WO2022079997A1

WO2022079997A1 - Anti-coronavirus virucide and method and usage employing same

Info

Publication number: WO2022079997A1
Application number: PCT/JP2021/030071
Authority: WO
Inventors: 成実岡崎
Original assignee: Ｄｒ．Ｃ医薬株式会社
Priority date: 2020-10-15
Filing date: 2021-08-17
Publication date: 2022-04-21
Also published as: JPWO2022079997A1

Abstract

Provided are: an anti-coronavirus virucide capable of destroying novel coronavirus in a simple, economic manner; and a method and usage employing the same.　The present invention provides an anti-coronavirus virucide containing composite particles, wherein the titanium oxide particle content is 70-96 mass% and the silver particle content is greater than 0.5 mass% and at most 3.6 mass% with respect to the total mass of the anti-coronavirus virucide.

Description

Coronavirus killing agent and methods and uses using it

The present invention relates to a coronavirus killing agent and a method and use using the same.

Currently, the new coronavirus (SARS-CoV-2), which causes unprecedented damage in the world, is one of the coronaviruses and the seventh virus that infects humans. Coronavirus is a type of RNA virus (single-stranded RNA virus) that has RNA as genetic information, and has a double membrane made of lipid called "envelope" on the outermost side of the particle. It does not increase by itself, but it can increase by adhering to cells such as mucous membranes. Regarding new coronavirus infections, knowledge about pathogens and diseases is gradually accumulating (see, for example, Non-Patent Documents 1 and 2).
The new coronavirus infection is generally said to be transmitted by droplet infection or contact transmission. In an environment such as talking to many people at close range in a closed space, there is a risk of spreading the infection even if there are no symptoms such as coughing or sneezing. According to a report from the World Health Organization (WHO), a five-minute conversation generally produces as many flies (about 3,000) as a single cough. Flying infection is when an infected person holds a sneeze or cough with his or her hand and then touches something around him with the virus, and when another person touches it, the virus attaches to his or her hand. Infection through the mucous membrane by touching the mouth or nose. According to a WHO report, the new coronavirus survives up to 72 hours on plastic surfaces and up to 24 hours on cardboard (see, for example, Non-Patent Documents 3 and 4).
As a clinical feature, it develops with fever, respiratory symptoms, general malaise, etc. after an incubation period of 1 to 14 days (mostly 5 days). Cold-like symptoms often persist for about a week. Some exhibit symptoms such as dyspnea and become severe. In addition, most of the affected people are considered to be mild, but strict caution is required because it may become serious especially in the elderly and those with underlying diseases. The increase in the number of seriously ill people should be avoided because it puts pressure on medical institutions and may lead to the collapse of medical care. Therefore, countermeasures against new coronavirus infectious diseases are required.

Development of new drugs and vaccines is required as one of the countermeasures against new coronavirus infectious diseases. However, in general, when developing new drugs, it is necessary to confirm their effectiveness and safety, and whether mass production is possible while ensuring a certain level of quality, and development is on an annual basis. Since it takes a long time, there is a problem that it takes time to reach the market in general. In addition, the development of new drugs requires enormous costs, and there is also the problem that legislation must be established for early approval.
It is expected that herd immunity will be obtained as an effect of vaccination. Herd immunity means that more than a certain percentage of the population has immunity, which makes it difficult for infected patients to infect other people and prevents the spread of infectious diseases. However, depending on the vaccine, even if vaccination has the effect of preventing aggravation, the effect of preventing infection is poor, and no matter how many people are vaccinated, the effect of herd immunity may not be obtained. In fact, it is not known if the new corona vaccine will have a herd immunity effect, and it is believed that it will take a considerable amount of time and effort to do so. In addition, there is concern that mutations in the new coronavirus may reduce the effects of immunity and vaccines. For example, it has been pointed out that the immune response / vaccine effect against the new coronavirus of Asian races is weakened in the "L452R" mutant strain, which is also called an Indian mutant virus. Under these circumstances, it is feared that herd immunity will not be obtained until a long time ago.
In this way, there are still issues that cannot be solved by the development of vaccines alone as a countermeasure against new coronavirus infectious diseases.

As another method for controlling new coronavirus infections, a member for preventing droplet infection and contact infection and a method using the same have been proposed, focusing on the above-mentioned infection route. As a concrete measure, wearing a mask is recommended. However, although wearing a mask could suppress the spread of the new coronavirus, it could not kill the new coronavirus. Therefore, as one of the countermeasures against infectious diseases of the new coronavirus, there has been a demand for a coronavirus killing agent that can be used for members such as masks.

From the above, an object of the present invention is to provide a coronavirus killing agent capable of killing a new type of coronavirus easily and economically, and a method and use using the same.

The present invention relates to the following contents.
<1> A coronavirus-killing agent comprising composite particles, wherein the coronavirus-killing agent contains 70% by mass to 96% by mass of titanium oxide particles and silver particles based on the total mass of the coronavirus-killing agent. A coronavirus killing agent containing an amount of more than 0.5% by mass and 3.6% by mass or less.
<2> The composite particle has a particle size of more than 0.1 μm and less than 0.3 μm as measured by a laser diffraction method, in which silver particles are bonded to the surface of titanium oxide particles. Viral agent.
<3> Based on the total mass of the coronavirus-killing agent, the content of titanium oxide particles is 78 to 96% by mass, and the content of silver particles is more than 0.5% by mass and 3.3% by mass or less <1>. Or the coronavirus killing agent according to <2>.
<4> The coronavirus killing agent according to any one of <1> to <3>, wherein the coronavirus is COVID-19.
<5> The coronavirus killing agent according to any one of <1> to <4>, further comprising calcium phosphate particles.
<6> The coronavirus killing agent according to any one of <1> to <5>, wherein the particle size of the composite particle is 0.2 μm or more and 0.29 μm or less.
<7> The coronavirus killing agent according to any one of <1> to <6>, wherein the particle size of the composite particle is 0.24 μm or more and 0.27 μm or less.
<8> A member containing the coronavirus-killing agent according to any one of <1> to <7>.
<9> The member according to <8>, wherein the member is any one selected from the group consisting of non-woven fabric, woven fabric, knitted fabric, resin film, plastic, metal, ceramics, and air filter.
<10> A medical device containing the coronavirus-killing agent according to any one of <1> to <7>.
<11> A mask containing the coronavirus-killing agent according to any one of <1> to <7>.
<12> A medical sheet containing the coronavirus-killing agent according to any one of <1> to <7>.
<13> A coronavirus-killing agent having silver particles bonded to the surface of titanium oxide particles and having a particle diameter of more than 0.1 μm and less than 0.3 μm as measured by a laser diffraction method, or a member containing the same. A coronavirus killing method that places the virus in a place where the presence of coronavirus is expected.
<14> The method for preventing the growth of coronavirus according to <13>, wherein the coronavirus killing agent or a member containing the agent is the one according to <8> or <9>.

According to the present invention, a coronavirus killing agent capable of killing a new type of coronavirus, and a method and use using the same are provided simply and economically.

