JP7537881B2 - Human norovirus inactivation agent - Google Patents

Description

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

Norovirus is a non-enveloped (membrane-like structure) RNA virus classified in the Caliciviridae family and Norovirus genus. It is known to have strong resistance to acidity (gastric acid) and to be infectious in small amounts (approximately 10 to 100 viruses).
At present, there is no vaccine or treatment for norovirus, so it is important to prevent infection by removing or inactivating the virus by cleaning and disinfecting cooking utensils, clothing, hands, etc., on which the virus may be attached.

Norovirus has strong physical and chemical resistance, so chlorine-based disinfectants (such as sodium hypochlorite), iodine agents (such as povidone-iodine), aldehyde agents (such as glutaral), and peracetic acid preparations are used to inactivate the virus. However, chlorine-based disinfectants have the effect of bleaching the colors and patterns of textiles, which limits their use on textile products and clothing. In addition, disinfectants containing ethanol or cationic surfactants, which are effective against many types of bacteria, may not be sufficiently effective against norovirus when used in the usual way. For this reason, a safe and more efficient norovirus inactivation technology is required.

Patent Documents 1 and 2 disclose cleaning sheets having virus removal or virus inactivation effects, in which a base sheet containing specified fibers is impregnated with a specified aqueous disinfectant containing a quaternary ammonium salt surfactant and an alkaline agent.
Furthermore, Patent Document 3 discloses a virus inactivating composition comprising 1,4-bis(3,3'-(1-decylpyridinium)methyloxy)butane dibromide as a virus inactivating component and one or more potency enhancers for the virus inactivating component selected from carbonates, hydrogencarbonates and alkanolamines dissolved in water, the content of the 1,4-bis(3,3'-(1-decylpyridinium)methyloxy)butane dibromide being 0.05% by mass or more and 1.0% by mass or less.
Furthermore, Patent Document 4 discloses a disinfectant for norovirus which contains (A) 50 to 70% by weight of ethanol and (B) a total of 0.05 to 4.50% by weight of an organic acid and/or an organic acid salt and an ethanolamine, and has a pH of 6 to 12, wherein the ethanolamine is at least one selected from the group consisting of monoethanolamine, diethanolamine, triethanolamine, isopropanolamine, and morpholine.
Furthermore, Patent Document 5 discloses a liquid germicidal detergent composition for laundry that contains (A) a quaternary ammonium salt which is at least one of dioctyldimethylammonium chloride and alkyldimethylbenzylammonium chloride, (B) an alkali agent, and (C) a chelating agent in predetermined ratios, and has a pH of 10.0 to 12.0 at 25° C. as a 0.1% by mass aqueous solution, and has inactivating properties against non-enveloped viruses.

In the past, since a laboratory culture system had not been established for norovirus that infects humans (hereinafter referred to as human norovirus), feline calicivirus and mouse norovirus, which belong to the same Caliciviridae family, were used as substitute viruses to evaluate the inactivation effect of norovirus. However, feline calicivirus is a virus that causes respiratory infections rather than diarrheal infections, and is weak against acids and alkalis, and many differences from human norovirus have been reported. It has also been reported that mouse norovirus is significantly weaker against alcohol than other enveloped viruses, including human norovirus (Non-Patent Document 1). In fact, Non-Patent Document 2 reports that alcohol, which has been thought to be effective for some substitute viruses in a recently established culture system for human norovirus, is insufficient to inactivate human norovirus. In other words, it has been revealed in recent years that it is not certain whether agents that have been confirmed to be effective for substitute viruses can actually inactivate human norovirus. Therefore, there is a strong demand for a more reliable inactivation technology 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.

JP 2019-112342 A JP 2018-27916 A JP 2018-127401 A JP 2016-210807 A JP 2016-60787 A

The present invention provides a new human norovirus inactivation agent that is highly effective in inactivating human norovirus and is easy to handle.

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 an alkanolamine as an active ingredient is brought into contact with an object containing human norovirus and allowed to stand for a predetermined period of time.

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

In the following, (a) alkanolamine will be explained as component (a).

The present invention provides a new human norovirus inactivation agent and a method for inactivating human norovirus that are highly effective in inactivating human norovirus and easy to handle.

