JP2022030481A - Antiviral agent - Google Patents

Abstract

To provide an antiviral agent to SARS-CoV-2 that can prevent or improve a SARS-CoV-2 infectious disease, and inactivate SARS-CoV-2.SOLUTION: An antiviral agent to SARS-CoV-2 has epigallocatechin gallate as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to antiviral agents, specifically antiviral agents against SARS coronavirus-2.

SARS coronavirus-2 (Severe acute respiratory syndrome 2,; SARS-CoV-2) belongs to the genus Beta coronavirus like SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV), and acute respiration. It is a SARS-related coronavirus that causes organ disease (COVID-19). The outbreak was first confirmed in 2019 near Wuhan City, Hubei Province, China, and has since caused a global pandemic of COVID-19.

At present, there are no vaccines or therapeutic agents for SARS-CoV-2, so infection can be prevented by removing the virus or inactivating the virus by cleaning and disinfecting the fingers, clothes, and various instruments and materials to which the virus can adhere. It is important to. Regarding disinfectants with anti-virus effect against SARS-CoV-2, substances expected to have anti-virus effect have been verified with reference to literature information on SARS-CoV and influenza virus, including ethanol with a certain concentration or higher. , Linear alkylbenzene sulfonate sodium, polyoxyethylene alkyl ether and other specific surfactants have been reported to be effective against SARS-CoV-2.

Epigallocatechin gallate (EGCg) is known as a major component of tea catechins, but EGCg has been reported to have bactericidal activity against enterohemorrhagic Escherichia coli such as O-157 (non-EGCg). Patent Document 1). In addition, it has been reported that tea polyphenol has an infection-preventing effect against porcine infectious gastroenteritis virus (TGE virus) and porcine epidemic diarrhea virus (PED virus), which are coronaviruses that infect domestic animals (PED virus). Patent Document 1).
On the other hand, theaflavin-3,3'-digalate (TF3), which is an oxidative condensate of catechin, has an inhibitory activity against 3C-like protease (3CLPro), which is considered to be important for viral replication of SARS-CoV. It has also been reported that catechins such as EGCg and epicatechin (EC) do not inhibit the 3CLPro activity (Non-Patent Document 2).

Japanese Unexamined Patent Publication No. 2000-44473

Okubo et al., Journal of the Japanese Association of Infectious Diseases, Vol. 72, No. 3: 211-217, March 20, 1998 Chia-Nan Chen et al., ECAM 2005; 2 (2) 209-215

The present invention relates to providing an antiviral agent against SARS-CoV-2, which enables prevention or amelioration of SARS-CoV-2 infection and inactivation of SARS-CoV-2.

Among highly safe natural materials, the present inventors have an effect that EGCg has an effect of suppressing the growth of SARS-CoV-2 and an effect of inactivating SARS-CoV-2, and has an effect on SARS-CoV-2. It was found to be useful as an antiviral agent.

That is, the present invention relates to the following 1) to 4).
1) An antiviral agent against SARS-CoV-2 containing EGCg as an active ingredient.
2) A preventive or ameliorating agent for SARS-CoV-2 infection containing EGCg as an active ingredient.
3) An antiviral food against SARS-CoV-2 containing EGCg as an active ingredient.
4) A food for preventing or improving SARS-CoV-2 infection containing EGCg as an active ingredient.

According to the antiviral agent against SARS-CoV-2 of the present invention, it is possible to prevent or ameliorate SARS-CoV-2 infectious diseases such as severe acute respiratory syndrome. Further, according to the antiviral agent against SARS-CoV-2 of the present invention, SARS-CoV-2 adhering to a hard / soft surface in a living environment can be inactivated, and the spread of SARS-CoV-2 infection can be prevented or spread. Can be reduced.

As used herein, SARS coronavirus-2 (SARS-CoV-2) is a SARS-related coronavirus that causes acute respiratory disease (COVID-19).
SARS-CoV-2 has a viral genome of about 29,903 bases and is a single-strand plus-strand RNA virus. Like a common coronavirus, it is composed of four proteins known as spike proteins, nucleoproteins, endogenous membrane proteins, and enveloped proteins, and RNA. Of these, nucleocapsids bind to RNA to form nucleocapsids, and lipid-bound spike proteins, endogenous membrane proteins, and enveloped proteins surround them to form envelopes (A pneumonia outbreak associated with a new coronavirus of probable). bat origin. Nature. 2020 Mar; 579 (7798): 270-273., A new coronavirus associated with human respiratory disease in China. Nature. 2020 Mar; 579 (7798): 265-269.).