1A and 1B are electron micrographs of cells used in the experiment. FIG. 1A shows VeroE6 / TMPRSS2 cells, and FIG. 1B shows VeroE6 cells. 2A to 2F are views showing the results of shape comparison (2nd day) with the eluate-added cells. FIG. 2A is hydrosilver titanium powder (low concentration), FIG. 2B is hydrosilver titanium powder (high concentration), FIG. 2C is A1 sheet (low concentration), FIG. 2D is A2 sheet (high concentration), and FIG. 2E is binder processing only. The sheet, FIG. 2F, is an additive-free sheet. 3A to 3F are views showing the results of shape comparison (4th day) with the eluate-added cells. FIG. 3A is a hydrosilver titanium powder (low concentration), FIG. 3B is a hydrosilver titanium powder (high concentration), FIG. 3C is an A1 sheet (low concentration), FIG. 3D is an A2 sheet (high concentration), and FIG. 3E is binder processing only. The sheet, FIG. 3F, is an additive-free sheet. FIG. 4 is a photograph before culturing (left of the screen) and after culturing (3rd day, right of the screen) when the virus was cultured in the VP-SFM medium. FIG. 5 is a virus infectious titer test crystal violet stained image. FIG. 6 is a list of virus liquid processing conditions. FIG. 7A is a conceptual diagram of the dipping method, and FIG. 7B is a conceptual diagram of the dropping method. FIG. 8 is a layout drawing of the dipping method plate. FIG. 9 is a layout drawing of the dropping method plate. FIG. 10 is a layout diagram of virus inoculation for measuring infectious titer. FIG. 11 is a virus infectious titer test plaque image (refrigerated 24 hr immersion treatment). FIG. 12 is a list of virus infectious titers and antiviral activity values (immersion method). FIG. 13 is a list of virus infectious titers and antiviral activity values (drop method). FIG. 14 is a list of antiviral activity of A1 sheet and A2 sheet. FIG. 15 is a diagram showing a configuration. FIG. 16 is an image diagram of the test. FIG. 17 is an image diagram of the test. FIG. 18 is a diagram showing the results of SDS-PAGE (+ βME), which is electrophoresis when the S domain sample solution is irradiated with artificial sunlight in the presence of composite particles M1 and composite particles M2. FIG. 19 is a diagram showing the results of SDS-PAGE (+ βME), which is an electrophoresis when an S domain sample solution is irradiated with artificial sunlight in the presence of an X1 sheet and an X2 sheet. FIG. 20A is a diagram showing the ratio of the amount of disintegrated protein over time to the control when the composite particles M1 and M2 are used. FIG. 20B is a diagram showing the ratio of the amount of disintegrated protein over time to the control when using the X1 and X2 sheets. FIG. 21 is a molecular dynamics (MD) simulation diagram of the new coronavirus spike protein. 21A to 21C show a view seen from the side, and FIGS. 21D to 21F show a view seen from above (outside). 22A-22D are molecular dynamics (MD) simulation diagrams focusing on the peplomer protein S domain and ACE2 receptor. FIG. 23A is a conceptual diagram of an electron microscope (SEM) photograph of a composite particle. FIG. 23B is a partially enlarged view of FIG. 23A. FIG. 24A is a conceptual diagram of the configuration of the coronavirus. FIG. 24B is a partially enlarged view of FIG. 24A. 25A-25G are schematic views of the coronavirus infection system. It is a partially cutaway perspective view of a mask as a medical instrument according to an embodiment. It is a perspective view of the sheet as a medical instrument which concerns on embodiment.

Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments.

As a result of diligent research to solve the above-mentioned problems, the present inventor has found that composite particles containing titanium oxide and silver at a predetermined concentration kill coronavirus at a mortality rate of 99.9% or more. This is amazing. The present invention is based on the above findings. An embodiment will be described below.

[Coronavirus killing agent]
The present invention is a coronavirus killing agent comprising composite particles having a particle size of more than 0.1 μm and less than 0.3 μm as measured by a laser diffraction method, wherein the coronavirus killing agent is all of the coronavirus killing agents. The present invention relates to a coronavirus killing agent containing 70% by mass to 96% by mass of titanium oxide particles and more than 0.5% by mass and 3.6% by mass or less of silver particles on a mass basis.
As will be described later, the composite particles have a morphology in which silver particles are bonded to the surface of titanium oxide particles.

(Titanium oxide particles)
The number of titanium oxide particles per composite particle may be one or two or more. The number of titanium oxide particles per composite particle is usually two or more.

The form of the titanium oxide particles contained in the composite particles is not particularly limited, and examples thereof include spherical, granular, needle-like, flaky, and indefinite shapes. The composite particles may contain two or more titanium oxide particles having different morphologies.

The particle size of the titanium oxide particles contained in the composite particles is not particularly limited as long as it is smaller than the particle size of the composite particles, and can be appropriately adjusted according to the particle size of the composite particles. The titanium oxide particles contained in the composite particles are, for example, nanoparticles or submicron particles.

Examples of the crystal structure of titanium oxide constituting the titanium oxide particles include anatase type, rutile type, brookite type and the like, of which anatase type is preferable.

(Silver particles)
The number of silver particles per composite particle may be one or two or more. The number of metal particles per composite particle is usually two or more.

The form of the silver particles contained in the composite particles is not particularly limited, and examples thereof include spherical, granular, needle-like, flaky, and indefinite shapes. The composite particle may contain two or more metal particles having different morphologies.

The particle size of the silver particles contained in the composite particles is not particularly limited as long as it is smaller than the particle size of the composite particles, and can be appropriately adjusted according to the particle size of the composite particles. The silver particles contained in the composite particles are, for example, nanoparticles or submicron particles.

(Component ratio)
The composite particles preferably have a content of titanium oxide particles of 70% by mass to 96% by mass and a content of silver particles of more than 0.5% by mass and 10% by mass or less based on the total mass of the composite particles. This is because the slaughtered coronavirus effect cannot be obtained if the range is out of the above range. It is more preferable that the content of the titanium oxide particles is 78 to 96% by mass and the content of the silver particles is more than 0.5% by mass and 3.3% by mass or less.

(Other ingredients)
The composite particles may further contain calcium phosphate particles. The number of calcium phosphate particles per composite particle may be one or two or more. The number of calcium phosphate particles per composite particle is usually two or more.

The morphology of the calcium phosphate particles contained in the composite particles is not particularly limited, and examples thereof include spherical, granular, needle-like, flaky, and indefinite shapes. The composite particles may contain two or more calcium phosphate particles having different morphologies.

The particle size of the calcium phosphate particles contained in the composite particles is not particularly limited as long as it is smaller than the particle size of the composite particles, and can be appropriately adjusted according to the particle size of the composite particles. The calcium phosphate particles contained in the composite particles are, for example, nanoparticles or submicron particles.

Examples of the calcium phosphate constituting the calcium phosphate particles include apatite (apatite), tricalcium phosphate, octacalcium phosphate and the like, and among these, apatite is preferable. Examples of the apatite include hydroxyapatite, fluorinated apatite, carbonated apatite and the like, and among these, hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) is preferable.

When the composite particles contain calcium phosphate particles, the content of the titanium oxide particles, the silver particles and the calcium phosphate particles per composite particle is not particularly limited, but the lower limit of the content of the titanium oxide particles is one mass of silver particles. It is usually 10 parts by mass, preferably 20 parts by mass, more preferably 25 parts by mass, still more preferably 30 parts by mass, and the upper limit of the content of titanium oxide particles is 1 part by mass of silver particles. On the other hand, it is usually 300 parts by mass, preferably 250 parts by mass, more preferably 200 parts by mass, and even more preferably 180 parts by mass.
The lower limit of the content of the calcium phosphate particles is usually 1 part by mass, preferably 2 parts by mass, more preferably 3 parts by mass with respect to 1 part by mass of the metal particles, and the upper limit of the content of the calcium phosphate particles is. , Usually 100 parts by mass, preferably 80 parts by mass, still more preferably 60 parts by mass, and even more preferably 50 parts by mass.

The composite particles may contain other metal particles in addition to the silver particles. The other metal particles can be selected from the group consisting of, for example, gold particles, platinum particles and copper particles. The composite particles may contain two or more metal particles (including silver particles) of different types.

(Particle size)
The particle size of the composite particles measured by the laser diffraction method is preferably 0.1 to 0.3 μm, more preferably 0.11 to 0.29 μm, and even more preferably 0.15 to 0.25 μm. The particle size is measured by the laser diffraction method using a commercially available particle size distribution measuring device, preferably a laser diffraction / scattering type particle size distribution measuring device Partica LA-960V2 series (manufactured by HORIBA).

(Construction)
In the composite particles, it is preferable that one or more titanium oxide particles and one or more silver particles are three-dimensionally and randomly arranged.

In one embodiment of three-dimensional and random arrangement, at least one silver particle is bonded to at least one titanium oxide particle.

In one embodiment of three-dimensional and random arrangement, one or more particles are present around one particle (one of the titanium oxide particles and one of the silver particles). .. In this embodiment, one or more particles of the same type may be adjacent to one or more particles of the same type, or one or more particles of different types may be adjacent to each other. Adjacent particles are preferably bonded to each other. Examples of the combination of adjacent particles include titanium oxide particles, silver particles, titanium oxide particles and silver particles, and the like.

In one embodiment of three-dimensional and random arrangement, a part of at least one of titanium oxide particles and silver particles is exposed on the surface of the composite particles.

In one embodiment of three-dimensional and random arrangement, a part of at least one titanium oxide particle and a part of at least one silver particle are exposed on the surface of the composite particle.

In one embodiment of three-dimensional and random arrangement, at least one particle selected from titanium oxide particles, metal particles and calcium phosphate particles is present inside the composite particles without being exposed on the surface of the composite particles. There is.