The inactivating agent of the present invention contains an alkanolamine as component (a) as an active ingredient. Component (a) has been used in various preparations in the past for the purpose of adjusting pH, etc. Patent Document 3 also proposes using component (a) as an antibacterial enhancer for a specific virus inactivating component. However, it was not known in the art that component (a) itself has an inactivating effect against human norovirus. In the present invention, the effect has been confirmed by actually using human norovirus as in the examples described below, and the inactivating effect of component (a) against human norovirus has been demonstrated more accurately than with conventional techniques.

Alkanolamines include alkanolamines with 1 to 9 carbon atoms.

As the alkanolamine, a monoalkanolamine is preferable. Furthermore, from the viewpoint of the inactivation effect of human norovirus, a monoalkanolamine having 1 to 6 carbon atoms, and further a monoethanolamine having 2 carbon atoms is preferable.
The alkanolamines may be used alone or in combination.

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

The pH of the inactivating agent can be adjusted by the type and amount of component (a) and, if necessary, by an alkaline compound other than component (a). Examples of alkaline compounds 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.

The inactivating agent of the present invention may contain water. The inactivating agent of the present invention may be an inactivating agent containing 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 inactivation effect even when the content of the cationic bactericide (b) [hereinafter referred to as component (b)] is small. The content of component (b) in the inactivating agent of the present invention may be less than 0.1% by mass, further less than 0.05% by mass, further less than 0.01% by mass, or even 0% by mass.

Component (b) may be 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 bactericide selected from a quaternary ammonium salt type bactericide and a pyridinium salt type bactericide may be within the above range, or the cationic bactericide may not be contained. Examples of quaternary ammonium salt type bactericides include quaternary ammonium salts in which one or two of the groups bonded to the quaternary nitrogen are alkyl groups having 8 to 14 carbon atoms, and the remaining groups are alkyl groups having 1 to 3 carbon atoms, hydroxyalkyl groups having 1 to 3 carbon atoms, or benzyl groups. Examples of pyridinium salt type bactericides include cetylpyridinium salt and 1,4-bis(3,3'-(1-decylpyridinium)methyloxy)butane di-salt. The counter anion of each salt is a halide ion, preferably a chloride ion or a bromide ion.

Ethanol is generally known as an inactivating agent for norovirus. For example, Non-Patent Document 2 describes a composition containing 50-70% ethanol, but this does not completely inactivate human norovirus. In addition, ethanol is highly volatile, has a pungent odor, and has a flash point, making it very difficult to handle. The present invention can achieve excellent human norovirus inactivation effects even when the content of (c) ethanol [hereinafter referred to as component (c)] is low or absent. The inactivating agent of the present invention may contain less than 10% by mass, or even 5% by mass or less, or even 0% by mass, of component (c).

The inactivating agent of the present invention can be applied to a cleaning agent. For this purpose, the inactivating agent of the present invention can contain one or more components selected from (d) (d1) a surfactant (excluding component (b)) (hereinafter referred to as component (d1)) and (d2) an organic solvent (excluding component (c)) (hereinafter referred to as component (d2)) (hereinafter referred to as component (d)).
Since the objects in which human norovirus is to be inactivated, such as vomit, often contain large amounts of contaminating substances, it is preferable for the agent to contain component (d) from the viewpoint of removing these contaminating substances.

Examples of the surfactant of the component (d1) include nonionic surfactants, amphoteric surfactants, and anionic surfactants.
The nonionic surfactant is preferably selected from alkyl alkoxylate type surfactants having an alkyl group having 10 to 18 carbon atoms (alkoxylate is one or more selected from ethoxylate and propoxylate, and the average number of moles added is 3 to 20), and alkyl glycoside type surfactants having an alkyl group having 10 to 18 carbon atoms. The amphoteric surfactant is preferably an amine oxide type surfactant having one alkyl group having 10 to 18 carbon atoms and two alkyl or hydroxyalkyl groups having 1 to 3 carbon atoms, a carbobetaine type surfactant, or a sulfobetaine type surfactant. The anionic surfactant is preferably selected from 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 alkyl aryl sulfonic acids having an alkyl group having 8 to 18 carbon atoms.