Peplomers are large transmembrane proteins of type I consisting of two subunits, S1 and S2, where S1 is primarily a receptor binding domain (RBD) that recognizes cell surface receptors and S2 is a membrane fusion. Contains the necessary elements. Known receptors that bind to S1 include ACE2 (angiotensin converting enzyme 2), DPP4 (dipeptidyl peptidase-4), APN (aminopeptidase N), CEACAM (cancer fetal antigen-related cell adhesion molecule 1), O- There is ac Sia (O-acetylated sialic acid), and SARS-CoV-2 has been reported to infect human respiratory epithelial cells via human ACE2 (tructure, Function, and Antigenicity of the SARS-CoV). -2 Spike Glycoprotein Cell. Volume 181, Issue 2, 16 April 2020, Pages 281-292.e6).

The antiviral effect of the antiviral agent against SARS-CoV-2 of the present invention is to suppress the growth of SARS-CoV-2 and suppress the amount of virus in cells infected with SARS-CoV-2 (host cells). Or the action of reducing (SARS-CoV-2 growth inhibitory action) and the action of reducing or eliminating the activity of SARS-CoV-2 and eliminating the infectivity to the host cell (SARS-CoV-2 inactivating action). Be included. That is, the antiviral agent against SARS-CoV-2 of the present invention includes a SARS-CoV-2 growth inhibitor and a SARS-CoV-2 inactivating agent.
The growth inhibitory effect of SARS-CoV-2 can be confirmed, for example, by measuring the viral load of SARS-CoV-2 in the host cell infected with the virus or observing the morphological change of the host cell. can. Here, examples of the host cell include Vero cells derived from African green monkeys, intestinal organoids derived from humans and bats, and the like.

In the present invention, epigallocatechin gallate (EGCg) is known as a main component of tea catechin. EGCg is commercially available, and examples of commercially available products include Theavigo (DSM Nutrition Japan) and Sanphenon EGCg (Taiyo Kagaku Co., Ltd.). Alternatively, EGCg may be chemically synthesized or prepared from a raw material derived from tea leaves.

As a method of chemical synthesis, Higuchi et al, Chem. Let. , 35 (9): The method described in 1006-1007, 2006 and the like.
When prepared from a raw material derived from tea leaves, for example, it may be purified from a tea leaf extract or a concentrate thereof. The tea extraction is from the genus Camellia, eg, C.I. sinensis, C.I. It can be carried out by adding water or hot water, or if necessary, an extraction aid to tea leaves made from tea leaves obtained from assamica or a hybrid thereof, and stirring and extracting the tea leaves. Further, a method of extracting in a so-called non-oxidizing atmosphere may be used in combination while removing dissolved oxygen by aerating an inert gas such as boiling degassing or nitrogen gas. Examples of the extraction aid include organic acids such as sodium ascorbate or salts thereof. The tea leaves made are green teas such as roasted tea, bancha, tamaro, tencha, and kettle roasted tea; semi-fermented teas such as iron Kannon, color species, golden katsura, and martial arts rock tea collectively called oolong tea; Includes fermented teas such as Darjeeling, Uva, and Keyman.

The tea extract can be concentrated by concentrating the extract, and the tea extract can be purified by using a solvent or a column. Examples of the form of the concentrate or purified product of the tea extract include solid, aqueous solution, slurry and the like. For example, the tea extract can be prepared by the method exemplified in detail in JP-A-59-219384, JP-A-4-20589, JP-A-5-260907, JP-A-5-306279 and the like. can. Commercially available products can also be used, and as such commercially available tea extracts, ITO EN Co., Ltd. "Theafran", Mitsui Norin Co., Ltd. "Polyphenone", Taiyo Kagaku Co., Ltd. "Sunphenon", Suntory Ltd. Examples include "Suntory".

EGCg can be purified by treating these tea extracts or their concentrates with a macroporous polar resin, for example, as described in Japanese Patent Application Laid-Open No. 2001-97968. Further, the content of EGCg can be measured by an analysis method suitable for the condition of the measurement sample among the commonly known measurement methods, and can be analyzed by, for example, liquid chromatography. Specifically, the method described in the examples described later can be mentioned. At the time of measurement, appropriate treatment is performed as necessary, such as freeze-drying the sample to match the detection range of the device and removing impurities in the sample to match the separation ability of the device. May be given.