Whether one particle has a particle morphology, has a membranous morphology, or has a membranous morphology integrally with or in conjunction with another one or more particles is a composite particle. It may be affected by the mixing ratio of particles when producing. Depending on the compounding ratio, one particle may no longer maintain its particle morphology and may take a membranous morphology that is present on at least a portion of the surface of the composite particle. For example, when particle compositing is carried out by a mechanical method such as a bead mill or a ball mill, particles composed of a material having a hardness lower than that of other particles (for example, silver particles) take such a film-like form. obtain.

Two or more of the above embodiments relating to three-dimensional and random arrangement may be combined.

When containing calcium phosphate particles, in one embodiment of three-dimensional and random arrangement, at least one particle selected from titanium oxide particles, silver particles and calcium phosphate particles has a film-like morphology and is on the surface of the composite particle. It exists at least in part.

In the case of containing calcium phosphate particles, in one embodiment of three-dimensional and random arrangement, two or more particles selected from titanium oxide particles, silver particles and calcium phosphate particles have a film-like morphology as one or a series of particles. However, it is present on at least a part of the surface of the composite particle.

(Production method)
The composite particles are obtained by mixing titanium oxide powder and silver powder in a liquid using, for example, a wet mill, and one or more titanium oxide particles contained in the titanium oxide powder and one or more titanium oxide particles contained in the silver powder. It can be manufactured by combining with silver particles. The composite particles thus produced are subsequently used in the present invention without being sintered.

The titanium oxide content (purity) in the titanium oxide powder is preferably 90% by weight or more, more preferably 95% by weight or more, and even more preferably 98% by weight or more. The upper limit is, for example, 99%.

The particle size of the titanium oxide particles (primary particles) contained in the titanium oxide powder is not particularly limited, but is, for example, 0.03 to 0.3 μm. Since the wet mill can disperse the particle agglomerates into individual particles, the titanium oxide powder may contain agglomerates of titanium oxide particles (secondary particles). The particle size of the aggregate of titanium oxide particles is, for example, 1 to 2 μm. The particle size of the titanium oxide particles or aggregates thereof is measured using, for example, a transmission electron microscope (TEM) or a scanning electron microscope (SEM).

The silver content (purity) in the silver powder is preferably 80% by weight or more, more preferably 95% by weight or more, and even more preferably 98% by weight or more. The upper limit is, for example, 99.9%.

The particle size of the silver particles (primary particles) contained in the silver powder is not particularly limited, but is, for example, 0.1 to 1.9 μm. Since the wet mill can disperse the particle agglomerates into individual particles, the metal powder may contain agglomerates of metal particles (secondary particles). By storing the silver powder in a freezer until use, it is possible to suppress the aggregation of silver particles contained in the silver powder. The particle size of silver particles or aggregates thereof is calculated, for example, based on the specific surface area.

When calcium phosphate is used, the calcium phosphate content (purity) in the calcium phosphate powder is preferably 90% by weight or more, more preferably 95% by weight or more, still more preferably 98% or more.
The particle size of the calcium phosphate particles (primary particles) contained in the calcium phosphate powder is not particularly limited, but is, for example, 0.1 to 2.0 μm. Since the wet mill can disperse the particle aggregates into individual particles, the calcium phosphate powder may contain aggregates (secondary particles) of calcium phosphate particles. The particle size of the aggregate of calcium phosphate particles is, for example, 4 to 5 μm. The particle size of the calcium phosphate particles or their aggregates is measured by, for example, a laser diffraction / scattering method.

The wet mill can combine one or more titanium oxide particles and one or more silver particles while dispersing and finely pulverizing the titanium oxide powder and the particles contained in the silver powder in a liquid. Examples of the wet mill include a bead mill, a ball mill and the like, and among these, a bead mill is preferable. Examples of the material of the crushing medium such as beads and balls used in a mill such as a bead mill and a ball mill include alumina, zircon, zirconia, steel and glass, and among these, zirconia is preferable. The size (diameter) of the pulverized media can be appropriately adjusted according to the particle size and the like of the composite particles to be produced, but is usually 0.05 to 3.0 mm, preferably 0.1 to 0.5 mm. As the pulverizing medium, for example, beads or balls having a size of about 0.1 mm and a mass of about 0.004 mg can be used.

The liquid used for mixing is, for example, an aqueous medium such as water. When the liquid used for mixing is water, the total blending amount of the titanium oxide powder and the silver powder is usually 25 to 45 parts by mass, preferably 30 to 40 parts by mass with respect to 65 parts by mass of water. Is adjusted to.

When mixing raw materials containing titanium oxide powder, silver powder and liquid with a wet mill, various conditions such as the total amount of raw material powder added, the flow rate of the liquid, the peripheral speed of the blades in the cylinder, the stirring temperature, the stirring time, etc. , It can be appropriately adjusted according to the particle size and the like of the composite particles to be produced. The total amount of the raw material powder (titanium oxide powder, metal powder and calcium phosphate powder) added is, for example, 4 kg or more, the cylinder volume is, for example, 0.5 to 4 L, and the flow rate of the liquid is, for example, 0.5 to 3 L / min. The peripheral speed of the blade is, for example, 300 to 900 m / min, the liquid temperature is, for example, 20 to 60 ° C., and the mixing time per 1 kg of raw material powder is, for example, 0.5 to 2 hours. The upper limit of the total addition amount of the raw material powder can be appropriately adjusted according to the cylinder volume and the like. The mixing time can be appropriately adjusted according to the total amount of the raw material powder added and the like.

It is preferable to add a dispersant to the raw material in addition to titanium oxide powder, silver powder and liquid. Examples of the dispersant include a polymer-type dispersant, a low-molecular-weight dispersant, an inorganic-type dispersant, and the like, and can be appropriately selected depending on the type of liquid used in the wet mixing. When the liquid used for mixing is an aqueous medium such as water, for example, an anionic polymer-type dispersant, a nonionic polymer-type dispersant, or the like can be used as the dispersant, and an anion can be used. Examples of the sex polymer type dispersant include a polycarboxylic acid type dispersant, a naphthalin sulfonic acid formalin condensation type dispersant and the like, and examples of the nonionic polymer type dispersant include polyethylene glycol and the like. .. The amount of the dispersant added can be appropriately adjusted, but is, for example, 0.01 to 5% by mass, preferably 0.05 to 3% by mass, based on 35 parts by mass of the total amount of the titanium oxide powder and the silver powder. %.

Using a wet mill, titanium oxide powder and metal powder are mixed in a liquid to combine one or more titanium oxide particles contained in the titanium oxide powder with one or more silver particles contained in the silver powder. By doing so, a suspension (slurry) of composite particles can be produced. Then, by removing the solvent in the suspension by evaporation or the like, an aggregate of composite particles (dry powder) can be produced. An aggregate of composite particles (dry powder) can also be produced from a suspension (slurry) of composite particles by a known granulation method such as a spray-dry granulation method.

The particle size of the aggregate of composite particles measured by the laser diffraction method is preferably more than 0.1 μm and less than 0.29 μm, and more preferably 0.15 μm or more and 0.25 μm or less. This is because if it is below the above lower limit, it may be absorbed by the human body. Further, if it is more than the above upper limit, the surface area of the composite particle becomes narrow and the action and effect of the present invention cannot be expected. The particle size of the composite particles is more preferably 0.2 μm or more and 0.29 μm or less, and further preferably 0.24 μm or more and 0.27 μm or less.
The median diameter (d50) of the aggregate of the composite particles measured by the laser diffraction method on a volume basis is, for example, 0.1 to 0.35 μm, preferably about 0.25 μm. The particle size by the laser diffraction method is measured by a commercially available particle size distribution measuring device, preferably a laser diffraction / scattering type particle size distribution measuring device Partica LA-960V2 series (manufactured by HORIBA).

The produced composite particles can be used as they are in the present invention, but the particle size may be adjusted before they are used in the present invention. The particle size can be adjusted, for example, by sieving the composite particles in the powder state or the suspension state.

In the aggregate of composite particles, the number of titanium oxide particles, silver particles and calcium phosphate particles per composite particle may be the same or different among the composite particles.