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

The organic solvent of component (d2) may be a monohydric or polyhydric alcohol solvent, or a glycol ether solvent. Preferred examples of the monohydric or polyhydric alcohol solvent include propanol, butanol, glycerin, ethylene glycol, propylene glycol, and butylene glycol. Examples of the glycol ether solvent include glycol ether solvents having an alkyl group with 1 to 6 carbon atoms and an added mole number of ethylene oxide or propylene oxide of 1 to 3, such as diethylene glycol monobutyl ether and dipropylene glycol monobutyl ether.

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

The inactivating agent of the present invention may further contain, as optional components, components such as a pH adjuster, a dye, a fragrance, a chelating agent, a dispersant, a stabilizer, and a preservative (excluding the components (a) to (d)).
The inactivating agent of the present invention may contain, as an optional component, for example:
(1) Polyvalent carboxylic acids such as citric acid, ethylenediaminetetraacetic acid, succinic acid, fumaric acid, methyliminodiacetic acid, iminodisuccinic acid, ethylenediaminedisuccinic acid, taurinediacetic acid, hydroxyethyliminodiacetic acid, β-alaninediacetic acid, hydroxyiminodisuccinic acid, methylglycinediacetic acid, glutamic acid diacetic acid, asparaginediacetic acid, and serinediacetic acid, and salts thereof (salts include alkali metal salts and alkaline earth metal salts, with sodium salts and potassium salts being preferred)
(2) Inorganic acids such as phosphoric acid, boric acid, carbonic acid, silicic acid, and salts thereof, and salts thereof (salts include alkali metal salts and alkaline earth metal salts, with sodium salts and potassium salts being preferred)
(3) It may contain non-carboxylic acid nitrogen-containing compounds such as tris(hydroxymethyl)aminomethane and diethylbarbituric acid. The inactivating agent of the present invention preferably contains a component selected from the group consisting of polyvalent carboxylic acids and salts thereof (1), and more preferably contains citric acid or a salt thereof.

In the present invention, the polycarboxylic acid and its salt are preferably contained in an amount of 0.01% by mass or more, more preferably 0.03% by mass or more, even more preferably 0.08% by mass or more, and preferably 1% by mass or less, more preferably 0.8% by mass or less, and even more preferably 0.5% by mass or less. In addition, in the present invention, the mass ratio of the content of the (a) component to the content of the polycarboxylic acid and its salt, that is, (a)/(polycarboxylic acid and its salt), is preferably 1 or more, more preferably 2 or more, even more preferably 3 or more, even more preferably 6 or more, and preferably 300 or less, more preferably 100 or less, and even more preferably 37.5 or less.

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

The inactivating agent of the present invention may be incorporated into disinfectants, cleaning agents, deodorants, etc. For example, the inactivating agent of the present invention may be incorporated into a cleaning agent for hard surfaces to obtain a cleaning agent composition for hard surfaces that has an inactivating effect against human norovirus. A contact-type human norovirus inactivating agent that is used by contacting the object is a preferred embodiment of the present invention.

The present invention provides a method for inactivating human norovirus, in which an effective amount of alkanolamine is applied to the human norovirus. The effective amount may be an amount at which inactivation of the human norovirus is observed upon application, for example, an amount that reduces the amount of human norovirus compared to when the virus is contacted, as determined by the evaluation method in the Examples described below. Specifically, the present invention provides a method for inactivating human norovirus, in which a human norovirus inactivating agent containing component (a) as an active ingredient is contacted with an object in which human norovirus is present, and allowed to stand for a predetermined period of time.