As shown in Examples described later, when EGCg is added to Vero cells infected with SARS-CoV-2, the proliferation of SARS-CoV-2 can be effectively suppressed at an EGCg concentration of 100 ppm or more. Also, the virus can be inactivated by contacting EGCg with SARS-CoV-2.
Therefore, EGCg serves as an antiviral agent against SARS-CoV-2 having a growth inhibitory effect on SARS-CoV-2 or an inactivating effect on SARS-CoV-2, or a preventive or ameliorating agent for SARS-COV-2 infection. obtain. Alternatively, EGCg can be used to produce an antiviral agent against SARS-CoV-2 or a prophylactic or ameliorating agent for SARS-COV-2 infection.
In addition, EGCg can be used for suppressing the growth of SARS-CoV-2, for inactivating SARS-CoV-2, or for preventing or ameliorating SARS-COV-2 infection. For example, by administering or ingesting EGCg to a subject, the growth of SARS-CoV-2 can be suppressed, SARS-COV-2 infection can be prevented or ameliorated, and by contacting the subject with EGCg. , SARS-CoV-2 can be inactivated.
Here, the use may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical practice, that is, a concept that does not include a method of operating, treating or diagnosing a human, and more specifically, a doctor or a person who has been instructed by a doctor to operate on a human. , A concept that does not include a method of performing treatment or diagnosis.

Here, the SARS-COV-2 infection refers to an acute respiratory disease (COVID-19) caused by infection with SARS-COV-2. The symptoms are not specific and range from asymptomatic (asymptomatic) to severe pneumonia. Typical symptoms / signs include fever, dry cough, fatigue, sputum, shortness of breath, sore throat, headache, diarrhea, etc., but the present invention is not limited to such symptoms.

In the present invention, "prevention" means preventing, suppressing or delaying SARS-COV-2 infection in an individual, or reducing the risk of developing SARS-COV-2 infection. Further, "improvement" means infection of SARS-COV-2 or improvement of symptoms, prevention or delay of worsening of symptoms, or reversal, prevention or delay of progression of symptoms, and includes "treatment".

The antiviral agent for SARS-CoV-2 or the preventive or ameliorating agent for SARS-COV-2 infection of the present invention may be in the form of using EGCg alone, or a composition containing the same (for example, a pharmaceutical product). It may be in the form of a composition, a food composition, a disinfectant composition, a sanitary product composition, etc.). That is, the antiviral agent against SARS-CoV-2 of the present invention is a drug, a non-medicinal product, or a food (that is, an effect of suppressing SARS-CoV-2 growth or preventing or ameliorating SARS-COV-2 infection). SARS-CoV-2 growth inhibitory food, SARS-COV-2 infection prevention or improvement food), SARS-CoV-2 inactivating effect, bactericidal disinfectant composition, sanitary product composition, or these Can be a material or formulation for compounding into.
In addition to general foods and drinks, foods for suppressing the growth of SARS-CoV-2 and foods for preventing or improving SARS-COV-2 infections include foods labeled to that effect, functional foods, and diseases as necessary. Includes foods for persons, foods for specified health use, foods with functional claims, and supplements.

The above-mentioned pharmaceutical products (including quasi-drugs) can be administered in any form, and may be orally or parenterally administered. Depending on the dosage form, the drug may be added to the active ingredient of the present invention as a solid or liquid pharmaceutical non-toxic carrier suitable for various dosage forms, for example, a stabilizer, a wetting agent, an emulsifier, a binder, or isotonicization. Conventional additives such as agents and excipients may be added as appropriate, and the preparation may be carried out by conventional means in the formulation. Examples of the form of the pharmaceutical composition include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, oral preparations such as sublingual tablets and troches, and nasal drops. Examples thereof include a spray agent, a spray agent, and a freeze-drying agent.

The above-mentioned food can be prepared as various pharmaceutical forms suitable for edible or drinking, for example, fine granules, tablets, granules, powders, capsules, syrups, liquids, pastes and the like. The types of foods are not limited, but for example, breads, noodles, pasta, jelly-like foods, various snacks, cakes, confectionery, ice creams, soups, dairy products, frozen foods, instant foods, and other processed foods. , Seasonings, supplements, various beverages (fruit juice beverages, carbonated beverages, tea-based beverages, coffee beverages, dairy beverages, alcoholic beverages, soft beverages, etc.), etc. Foods that stay in the oral cavity for a long time or stay in the digestive tract for a long time, such as supplements, are preferable.

Such pharmaceuticals, quasi-drugs, and foods may be appropriately blended with bactericides, anti-inflammatory agents, water-soluble vitamins, plant extracts, and other medicinal ingredients as long as the effects of the present invention are not impaired.

