks, disposable gloves, cloth products, etc. used for cleaning excrement and vomitus of a patient infected with human norovirus. It can also be sealed in a bag or the like.

The predetermined time for which the inactivating agent of the present invention is left in contact with the object is 1 minute or more, further 3 minutes or more, further 5 minutes or more, and 300 minutes or less, further 60 minutes or less, further 30 minutes or less. can do.

The portion treated with the inactivating agent of the present invention can be inactivated and suppress the growth even if human norovirus is attached. Therefore, the inactivating agent of the present invention is also useful for preventing human norovirus infection. INDUSTRIAL APPLICABILITY The present invention provides, for example, a method for treating an object by attaching a human norovirus inactivating agent containing an alkanolamine as an active ingredient to the object to form a portion of the object that inactivates human norovirus.

<Evaluation method>
(1) Culture of Human Industrial Enteroid (HIE) HIE was embedded in Matrigel (Corning, 356231) on a 48-well plate and cultured three-dimensionally. As the medium, IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies, ST-06010) was used. The procedure for medium exchange, passage, and monolayering using a 96-well plate was in accordance with the user manual. CultureSure Y-27632 (Fuji Film Wako Pure Chemical Industries, Ltd.), which is a ROCK (Rho-associated coiled-coil forming kinase) inhibitor so that the final concentration is 10 μM in the medium to inhibit anoikis for 2 days after trypsin treatment. , 036-24023) was added. Add 5 mL of GlutaMAX I (100 ×) (Gibco, 35050-061), 5 mL of 1M HEPES (Gibco, 15630080), and 5 mL of Penicillin-Streptomycin (Gibco) to 500 mL of Advanced DMEM / F12 (Gibco, 12634010). The basal medium was prepared by doing so. A differentiation medium was prepared by mixing an equal amount of the basal medium and the component A of the IntestiCult Organoid Growth Medium. Differentiation was induced in cells monolayered with a 96-well plate for a total of 6 days while exchanging 200 μL of differentiation medium per well at 2-day intervals.

(2) Preparation of 10% Emulsion of Human Norovirus (HNV) -Containing Feces The 10% emulsion of feces is GII. It was prepared from the feces of a type 4 HNV affected person. One tablet of the protease inhibitor Complete protein inhibitor cocktail tablets (Sigma-Aldrich, 116979489001) was suspended in 50 mL of D-PBS (-). 1 g of stool was suspended in 10 mL of D-PBS (-) containing Complete and mixed well with a test tube mixer. After allowing to stand at 4 ° C. for 20 minutes, the mixture was centrifuged at 2000 × g at 4 ° C. for 10 minutes. The supernatant was collected in a new tube and stored at -80 ° C until it was used for infection experiments.

(3) Inactivation treatment of HNV and infection with differentiated HIE The 10% fecal emulsion containing HNV was diluted 10-fold with a differentiation medium and filtered using a 1 mL syringe and Millex HV Filter unit (Millipore, SLHVR04NL). 45 μL of the human norovirus inactivating agent shown in Tables 1 and 2 was mixed with 5 μL of the fecal solution filtered in a PA microcentrifuge tube (Beckman coulter, 357448) and contacted at 25 ° C. for 20 minutes. Next, 1.45 mL of FBS (Fetal bovine serum; BIOWEST) deactivated at 56 ° C. for 30 minutes was added to the drug-treated fecal solution. Set the centrifuge tube on the fixed angle rotor TLA-55 (Beckman coulter) and use Optima MAX-TL (Beckman coulter) at the maximum turning radius (Rmax) at a centrifugal force of 186047 × g (equivalent to 55000 rpm) for more than 1.5 hours. The supernatant was removed after centrifugation. To the precipitate, 1.45 mL of FBS deactivated at 56 ° C. for 30 minutes was added again, suspended, and ultracentrifuged under the same conditions, and then the supernatant was removed.
The pellet was suspended in 100 μL of differentiation medium and applied to the post-differentiation HIE from which the existing medium in the wells had been removed. Incubation was carried out at 37 ° C. for 3 hours. After washing 3 times with 300 μL of basal medium, 250 μL of differentiation medium containing porcine bile extract (Sigma-Aldrich, B8631-100G) was added so that the final concentration was 125 ppm, and the conditions _{were 37 ° C. and 5% CO 2.} It was cultured underneath until the timing of sampling. The test was started at the timing when the suspension of the pellet was applied to HIE, and 10 μL of the supernatant was collected 7 days after the start of the test (day 7). The collected supernatant was stored at −80 ° C. until it was subjected to RT-qPCR. In addition, a sample that was similarly cultured for 7 days using a fecal solution that was not contacted with a human norovirus inactivating agent was designated as a "drug-untreated group" and the supernatant was collected.