In addition to the composite particles containing one or more titanium oxide particles and one or more silver particles, the aggregate of the composite particles contains other particles that may be by-produced in the production of the composite particles. May be. Examples of other particles include single titanium oxide particles, single silver particles, single metal particles, single calcium phosphate particles, conjugates of titanium oxide particles (excluding metal particles and calcium phosphate particles), and metal particles of each other. (Excluding titanium oxide particles and calcium phosphate particles), conjugates of calcium phosphate particles (excluding titanium oxide particles and metal particles), and conjugate of titanium oxide particles and metal particles (excluding calcium phosphate particles) , A conjugate of titanium oxide particles and calcium phosphate particles (excluding metal particles), a conjugate of metal particles and calcium phosphate particles (excluding titanium oxide particles), and the like.

The composite particles can be applied to coronavirus, but among coronaviruses, it is preferably applied to COVID-19 (SARS-CoV-2). This is from the viewpoint that the mortality rate of SARS-CoV-2 reaches 99.9%.

[Coronavirus infection process]
For the purpose of facilitating the understanding of the action and effect of the present invention, the coronavirus infection process will be briefly described.
FIG. 24A is a schematic diagram of the coronavirus (SARS-CoV-2), and FIG. 24B is a partially enlarged view of FIG. 24A. The coronavirus is surrounded by an envelope protein (Envelope (E)), which is the outer shell, and RNA (ribonucleic acid) and N protein are stored inside it. Further, on the surface of the coronavirus, there are protruding spike proteins (Spike, S) and the like as shown in FIG. 24B. On the other hand, human cells have a molecular mechanism that allows coronavirus to efficiently invade. The Ace2 receptor used as an entrance to cells and the proteolytic enzymes TMPRSS2 and FURIN. The coronavirus infection process is thought to be as follows.
As shown in FIG. 25A, the coronavirus enters the body via the oral cavity and reaches the lungs. The coronavirus then binds superficial protruding peplomer proteins (Spike, S) to the Ace2 receptor on the host cell, as shown in FIG. 25B. Then, as shown in FIG. 25C, the protein-degrading enzymes TMPRSS2 and FURIN in the cell membrane cleave the head of the viral spike protein. As shown in FIG. 25D, the fusion mechanism begins to develop. As shown in FIG. 25E, the tip of the fusion mechanism penetrates the lung cells of the coronavirus. As shown in FIG. 25F, the fusion mechanism grabs and attracts lung cells. As shown in FIG. 25G, the coronavirus and lung cells fuse to open a channel through which N protein and RNA invade the lung cells. After that, the RNA that entered the lung cells is translated into protein, and the coronavirus proliferates.
According to the above-mentioned coronavirus infection process, if the coronavirus spike protein (S) or envelope protein (E) fails, it is considered that the coronavirus cannot infect human cells. Although the reason is not clear, the coronavirus killing agent according to the present invention has, for example, (1) destroys the spike protein (S), (2) inhibits the function of the spike protein (S), and (3) the envelope protein. It is believed that (E) is destroyed, or (4) a combination thereof kills the coronavirus.
Specifically, the spike protein (S) is considered to be destroyed as follows.
First, when titanium dioxide (TiO ₂ ) is irradiated with light (ultraviolet rays), electrons (e-) are ejected from its surface. At this time, by coexisting a metal catalyst (for example, Ag) with titanium dioxide, the emission of electrons is further promoted. The holes from which the electrons have escaped are called holes, which are positively charged. Since these holes have strong oxidizing power, they take electrons from hydroxide ions (OH-) in water, and the deprived hydroxide ions are OH radicals [・ OH] in a very unstable state. ]become. On the other hand, the electrons (e-) ejected from the surface of titanium dioxide reduce oxygen (O ₂ ) to generate oxygen radicals [O ₂ · ^- ]. It is considered that the spike protein (S) is destroyed by the OH radicals and oxygen radicals thus obtained.

[Members containing coronavirus killing agent]
The present invention also relates to a member containing the above-mentioned coronavirus killing agent.
Examples of the member include non-woven fabric, woven fabric, woven fabric, resin film, plastic, metal, ceramics, air filter and the like. Specific products include, for example, masks, towels, gauze, socks, underwear, shirts, eyeglasses and the like.

[Medical equipment]
The present invention also relates to a medical device containing the above-mentioned coronavirus killing agent. The medical device according to the present invention is a medical device capable of killing a new type of coronavirus. Hereinafter, a mask will be described as an example as a medical device according to an embodiment based on the drawings.

(mask)
FIG. 26 is a partially cutaway perspective view of a mask as a medical instrument according to an embodiment.
As shown in FIG. 26, the mask 10 includes a substantially rectangular mask body 11 that covers the nostrils of the target, ring-shaped ear hooks 12a and 12b provided on both short sides of the mask body 11, and a mask. A composite particle 13 detachably attached to the inside of the main body 11 is provided. Here, the composite particles include one or more titanium oxide particles and one or more metal particles.

When the mask 10 is attached to the target, the ear hook portion 12a is hung on one ear of the target, the ear hook portion 12b is hung on the other ear of the target, and at least the nostrils of the target face are covered with the mask body 11. .. When the mask 10 is attached to the subject, the mouth of the subject may be covered with the mask body 11 in addition to the nostrils of the subject.

As shown in FIG. 26, the form of the mask 10 is the form of the flat mask, but the form of the mask 10 is not limited to the form of the flat mask, and the pleated type mask, the three-dimensional type mask, etc. It may be in the form of other masks.

The mask body 11 is a mask body that covers the nostrils of the target and has breathability. The air permeability of the mask body 11 can be appropriately adjusted within a range in which the subject wearing the mask 10 can breathe. The air permeability of the mask body 11 is, for example, 5 to 150 cm ³ / cm ² · sec, preferably 30 to 100 cm ³ / cm ² · sec. The measurement of the air permeability is carried out in accordance with, for example, JIS L10968.27.1A method (Frazil type method).

The mask body 11 is formed of a plurality of laminated breathable sheet members. The edge portions of the plurality of breathable sheet members are joined by a known joining method such as heat welding, ultrasonic welding, or an adhesive. The mask main body 11 includes a first breathable sheet member 111, a second breathable sheet member 112, and a third breathable sheet member 113, which are stacked in this order. When the mask 10 is attached to the target, the first breathable sheet member 111 is arranged on the face side of the target, and the third breathable sheet member 113 is arranged on the outside air side. The number of breathable sheet members constituting the mask body 11 can be appropriately changed. For example, one or more breathable sheet members may be provided between the first breathable sheet member 111 and the second breathable sheet member 112. Further, one or more breathable sheet members may be provided between the second breathable sheet member 112 and the third breathable sheet member 113.

Each breathable sheet member can be formed of, for example, a non-woven fabric, a woven fabric, a knitted fabric, or the like. Examples of the fiber constituting each breathable sheet member include synthetic fiber, regenerated fiber, natural fiber and the like. Examples of the synthetic fiber include polyolefin fibers such as polyethylene and polypropylene, polyester fibers such as polyethylene terephthalate and polybutylene terephthalate, and polyamide fibers such as nylon. The synthetic fiber may be a composite fiber such as a core-sheath type fiber. Examples of the recycled fiber include rayon, acetate and the like. Examples of natural fibers include cotton and the like. Examples of the non-woven fabric include spunbond non-woven fabric, thermal bond non-woven fabric, spunlace non-woven fabric, air-through non-woven fabric, melt blow non-woven fabric, needle punch non-woven fabric and the like. Examples of the woven fabric include gauze and the like. The nonwoven fabric may have a multilayer structure having two or more layers. Examples of such a multi-layer structure include an SS structure (spun bond-spun bond two-layer structure), SMS (spun bond-melt blow-spun bond three-layer structure), and the like.

The basis weight of each breathable sheet member can be adjusted to the same extent as the breathable sheet member used in commercially available household or medical masks. The basis weights of the first breathable sheet member 111 and the third breathable sheet member 113 can be adjusted from the viewpoint of breathability, for example. The basis weight of the second breathable sheet member 112 can be adjusted, for example, from the viewpoint of filterability. When one or more breathable sheet members are provided between the first breathable sheet member 111 and the second breathable sheet member 112, or between the second breathable sheet member 112 and the third breathable sheet member 112. When one or more breathable sheet members are provided between the sex sheet member 113, the basis weight of these breathable sheet members can be adjusted from the viewpoint of breathability or filterability, for example.