The above-mentioned bactericidal disinfectant composition includes antibacterial substances such as hypochlorite, hydrogen peroxide, and silver ionization, cationic antibacterial agents (benzethonium chloride, etc.), bactericidal agents (tricrosan, isopropylmethylphenol, etc.), and ethanol. , Surfactant, etc., chelating agent, moisturizing agent, lubricant, builder, buffering agent, abrasive, electrolyte, bleaching agent, fragrance, dye, foam control agent, corrosion inhibitor, essential oil, thickening It is prepared by appropriately blending additives such as agents, pigments, gloss improvers, enzymes, detergents, solvents, dispersants, polymers, silicones, and water-soluble substances. The sterilizing and disinfecting agent composition is in the form of liquid, milky liquid, cream, lotion, paste, gel, sheet (supporting a base material), aerosol, spray, oil, gel, etc. It is possible, but not limited to these.

Examples of the hygiene product composition include lotions, creams, shampoos, hair conditioners, hand soaps, body shampoos, mouthwashes, bathing agents, foams, antiperspirants, deodorants, axillary odor inhibitors, and oral hygiene products (mouthwash). Liquids, toothpastes, mouthwashes, mouthwashes, etc.).
The composition is a carrier (for example, a diluent, a dispersant, a buffer, a pH adjuster, a dispersant, an emulsifier, a surfactant, a preservative, a stabilizer, an antioxidant, a colorant) that is acceptable as cosmetics and the like. , Moisturizer, thickener, bactericide, fragrance, etc.) can be appropriately combined and prepared by a conventional method.

The content of the active ingredient in the embodiment in which the antiviral agent for SARS-CoV-2 of the present invention or the preventive or ameliorating agent for SARS-COV-2 infection is used as a composition is appropriately determined according to the form of the composition. can. For example, the content of the active ingredient with respect to the total amount of the composition is preferably 0.005% by mass or more, more preferably 0.008% by mass or more, still more preferably 0.01% by mass or more in terms of EGCg. Further, 10% by mass or less is preferable, 8% by mass or less is more preferable, and 5% by mass or less is further preferable. Further, the numerical range of the content of the active ingredient is preferably 0.005 to 10% by mass, more preferably 0.008 to 8% by mass, still more preferably 0.01 to 5% by mass.

When the antiviral agent for SARS-CoV-2 of the present invention or the preventive or ameliorating agent for SARS-COV-2 infection is used as a drug, a non-medicinal product or a food, the target of administration or inoculation is preferably a mammal. And includes non-human mammals, but is preferably human. In addition, the subject of administration or inoculation is preferably a human who desires prevention of SARS-CoV-2 infection, symptoms of SARS-CoV-2 infection (fever, dry cough, fatigue, sputum, shortness of breath, sore throat, headache, etc. Humans who desire to improve diarrhea, etc.) can be mentioned.
The administration or inoculation amount in this case can be appropriately determined depending on the individual's condition, body weight, sex, age, material activity, administration route, administration schedule, pharmaceutical form or other factors. For example, 20 mg / day or more is preferable, and 100 mg / day or more is more preferable in terms of EGCg per adult (body weight 60 kg). Further, 3000 mg / day or less is preferable, 1000 mg / day or less is more preferable, and 800 mg / day is more preferable. Further, the numerical range of the dose of the active ingredient is preferably 20 to 3000 mg / day, more preferably 100 to 1000 mg / day.
The active ingredient may be ingested and administered once to several times a day, or at arbitrary periods and intervals.

Further, the antiviral agent against SARS-CoV-2 of the present invention can be used as a disinfectant composition or a sanitary product composition to contaminated the skin or mucous membrane of an animal or an inanimate object contaminated with SARS-CoV-2. It is possible to inactivate the virus adhering to the hard or soft surface of the virus. Here, as the surface of an inanimate object, for example, a hard surface such as a counter, a sink, a dressing room, a toilet, a bathtub, a shower stand, a floor, a window, a door knob, a wall, a sewage outlet, and a pipe in a home or business facility; Hard surfaces such as kitchen utensils, furniture, telephones, toys and other utensils, tools, miscellaneous goods; soft surfaces such as textile products (carpets, area rugs, curtains, cloth furniture, clothing, etc.).
The composition is used by contacting it with a treatment target, but the embodiment thereof is not particularly limited, and a method of applying the composition to the treatment target as it is, a pump spray, an aerosol, a pressurized liquid spray spray, or the like. A method of spraying or spraying in an atomized state using an atomizer such as a pressurized air atomizer, or a sheet, gauze, towel, hand towel, tissue, wet tissue, etc. impregnated with the composition. Any method such as wiping the surface may be used.
The concentration of EGCg when treating the treatment target is 100 ppm or more, preferably 150 ppm or more, and more preferably 200 ppm or more from the viewpoint of SARS-CoV-2 inactivation.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Analysis of EGCg
The sample solution is filtered with a filter (0.45 μm), and a packed column for an octadecyl group-introduced liquid chromatograph (L-column TM ODS 4.6 mm φ × 250 mm) is used using a high performance liquid chromatograph (model SC L-10AVP, manufactured by Shimadzu Corporation). : Chemical Substance Evaluation and Research Institute) was attached and analyzed by the gradient method at a column temperature of 40 ° C. As a standard product of EGCg, one manufactured by Kurita Water Industries was used and quantified by the calibration curve method. Mobile phase A solution was a distilled aqueous solution containing 0.1 mol / L acetic acid, solution B was an acetonitrile solution containing 0.1 mol / L acetic acid, the sample injection amount was 10 μL, and the UV detector wavelength was 280 nm. .. The conditions for the gradient are as follows.