(4) RT-qPCR
A norovirus detection kit G1 / G2 (Toyobo, FIK-273) was used to quantify the number of HNV genome copy in the collected supernatant. The operation followed the protocol. Light Cycler 480II (Roche) was used for PCR amplification and data measurement. Based on the measured amount of human norovirus, the difference between the amount of human norovirus in the drug-treated group and the amount of human norovirus in the drug-untreated group 7 days after the start of the test was calculated, and the difference in the amount of reduction in human norovirus was evaluated in the following four stages. bottom. The results are shown in Tables 1 and 2.
A: Inactivate HNV to the lower limit of detection B: HNV is detected, but HNV is reduced by 2 log or more compared to "drug untreated group" C: HNV is less than 2 log compared to "drug untreated group" 1 log or more decrease D: The amount of decrease in HNV is less than 1 log compared to the "drug-untreated group"

In Table 1, the quaternary ammonium salt is benzalkonium chloride (mixed alkyldimethylbenzylammonium chloride having 12 to 16 carbon atoms, Sanizol C manufactured by Kao Corporation).
In Table 1, "with" a flash point means that there is a flash point below 95 ° C., and "without" means that there is no flash point below 95 ° C.
In Table 1, "Yes" for pungent odor means that the user feels an unpleasant odor during use in the evaluation of one panelist who is skilled in judging odor, and "No" means that the user feels unpleasant odor with the same evaluation. It means that you don't smell anything.

From the results in Table 1, it can be seen that when the content of ethanol is high, the flash point is low and there is a pungent odor, so it is desirable not to contain ethanol in terms of handleability. Further, it can be seen that even if the quaternary ammonium salt is used, the inactivating effect on HNV is not exhibited when the amount of monoethanolamine is small or not contained.

In Table 2, the fatty acid is "Lunac L-55A" manufactured by Kao Corporation.
In Table 2, the amine oxide is "Anchor 20N" manufactured by Kao Corporation.

The human norovirus inactivating agent shown in Table 2 contains the component (d) and can be used as a cleaning agent. It can be seen that even in these cases, the inactivating effect on HNV is exhibited by containing the component (a) of the present invention.

<Formulation example>
Table 3 shows a formulation example of the human norovirus inactivating agent of the present invention.

Claims (11)

(A) A human norovirus inactivating agent containing an alkanolamine [hereinafter referred to as a component (a)] as an active ingredient. The inactivating agent according to claim 1, wherein the pH at 25 ° C. is 10 or more and 14 or less. (A) The inactivating agent according to claim 1 or 2, which contains 0.5% by mass or more of the component. (A) The inactivating agent according to any one of claims 1 to 3, wherein the component is monoalkanolamine. The inactivating agent according to any one of claims 1 to 4, wherein the component (a) is a monoalkanolamine having a total carbon number of 1 or more and 9 or less. (B) The inactivating agent according to any one of claims 1 to 5, wherein the content of the cationic bactericidal agent [hereinafter referred to as the component (b)] is less than 0.1% by mass. (C) The inactivating agent according to any one of claims 1 to 6, wherein the content of ethanol [hereinafter referred to as the component (c)] is less than 10% by mass. From (d) (d1) surfactant [however, excluding component (b)] [hereinafter referred to as (d1) component] and (d2) organic solvent [however, excluding component (c)] [hereinafter referred to as (d2) component] The inactivating agent according to any one of claims 1 to 7, which contains one or more selected components. A method for inactivating human norovirus in which a human norovirus inactivating agent containing alkanolamine as an active ingredient is brought into contact with an object in which human norovirus is present and left for a predetermined time. The method for inactivating human norovirus according to claim 9, wherein the predetermined time is 1 minute or more and 300 minutes or less. A method for treating an object, in which a human norovirus inactivating agent containing alkanolamine as an active ingredient is attached to the object to form a portion of the object that inactivates human norovirus.