The

ear hook portions

12a and 12b are formed of, for example, a string-shaped member. The string-shaped member preferably has elasticity. The string-shaped member is, for example, a stretchable rubber or plastic string-shaped member or the like. Both ends of the ear hooks 12a and 12b are fixed to the mask body 11 by a joining method such as sewing, whereby a ring that can be hung on the target ear is formed.

The composite particle 13 contains one or more titanium oxide particles and one or more silver particles. The above description applies to composite particles.

The composite particles 13 are attached to the mask body 11 so that they can be detached by breathing of the subject wearing the mask 10. Therefore, when the subject wearing the mask 10 breathes, a part of the large number of composite particles 13 adhering to the mask body 11 is detached and administered to the mucous membrane in the nasal cavity of the subject. That is, the composite particles 13 can be administered to the intranasal mucosa of the subject by utilizing the respiration of the subject wearing the mask 10, whereby the subject can be protected from coronavirus infection.

The target to which the mask 10 is applied is not limited to those who use it for the purpose of preventing coronavirus infection. For example, those in need of prevention or treatment of rhinitis, specifically rhinitis patients are also included.

In the present embodiment, the composite particles 13 are detachably attached to the second breathable sheet member 112 among the plurality of sheet members constituting the mask main body 11. The sheet member to which the composite particles 13 are detachably attached is not limited to the second breathable sheet member 112, and may be another breathable sheet member. Further, the composite particles 13 may be detachably attached to two or more breathable sheet members.

The total amount of composite particles attached per unit area of the breathable sheet member is not particularly limited, but the lower limit of the total amount of adhesion is usually 1 g / m ² , preferably 2 g / m ² , and more preferably 3 g / m ² . , Even more preferably 4 g / m ² , even more preferably 5 g / m ² , and the upper limit of the total adhered amount is usually 20 g / m ² , preferably 15 g / m ² , still more preferably 10 g / m ² . be.

The amount of the binder resin contained in the mixed solution is preferably 20 to 90 parts by mass, more preferably 30 to 85 parts by mass, and even more preferably 40 to 80 parts by mass with respect to 100 parts by mass of the composite particles. The same applies to the amount of binder resin contained in the breathable sheet member.

The total adhesion amount of the composite particles and the binder resin per unit area of the breathable sheet member is not particularly limited, but the lower limit of the total adhesion amount is usually 2 g / m ² , preferably 3 g / m ² , and more preferably 4 g. / M ² , even more preferably 5 g / m ² , even more preferably 6 g / m ² , and the upper limit of the total adhered amount is usually 30 g / m ² , preferably 25 g / m ² , still more preferably 20 g / m 2. m ² , even more preferably 15 g / m ² .

As the binder resin, a known resin having adhesiveness can be used alone or in combination of two or more. Examples of the binder resin include natural glues or natural resins such as gelatin, gum arabic, shellac, dammar, elemy, and thunderac; semi-synthetic glues or semi-synthetic such as methyl cellulose, ethyl cellulose, nitrocellulose, carboxymethyl cellulose, and acetate. Resins; Isocyanate-based, terephthalic acid-based, bisphenol-based, vinyl ester-based polyester resins; Acrylic copolymer resins such as ethylene-acrylic acid, ethylene-acrylic acid ester, acrylic ester-vinyl, and methacrylic acid ester-vinyl; Isocyanate derivatives or isocyanurate derivatives such as tolylene diisocyanate, 4,4'-diphenylmethane diisocyanate, lysine ester triisocyanate, isocyanate derivatives such as tolylene diisocyanate or isocyanurate derivatives, polyester polyols, polyether polyols, acrylic loylol , Urethane-based resin formed by reaction with polyol such as phenolic polyol; halogenated polymer such as polyvinyl chloride and polyvinylidene chloride; polyvinyl acetate, ethylene-vinyl acetate copolymer, vinyl chloride-vinyl acetate copolymer , Polyacrylic ester, polystyrene, polyvinyl acetal and other acetal resins; polycarbonate resins; cellulose acetate and other cellulose resins; polyolefin resins; urea resins, melamine resins, benzoguanamine resins and other amino resins and other synthetic glues or synthetics. Resins: Silicone resins such as polyalkylsiloxane, polyalkylhydrogensiloxane, polyalkylalkenylsiloxane, polyalkylsyliconate, polyalkalialkylsiliconate, and polyalkylphenylsiloxane, as well as epoxy-modified, amino-modified, urethane-modified, and alkyd-modified. , A modified product such as acrylic modification, a silicone resin containing a copolymer, etc .; or a polymer such as tetrafluoroethylene or vinylidene fluoride, a fluororesin such as a copolymer of these monomers and other kinds of monomers, etc. Be done. Of these, urethane-based resins and silicone resins are preferable, and urethane-based resins are particularly preferable, from the viewpoint of adhesiveness and the like.

The inorganic binder may be used alone or in combination of two or more in place of the binder resin or in combination with the binder resin. Examples of the inorganic binder include alkyl silicates, silicon halides, products obtained by decomposing hydrolyzable silicon compounds such as partial hydrolysates thereof, organic polysiloxane compounds and their polycondensates, silica, colloidal silica. , Water glass, silicon compounds, phosphates such as zinc phosphate, metal oxides such as zinc oxide and zirconium oxide, heavy phosphates, cement, gypsum, lime, frit for brooms and the like.

(Medical sheet)
FIG. 27 is a perspective view of a medical sheet as a medical device according to an embodiment.

The medical sheet according to the embodiment includes a sheet portion inserted into the nasal cavity of the target and composite particles attached to the sheet portion, and the composite particles include one or more titanium oxide particles and one or more silver particles. Including particles.

As shown in FIG. 27, the medical sheet 20 according to the present embodiment includes a sheet portion 21 inserted into the nasal cavity of the target and composite particles 22 attached to the sheet portion 21.

The medical sheet 20 is used by inserting it into the target nasal cavity. When the medical sheet 20 is inserted into the nasal cavity of the subject, the medical sheet 20 may be deformed so as to be easy to insert. For example, the medical sheet 20 can be twisted, deformed into a paper twist, and then inserted into the nasal cavity of the subject. As long as the portion of the sheet portion 21 to which the composite particles 22 are attached is inserted into the target nasal cavity, the entire medical sheet 20 may be inserted into the target nasal cavity, or a part of the medical sheet 20 may be inserted. It may be inserted into the nasal cavity of the subject, but from the viewpoint of ease of removal from the nasal cavity, a part of the medical sheet 20 is inserted into the nasal cavity of the subject and the rest is held outside the nasal cavity of the subject. Is preferable. The medical sheet 20 is preferably inserted up to the posterior part of the nasal cavity (at a distance of, for example, 1 to 10 cm, preferably 1 to 8 cm from the lower nose point). Further, it is preferable that the medical sheet 20 is inserted into the target nasal cavity so that the portion of the sheet portion 21 to which the composite particles 22 are attached comes into contact with the target intranasal mucosa.

The seat portion 21 has a size that can be inserted into the nasal cavity of the subject. The length of the sheet portion 21 is usually 50 to 300 mm, preferably 100 to 200 mm, and the width of the sheet portion 21 is usually 5 to 40 mm, preferably 10 to 20 mm. When the medical sheet 20 is inserted into the target nasal cavity, for example, a part of the sheet portion 21 (for example, a portion having a length of 1 to 5 cm) is held outside the target nasal cavity without being inserted into the target nasal cavity. , The rest of the sheet portion 21 is inserted into the nasal cavity of the subject. The sheet portion 21 is, for example, in the shape of a strip. The sheet portion 21 does not have to be breathable, but is preferably breathable. The air permeability of the seat portion 21 can be appropriately adjusted within a range in which the subject into which the medical sheet 20 is inserted can breathe. The air permeability of the sheet portion 21 is, for example, 5 to 150 cm ³ / cm ² · sec, preferably 30 to 100 cm ³ / cm ² · sec. The measurement of the air permeability is carried out in accordance with, for example, JIS L10968.27.1A method (Frazil type method).