Example 2
(1) SARS-CoV-2 growth inhibitory effect by EGCg 1) Test substance EGCg (epigallocatechin hydrate epigallocatechin gallate): Tokyo Kasei Kogyo Co., Ltd. (purity 98% or more)

2) Test method As SARS-CoV-2, SARS-CoV2 (KUH003) isolated at Kitasato University was used. Vero E6 / TMPRSS2 cells (derived from African green monkey kidney) were used as the cultured cells, and Pen-Strep (manufactured by Cosmo Bio Co., Ltd.) and G418 (manufactured by Sigma Aldrich) were added to the medium to contain 5% FBS-containing DMEM medium (Nakalitesk). The cells were cultured at 37 ° C. in the presence of 5% CO ₂ (manufactured by the same company). In the state of being 100% confluent, the medium was removed and washed twice with 2% FBS-containing DMEM on a 96-well plate, and finally 50 uL each of 2% FBS-containing DMEM was added.
50 μL of SARS-CoV2 solution (2.5 × ¹⁰⁶ RNA copy / well) was added thereto so that MOI = 50, and the cells were cultured in a 37 ° C. 5% CO ₂ environment for 1 hour to infect the virus. After infection, the virus solution was removed and washed once with DMEM containing 2% FBS. Then, a solution in which EGCg dissolved in dimethyl sulfoxide was serially diluted 2-fold from 2000 μg / mL was added in increments of 100 μL / well, and the cells were cultured at 37 ° C. at 5% CO ₂ . After 24 hours of culturing, 3 μL of the supernatant was collected and subjected to RT-qPCR. RT-qPCR was quantified using Toyobo's SARS-CoV-2 Detection Kit.

3) Results The results are shown in Table 2.

As a result, it was found that the growth of SARS-CoV-2 was suppressed to less than 1/100 when EGCg was 100 ppm or more.

(2) SARS-CoV-2 inactivation test with EGCg 1) Test method 3 μL of SARS-CoV-2 isolated in the same manner as in Example 1 and 27 μL of EGCg aqueous solution solution were uniformly mixed and allowed to stand for 1 minute. Then, 270 uL of 2% FBS medium was added to stop the reaction, and pipetting was performed. From there, 20 μL each was taken, added to 100% confluent Vero-E6 / TMPRSS2 cells (previously added with 180 μL of 2% FBS medium) to 96-well plates, lightly pipetting and then infected for 1 hour. Then, after washing twice with 200 μL of DMEM medium containing 2% FBS, 200 μL of DMEM medium containing 2% FBS was added and cultured under 37 ° C./5% CO ₂ , and 3 days later, by the same method as in Example 1. RT-qPCR and host cell mutation observation (CPE) were performed.

2) Results The results are shown in Table 3.

Mutation observation (CPE) in the table shows no cell mutations, in other words, not infected with virus. In +, the cell is mutated.
Regarding the virus inactivating effect, those having a CPE of-and a viral load below the detection limit by PCR were considered to be effective.

As a result, it was clarified that SARS-CoV-2 can be completely inactivated when EGCg is 200 ppm or more.

Claims (6)

An antiviral agent against SARS coronavirus-2 containing epigallocatechin gallate as an active ingredient. A preventive or ameliorating agent for SARS coronavirus-2 infection containing epigallocatechin gallate as an active ingredient. An antiviral food against SARS coronavirus-2 containing epigallocatechin gallate as an active ingredient. A food for preventing or ameliorating SARS coronavirus-2 infection containing epigallocatechin gallate as an active ingredient. The antiviral agent according to claim 1, which is a disinfectant composition or a sanitary product composition. The antiviral agent according to claim 5, wherein EGCg is used at a concentration of 100 ppm or more with respect to the treatment target.