The object may be selected from an article, an excreta from the human body, and the like.
Examples of articles include: (1) textile products such as clothing, gloves, hats, bedding, and curtains; (2) hard articles such as toilets, washbasins, bathtubs, tables, chairs, tableware, refrigerators, bags, electronic devices, doorknobs, and switches; and (3) structures such as floors, walls, ceilings, pillars, and windows.
Furthermore, examples of excretions from the human body include feces and vomit from individuals 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, immersion, spraying, etc. For example, when the inactivating agent of the present invention is sprayed to contact with an object, the inactivating agent of the present invention can be stored in a general-purpose container such as a trigger sprayer or a dispenser and used.
When the inactivating agent of the present invention is applied to an object, the object can be inactivated by, for example, wiping it with a base sheet carrying the inactivating agent of the present invention. Examples of materials for the base sheet include nonwoven fabrics, woven fabrics, mesh fabrics, etc. made of recycled fibers, synthetic fibers, plant fibers, or mixtures of these fibers.

In addition, for example, when disposing of clothing, disposable masks, disposable gloves, cloth products, etc. used in cleaning up the excrement or vomit of a patient infected with human norovirus, they can be sealed in a plastic bag sprayed with the inactivating agent of the present invention.

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

Even if human norovirus adheres to a portion treated with the inactivating agent of the present invention, it is inactivated and its proliferation is suppressed. Therefore, the inactivating agent of the present invention is also useful for preventing infection with human norovirus. The present invention provides a method for treating an object, for example, by adhering a human norovirus inactivating agent containing an alkanolamine as an active ingredient to the object, and forming a portion of the object that inactivates human norovirus.

<Evaluation method>
(1) Culture of Human Intestinal Enteroid (HIE) HIE was embedded in Matrigel (Corning, 356231) on a 48-well plate and cultured three-dimensionally. The medium used was IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies, ST-06010). The procedures for medium replacement, subculture, and monolayer formation using a 96-well plate were performed according to the user manual. For two days after trypsin treatment, CultureSure Y-27632 (Fujifilm Wako Pure Chemical Industries, 036-24023), a ROCK (Rho-associated coiled-coil forming kinase) inhibitor, was added to the medium at a final concentration of 10 μM to inhibit anoikis. A basic medium was prepared by adding 5 mL of GlutaMAX I (100x) (Gibco, 35050-061), 5 mL of 1 M HEPES (Gibco, 15630080), and 5 mL of Penicillin-Streptomycin (Gibco, 15140122) to 500 mL of Advanced DMEM/F12 (Gibco, 12634010). A differentiation medium was prepared by mixing equal amounts of the basic medium and component A of IntestiCult Organoid Growth Medium. Differentiation was induced for a total of 6 days by replacing the differentiation medium with 200 μL per well at 2-day intervals in cells monolayered in a 96-well plate.

(2) Preparation of 10% emulsion of feces containing human norovirus (HNV) A 10% emulsion of feces was prepared from feces of a patient with GII.4 type HNV. One tablet of Complete protease inhibitor cocktail tablets (Sigma-Aldrich, 11697498001), a protease inhibitor, was suspended in 50 mL of D-PBS(-). 1 g of feces was suspended in 10 mL of Complete-containing D-PBS(-) and mixed well with a test tube mixer. After standing at 4°C for 20 minutes, the mixture was centrifuged at 2000 x g for 10 minutes at 4°C. The supernatant was collected in a new tube and stored at -80°C until used in an infection experiment.

(3) Inactivation of HNV and infection of differentiated HIE 10% fecal emulsion containing HNV was diluted 10-fold with differentiation medium and filtered using a 1 mL syringe and Millex HV Filter unit (Millipore, SLHVR04NL). 5 μL of fecal solution filtered in a PA microcentrifuge tube (Beckman Coulter, 357448) was mixed with 45 μL of a human norovirus inactivating agent shown in Tables 1 and 2, and allowed to come into contact at 25° C. for 20 minutes. Next, 1.45 mL of FBS (fetal bovine serum; BIOWEST) inactivated at 56° C. for 30 minutes was added to the drug-treated fecal solution. The centrifuge tube was placed in a fixed angle rotor TLA-55 (Beckman Coulter) and ultracentrifuged for 1.5 hours at a centrifugal force of 186047×g (equivalent to 55000 rpm) at the maximum rotation radius (Rmax) using an Optima MAX-TL (Beckman Coulter), and the supernatant was then removed. 1.45 mL of FBS that had been inactivated at 56° C. for 30 minutes was added to the precipitate, which was then suspended, and ultracentrifuged under the same conditions, after which the supernatant was removed.
The pellet was suspended in 100 μL of differentiation medium and applied to the HIE after differentiation induction in which the existing medium in the well had been removed. Incubation was performed at 37 ° C. for 3 hours. After washing three times with 300 μL of basic medium, 250 μL of differentiation medium containing porcine bile extract (Sigma-Aldrich, B8631-100G) added to a final concentration of 125 ppm was added, and the cells were cultured under conditions of 37 ° C. and 5% CO ₂ until the timing of sampling. The timing when the suspension of the pellet was applied to the HIE was set as the start of the test, 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 subjected to RT-qPCR. In addition, the supernatant was collected from a sample similarly cultured for 7 days using a fecal solution that had not been contacted with a human norovirus inactivating agent as a "drug-untreated group".