The sheet portion 21 can be formed of, for example, a non-woven fabric, a woven fabric, a knitted fabric, a plastic film having ventilation holes, or the like. Examples of the fiber constituting the sheet portion 21 include synthetic fiber, regenerated fiber, natural fiber and the like. Examples of the synthetic fiber include polyolefin fibers such as polyethylene and polypropylene, polyester fibers such as polyethylene terephthalate and polybutylene terephthalate, and polyamide fibers such as nylon.
The synthetic fiber may be a composite fiber such as a core-sheath type fiber. Examples of the recycled fiber include rayon, acetate and the like. Examples of natural fibers include cotton and the like. Examples of the non-woven fabric include spunbond non-woven fabric, thermal bond non-woven fabric, spunlace non-woven fabric, air-through non-woven fabric, melt blow non-woven fabric, needle punch non-woven fabric and the like. Examples of the woven fabric include gauze and the like. The nonwoven fabric may have a multilayer structure having two or more layers. Examples of such a multi-layer structure include an SS structure (spun bond-spun bond two-layer structure), SMS (spun bond-melt blow-spun bond three-layer structure), and the like.

The composite particle 22 includes one or more titanium oxide particles, one or more metal particles, and one or more calcium phosphate particles. The above description applies to composite particles.

The composite particles 22 may be attached to the sheet portion 21 so that the medical sheet 20 can be detached by breathing of the object inserted into the nasal cavity, or the medical sheet 20 is inserted into the nasal cavity. It may be attached to the sheet portion 21 so as not to be detached by breathing.

When the medical sheet 20 is inserted into the target nasal cavity, the composite particles 22 attached to the sheet portion 21 come into contact with or adhere to the target intranasal mucosa. Therefore, the composite particles 22 can be administered to the intranasal mucosa of the subject without utilizing the subject's respiration.

The total adhesion amount of the composite particles per unit area of the sheet portion 21 is not particularly limited, but the lower limit of the total adhesion amount is usually 1 g / m ² , preferably 2 g / m ² , and more preferably 3 g / m ² . It is even more preferably 4 g / m ² , even more preferably 5 g / m ² , and the upper limit of the total adhered amount is usually 20 g / m ² , preferably 15 g / m ² , and even more preferably 10 g / m ² . ..

The composite particles 22 can be attached to the sheet portion 21 via, for example, a binder resin. For example, by supplying the sheet member with a mixed solution containing the composite particles 22 and the binder resin, or by immersing the sheet member in the mixed solution containing the composite particles 22 and the binder resin, and then drying the sheet member. , The sheet portion 21 to which the composite particles 22 are attached via the binder resin can be manufactured. Further, after the mixed liquid containing the composite particles 22 and the binder resin is supplied to the raw material of the sheet, or after the raw material of the sheet is immersed in the mixed liquid containing the composite particles 22 and the binder resin, the raw material of the sheet is used. By drying the fabric and then cutting out the sheet member from the original fabric of the sheet, the sheet portion 21 to which the composite particles 22 are attached via the binder resin can be manufactured.

The amount of the binder resin contained in the mixed solution is preferably 20 to 90 parts by mass, more preferably 30 to 85 parts by mass, and even more preferably 40 to 80 parts by mass with respect to 100 parts by mass of the composite particles. The same applies to the amount of binder resin contained in the sheet portion 21.

The total adhesion amount of the composite particles and the binder resin per unit area of the sheet portion 21 is not particularly limited, but the lower limit of the total adhesion amount is usually 2 g / m ² , preferably 3 g / m ² , and more preferably 4 g / m 2. m ² , even more preferably 5 g / m ² , even more preferably 6 g / m ² , and the upper limit of the total adhesion amount is usually 30 g / m ² , preferably 25 g / m ² , still more preferably 20 g / m. ² , even more preferably 15 g / m ² .

[Coronavirus killing method]
Further, in the present invention, the particle size when silver particles are bonded to the surface of titanium oxide particles and measured by a laser diffraction method is more than 0.1 μm and less than 0.3 μm, based on the total mass of the coronavirus killing agent. , A corona-killing agent or a member containing the composite particles, wherein the content of titanium oxide particles is 70 to 96% by mass, the content of silver particles is more than 0.5% by mass and 3.6% by mass or less. Is also related to the method of killing coronaviruses, which places the particles in places where the presence of coronaviruses is expected.
As the coronavirus-killing agent or a member containing the same, for example, the above-mentioned medical sheet is preferably used.
In medical institutions, materials that are expected to be infected by droplets or contact from patients, such as clothing such as underwear and socks; textiles such as sheets and duvet covers; air conditioning equipment such as air purifiers and air conditioner filters; door knobs and wallpaper Indoor equipment such as; By applying a coronavirus killing agent or a member containing it to infection protection products such as masks, face shields, protective clothing, etc., the new coronavirus can be killed in a short time, which is effective. It is possible to prevent the spread of the new coronavirus.
In addition, the new coronavirus is killed by applying a coronavirus-killing agent or a member containing it to, for example, seats, handrails, straps, buzzers, air-conditioning equipment, etc. in a car of public transportation. be able to.
When the coronavirus-killing agent is applied to each member, the service life of the coronavirus-killing agent becomes longer by applying it using a predetermined binder.

(Other embodiments)
As mentioned above, the invention has been described by embodiment, but the statements and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure will reveal to those skilled in the art various alternative embodiments, examples and operational techniques. For example, the following pharmaceutical formulations are included.

[Pharmaceutical product]
The pharmaceutical preparation of the present invention is a pharmaceutical preparation administered to the mucous membrane in the nasal cavity, and is a composite particle containing one or more titanium oxide particles, one or more calcium phosphate particles, and one or more metal particles. contains. The above description applies to composite particles.

The pharmaceutical preparation of the present invention can be produced by blending a pharmaceutically acceptable additive in addition to the composite particles as an active ingredient. Examples of such additives include pH adjusters, preservatives, fragrances, dispersants, wetting agents, stabilizers, preservatives, suspending agents, surfactants and the like.

The pH adjuster can be appropriately selected and used from those generally used for external use. The blending amount of the pH adjuster can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the pH adjuster include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid and phosphoric acid, acetic acid, succinic acid, fumaric acid, malic acid, oxalic acid, lactic acid, glutaric acid, salicylic acid and tartrate acid. Examples thereof include organic acids and salts of these acids. As the pH adjuster, one type may be used alone, or two or more types may be used in combination.

The preservative can be appropriately selected and used from those generally used for external use. The blending amount of the preservative can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the preservative include paraoxybenzoic acid alkyl esters such as paraoxybenzoic acid, methylparaben, ethylparaben, propylparaben, chlorobutanol, benzyl alcohol, methyl paraoxybenzoate, and propyl paraoxybenzoate. As the preservative, one type may be used alone, or two or more types may be used in combination.

The fragrance can be appropriately selected and used from those generally used for external use. The blending amount of the flavoring agent can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the flavoring agent include menthol, rose oil, eucalyptus oil, d-camphor and the like. As the flavoring agent, one type may be used alone, or two or more types may be used in combination.

The dispersant can be appropriately selected and used from those generally used for external use. The blending amount of the dispersant can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the dispersant include sodium metaphosphate, calcium polyphosphate, silicic acid anhydride and the like. As the dispersant, one type may be used alone, or two or more types may be used in combination.

The wetting agent can be appropriately selected and used from those generally used for external use. The blending amount of the wetting agent can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the wetting agent include propylene glycol, butylene glycol, glycerin, sorbitol, sodium lactate, sodium hyaluronate and the like. As the wetting agent, one type may be used alone, or two or more types may be used in combination.

The stabilizer can be appropriately selected and used from those generally used for external use. The blending amount of the stabilizer can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the stabilizer include sodium bisulfite, tocopherol, ethylenediaminetetraacetic acid (EDTA), citric acid and the like. As the stabilizer, one type may be used alone, or two or more types may be used in combination.

The preservative can be appropriately selected and used from those generally used for external use. The blending amount of the preservative can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the preservative include ethyl paraoxybenzoate, propyl paraoxybenzoate, benzalkonium hydrochloride, sorbic acid and the like. As the preservative, one kind may be used alone, or two or more kinds may be used in combination.

The suspending agent can be appropriately selected and used from those generally used for external use. The blending amount of the suspending agent can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the suspending agent include tragant powder, gum arabic powder, bentonite, sodium carboxymethyl cellulose and the like. As the suspending agent, one type may be used alone, or two or more types may be used in combination.

The surfactant can be appropriately selected and used from those generally used for external use. The blending amount of the surfactant can be appropriately adjusted according to the dosage form, the base component and the like. Examples of the surfactant include polyoxyethylene hydrogenated castor oil, sorbitan fatty acid ester such as sorbitan sesquioleate, and polyoxyl stearate. As the surfactant, one type may be used alone, or two or more types may be used in combination.

The dosage form of the pharmaceutical preparation of the present invention is not particularly limited as long as it can be administered to the intranasal mucosa, and for example, nasal drops, sprays, aerosols, ointments, creams, lotions, liniments, and paps. , Plasters, patches, plasters, gels, liquids, tapes, powders, granules and the like. The formulation into a desired dosage form can be carried out by using an additive, a base or the like suitable for each dosage form according to the usual method described in the general formulation rules of the Japanese Pharmacopoeia. Examples of the base material used in the dosage form of patches, tapes, etc. include woven fabrics such as cotton, sufu, linen, and chemical fibers; non-woven fabrics such as rayon, polyester, and nylon; soft polyvinyl chloride, polyethylene, and the like. Examples thereof include plastic films such as polyurethane. The base material may be a laminated sheet composed of two or more layers.

When the dosage form is an ointment or a cream, for example, an oily base or an emulsion base can be used as the base.

Examples of the oily base include hydrocarbons, higher alcohols, higher fatty acids, higher fatty acid esters, glycols, vegetable oils, animal oils and the like. The oily base may be used alone or in combination of two or more.

Hydrocarbons that can be used as an oily base include, for example, hydrocarbons having 12 to 32 carbon atoms, liquid paraffin, which is a mixture of various hydrocarbons, branched paraffin, solid paraffin, white petrolatum, yellow petrolatum, and squalane. , Squalane, paraffin base, etc.

Higher alcohols that can be used as oily bases include, for example, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol (cetanol), hexadecyl alcohol, heptadecyl alcohol, stearyl alcohol, oleyl alcohol, and nona. Examples thereof include aliphatic monohydric alcohols having 12 to 30 carbon atoms such as decyl alcohol, eicosyl alcohol, ceryl alcohol, mericyl alcohol and cetostearyl alcohol.

Higher fatty acids that can be used as oily bases include, for example, caproic acid, enanthic acid, capric acid, pelargonic acid, capric acid, undesic acid, lauric acid, tridecyl acid, myristic acid, pentadecic acid, palmitic acid and heptadecic acid. , Stearic acid, oleic acid, nonadecanic acid, araquinic acid, arachidonic acid, linoleic acid, linolenic acid, behenic acid, lignoseric acid, cellotic acid, heptacosanoic acid, montanic acid, melicic acid, laxeric acid, ellagic acid, brushzic acid, etc. Examples thereof include saturated or unsaturated fatty acids having 6 to 32 carbon atoms.

Examples of higher fatty acid esters that can be used as an oily base include fatty acid esters such as myristyl palmitate, stearyl stearate, myristyl myristate, ceryl lignoserate, luxeryl serotinate, and luxeryl laxelate; Esters of fatty acids having 10 to 32 carbon atoms and fatty monovalent alcohols having 14 to 32 carbon atoms such as natural waxes derived from animals such as cellac wax, carnauba wax, and natural waxes derived from plants such as candelilla wax; glyceryl monolaurylate, glyceryl Monomyristylate, glyceryl monooleate, glyceryl monostearate, glyceryl dilaurylate, glyceryl dimyristylate, glyceryl distearate, glyceryl trilaurylate, glyceryl trimylstyrate, glyceryl tristearate, etc. 10 carbon atoms Examples thereof include esters of saturated or unsaturated fatty acids of ~ 22 and glycerin, or hydrogenated additives thereof.

Examples of glycols that can be used as an oily base include ethylene glycol, diethylene glycol, propylene glycol, 1,3-butanediol, and polyethylene glycol.

Vegetable oils that can be used as oily bases include, for example, camellia oil, castor oil, olive oil, cacao oil, palm oil, palm oil, macadamia nut oil, soybean oil, brown seed oil, sesame oil, gentle oil, and saflower oil. Examples thereof include cottonseed oil, terepine oil, and vegetable oils and fats obtained by hydrogenating these vegetable oils.

Examples of animal oils that can be used as oily bases include mink oil, egg yolk oil, squalane, squalene, lanolin, and animal oil derivatives.

Examples of the emulsion base include an oil-in-water base, a water-in-oil base, and a suspension base. The emulsion base may be used alone or in combination of two or more.

As the oil-in-water base, components such as lanolin, propylene glycol, stearyl alcohol, petrolatum, silicone oil, liquid paraffin, glyceryl monostearate, and polyethylene glycol are contained in the aqueous phase in the presence or absence of a surfactant. Examples include emulsified and dispersed bases. The oil-in-water base can be suitably used when preparing a cream or the like.

Examples of the water-in-oil base include a base obtained by adding water to components such as petrolatum, higher fatty alcohol, and liquid paraffin in the presence of a nonionic surfactant, and emulsifying and dispersing the base.

The oil-in-water base and the water-in-oil base can be suitably used in a dosage form containing water, for example, a liquid containing water, a lotion, a poultice, an ointment, and the like.

Examples of the suspendable base include an aqueous base obtained by adding a suspending agent such as starch, glycerin, high-viscosity carboxymethyl cellulose, and carboxyvinyl polymer to water to form a gel.

The pharmaceutical preparation of the present invention can be produced according to a generally adopted method for preparing an external preparation. For example, an ointment or cream is produced by kneading, emulsifying or suspending a base material according to each dosage form to prepare a base, and then adding an active ingredient and various additives and mixing them. can do. For mixing, a commonly used mixer such as a screw mixer, a homomixer, a kneader, or a roll mill can be used.

When the dosage form is a lotion, it may be any type of suspension type, emulsion type and solution type.

Examples of the base of the suspension type lotion include rubbers such as Arabica rubber and tragant rubber, celluloses such as methyl cellulose, hydroxyethyl cellulose and hydroxyethyl starch, and a mixture of clay suspension such as bentonite and bee gum HV and water. Can be mentioned. The base of the suspension lotion can usually be used alone or in admixture of two or more.

Examples of the base of the emulsion-type lotion include water and a base obtained by emulsifying an oily substance such as fatty acid such as stearic acid, behenic acid and oleic acid, and higher alcohol such as stearyl alcohol, cetanol and behenic alcohol. The base of the emulsion lotion can usually be used alone or in admixture of two or more.

Examples of the base of the solution type lotion include water, ethanol, glycerin, alcohol such as propylene glycol, and the like. The base of the solution lotion can usually be used alone or in combination of two or more.

The lotion can be produced, for example, by adding various base components to purified water, mixing and stirring, then adding the active ingredient and additives, mixing, and filtering if desired. ..

When the dosage form is a liniment agent, the base thereof is, for example, vegetable oils such as olive oil, sesame oil, prunus dulcis oil, cottonseed oil, and terepine oil, alcohols such as ethanol, propanol and isopropanol, and a mixture thereof with water. And so on. The base of the liniment agent can usually be used alone or in combination of two or more.

The liniment agent can be produced by dissolving the active ingredient in the base, further adding the desired ingredient, and mixing.

When the dosage form is a poultice, the base thereof is, for example, a water-soluble polymer compound such as polyacrylic acid and a salt thereof, polyvinyl alcohol, polyvinylpyrrolidone, etc., and the water-soluble polymer compound is a polyvalent metal such as myoban. Examples thereof include a base crosslinked with a salt, a crosslinked body such as a base crosslinked by subjecting the water-soluble polymer compound to a physical treatment such as irradiation. The base of the poultice can usually be used alone or in admixture of two or more.

The poultice can be produced by mixing the active ingredient, the base and the desired additive, heating and then cooling.

In the case of plasters, patches and plasters, supports such as non-woven fabrics, natural rubber, styrene-butadiene rubber (SBR), butyl rubber, polyisobutylene, polyvinyl alkyl ether, polyurethane, dimethylpolysiloxane, styrene-isoprene-styrene. Elastic materials such as rubber and isoprene rubber, fillers such as zinc flower, titanium oxide, silica, adhesives such as terpene resin, rosin or its ester, phenol resin, etc., which have good compatibility with elastic materials, vinyl acetate, silicone Examples thereof include a stripping agent such as resin and polyvinyl chloride, a softening agent such as liquid paraffin and process oil, and an antiaging agent such as dibutylhydroxytoluene (BHT). These components can be used alone or in admixture of two or more.

Plaster agents, patches, plasters, etc. can be manufactured by conventional methods such as solution method and thermal pressure method. Specifically, for example, in the case of a thermal pressure type, the active ingredient and each ingredient are uniformly kneaded with a roll machine or the like, and applied to a uniform thickness on a release paper using a calendar to which heat and pressure are applied. This can be used to form a drug-containing layer, which can be laminated on the surface of the support and brought into close contact with the support.