(4) RT-qPCR
The number of HNV genome copies in the collected supernatant was quantified using a Norovirus Detection Kit G1/G2 (Toyobo, FIK-273). The operation was performed according to the protocol. PCR amplification and data measurement were performed using a LightCycler 480II (Roche). 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 was calculated 7 days after the start of the test, and the following four levels were assigned according to the difference in the amount of human norovirus reduction. The results are shown in Tables 1 and 2.
A: HNV is inactivated to the lower limit of detection. B: HNV is detected, but HNV is reduced by more than 2 logs compared to the "drug-naïve group." C: HNV is reduced by less than 2 logs compared to the "drug-naïve group." D: Reduction in HNV by less than 1 log compared to the "non-drug treated group"

In Table 1, the quaternary ammonium salt is benzalkonium chloride (mixed alkyldimethylbenzylammonium chloride having 12 to 16 carbon atoms, Sanisol C manufactured by Kao Corporation).
In Table 1, "Yes" for the flash point means that there is a flash point below 95°C, and "No" means that there is no flash point below 95°C.
In Table 1, "present" for irritating odor means that the user feels an unpleasant odor when using the product, based on the evaluation by one panelist skilled in judging odors, and "not present" means that the user does not feel an unpleasant odor, based on the same evaluation.

The results in Table 1 show that a high ethanol content leads to a low flash point and a pungent odor, making it preferable from the standpoint of ease of handling to not include ethanol. It is also clear that even if a quaternary ammonium salt is used, if the amount of monoethanolamine is low or not present, the inactivation effect against HNV is not achieved.

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

The human norovirus inactivating agents in Table 2 contain component (d) and can be used as cleaning agents. It can be seen that these also have an inactivating effect against HNV by containing component (a) of the present invention.

<Composition Example>
Table 3 shows examples of formulations of the human norovirus inactivating agent of the present invention.

Claims (5)

(a) a human norovirus inactivating agent comprising, as an active ingredient, a monoalkanolamine having a total carbon number of 2 (hereinafter referred to as ingredient (a)),
Contains water and a component selected from citric acid and its salts,
The content of the component (a) is 1.1% by mass or more and 10% by mass or less ,
(b) the content of a cationic bactericide (hereinafter referred to as component (b)) is less than 0.1% by mass;
(c) the content of ethanol [hereinafter referred to as component (c)] is less than 10% by mass,
a mass ratio of the content of the component (a) to the content of the component selected from citric acid and a salt thereof , (a)/( a component selected from citric acid and a salt thereof), is 3 or more and 37.5 or less;
A human norovirus inactivation agent having a pH of 10 or more and 14 or less at 25°C. The inactivating agent according to claim 1, which contains one or more components selected from (d) (d1) a surfactant [excluding component (b)] [hereinafter referred to as component (d1)] and (d2) an organic solvent [excluding component (c)] [hereinafter referred to as component (d2)]. A method for inactivating human norovirus, comprising contacting an object containing human norovirus with the human norovirus inactivating agent according to claim 1 or 2 and leaving the object for a predetermined period of time (however, humans are excluded from the object) . The method for inactivating human norovirus according to claim 3, wherein the predetermined time is 1 minute or more and 300 minutes or less. A method for treating an object (excluding humans), comprising attaching the human norovirus inactivating agent according to claim 1 or 2 to the object to form a portion on the object that inactivates human norovirus.