Even in the case of gels, liquids, tapes, etc., the base may be any as long as it is used as a normal external preparation, and is not particularly limited.

As described above, it goes without saying that the present invention includes various embodiments not described here. Therefore, the technical scope of the present invention is defined only by the matters specifying the invention relating to the reasonable claims from the above description.

Hereinafter, the present invention will be described in more detail based on manufacturing examples and test examples. However, the scope of the present invention is not limited to these production examples and test examples.

[Production Example 1]: Production of Composite Particles In this production example, titanium oxide powder, silver powder and hydroxyapatite powder are used as raw material powders, and one or more titanium oxide particles, one or more silver particles and one or more. A composite particle comprising the hydroxyapatite particles of the above was produced.

In this production example, two types of composite particles M1 and M2 were produced. The composite particles M1 and M2 differ in the content ratio of titanium oxide, silver and hydroxyapatite.

The raw material powder shown in Table 1 was prepared. The particle size of the titanium oxide powder is a value measured using a transmission electron microscope (TEM) or a scanning electron microscope (SEM), and the particle size of the silver powder is a value calculated based on the specific surface area. The particle size of the hydroxyapatite powder is a value measured by a laser diffraction / scattering method. Since the silver powder was stored frozen until use, the aggregation of silver particles contained in the silver powder was suppressed.

Titanium oxide powder by mixing titanium oxide powder, silver powder, hydroxyapatite powder and polycarboxylic acid-based dispersant in water using a commercially available wet bead mill (“Star Mill LME” manufactured by Ashizawa Finetech Co., Ltd.). One or more titanium oxide particles contained in the above, one or more silver particles contained in the silver powder, and one or more hydroxyapatite particles contained in the hydroxyapatite powder are combined to form a suspension of the composite particles. Slurry) was produced. The wet bead mill used can be finely pulverized while dispersing titanium oxide particles, silver particles and hydroxyapatite particles contained in the raw material powder, and can be finely divided into nanoparticles or submicron particles, and the finely divided particles are combined. Can be transformed into.

The conditions for particle compositing using a wet bead mill are as follows.
Total amount of raw material powder added: 4 kg or more Cylinder volume: 3.3 L
Beads: Zirconia beads (diameter 0.5 mm, mass 0.37 mg)
Liquid flow rate: 2 L / min Peripheral speed of blades in cylinder: 540 m / min Liquid temperature: 35-45 ° C
Mixing time per 1 kg of raw material powder: 30-40 minutes (about 36 minutes)

The total amount of titanium oxide powder, silver powder and hydroxyapatite powder was adjusted to 35 parts by mass with respect to 65 parts by mass of water. The blending amount of the polycarboxylic acid-based dispersant was adjusted to 0.5 parts by mass with respect to 35 parts by mass of the total blending amount of titanium oxide powder, silver powder and hydroxyapatite powder.

In the production of the composite particle M1, the blending amount of the titanium oxide powder is adjusted to about 160 parts by mass (155 to 165 parts by mass) with respect to 1 part by mass of the silver powder, and the blending amount of the hydroxyapatite powder is 1 mass by mass of the silver powder. It was adjusted to about 40 parts by mass (39 to 41 parts by mass) with respect to the part.

In the production of the composite particle M2, the blending amount of the titanium oxide powder is adjusted to about 30 parts by mass (29 to 31 parts by mass) with respect to 1 part by mass of the silver powder, and the blending amount of the hydroxyapatite powder is 1 mass by mass of the silver powder. It was adjusted to about 3 parts by mass (2.5 to 3.5 parts by mass) with respect to the part.

Composite particles M1 and M2 were produced by drying a suspension (slurry) of composite particles. The particle diameters of the composite particles M1 and M2 measured by the laser diffraction method were 200 to 500 nm. The median diameter (d50) of the composite particles M1 and M2 measured by the laser diffraction method on a volume basis was about 300 nm. The particle size is measured by the laser diffraction method using a commercially available particle size distribution measuring device, specifically, a laser diffraction / scattering type particle size distribution measuring device Partica LA-960V2 series (manufactured by HORIBA). bottom.

[Production Example 2]: Production of composite particle-adhered nonwoven fabric A binder resin is added to the suspension (slurry) of the composite particles M1 obtained in Production Example 1 to prepare a mixed solution, and then a polyester spunpond nonwoven fabric is mixed. The non-woven fabric was impregnated with the mixed solution. After the immersion, the non-woven fabric was taken out from the mixed solution and pressed with a roller to squeeze out the excess mixed solution. After pressing, the nonwoven fabric was dried at about 130 ° C. for about 1 minute to produce a composite particle-adhered nonwoven fabric A1. As the binder resin, a urethane resin (C ₃ H ₇ NO ₂ / NH ₂ COOC ₂ H ₅ ) was used.

By adjusting the concentrations of the composite particles M1 and the binder resin in the mixed solution, the total adhesion amount (total fixed amount) of the composite particles M1 and the binder resin per unit area of the composite particle-adhered nonwoven fabric A1 is adjusted to 4 g / m ² . bottom.

The breakdown of 4 g / m ² was titanium oxide 2.27 g / m ² , hydroxyapatite 0.571 g / m ² , silver 0.014 g / m ² , and binder resin 1.14 g /.

The composite particle-adhered nonwoven fabric A2 was produced in the same manner as above, except that the suspension of the composite particles M2 was used instead of the suspension of the composite particles M1.

By adjusting the concentrations of the composite particles M2 and the binder resin in the mixed solution, the total adhesion amount (total fixed amount) of the composite particles M2 and the binder resin per unit area of the composite particle-adhered nonwoven fabric A2 is 10.5 g / m ² . Adjusted to.

The breakdown of 10.5 g / m ² was titanium oxide 6.525 g / m ² , hydroxyapatite 0.750 g / m ² , silver 0.225 g / m ² , and binder resin 3.00 g / m ² .

The composite particle-adhered nonwoven fabric A1 having a total adhered amount (total fixed amount) of 4 g / m ² of the composite particles M1 and the binder resin was observed with an electron microscope.
A schematic diagram of the electron microscope observation results is shown in FIG. 23A, and a partially enlarged view thereof is shown in FIG. 23B. In the figure, the white portion is titanium oxide particles, and the hatch portion is silver particles. As shown in FIGS. 23A and 23B, it was confirmed that the composite particles have a morphology in which silver particles are bonded to the surface of titanium oxide particles.

[Production Example 3]: Production of Composite Particle Adhesive Mask A polypropylene spunbonded nonwoven fabric, a polypropylene melt blow nonwoven fabric, a composite particle adhered nonwoven fabric, and a polypropylene spunbonded nonwoven fabric were laminated in this order from the outside air side to produce a composite particle adhesion mask. As the composite particle-adhered nonwoven fabric, the composite particle-attached nonwoven fabric A2 having a total adhered amount (total fixed amount) of 10.5 g / m ² of the composite particles M2 and the binder resin was used.

[Example 1]
Bioshoot Co., Ltd. was commissioned to evaluate the antiviral activity of the hydrosilver-titanium coated product against the new coronavirus (SARS-CoV-2). This experiment was conducted at the Yasuda Laboratory in the Department of Emerging Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, under the supervision of Professor Jiro Yasuda of the same laboratory. Cells were handled in the BSL-2 area and viruses were handled in the BSL-3 area.

<Materials and experimental methods>
1. Test material The following materials were used as the test material.
1.1 (Test material 1)
Processed non-woven fabric with composite particles (hereinafter also referred to as "A1 sheet") (low concentration)
(Titanium oxide (79.5%), silver (0.5%), apatite (20%), urethane binder; 4g / m ² )
1.2 (Test material 2)
Processed non-woven fabric with composite particles (hereinafter also referred to as "A2 sheet") (high concentration)
(Titanium oxide (87%), silver (3%), apatite (10%), urethane binder; 10.5 g / m ² )
1.3 (Reference material 1)
Hydro Silver Titanium Processed Powder (Hydro Silver Titanium Powder) (Low Concentration)
(Titanium oxide, silver, apatite; for 4g / m ² )
1.4 (Reference material 2)
Hydro Silver Titanium Processed Powder (Hydro Silver Titanium Powder) (High Concentration)
(Titanium oxide, silver, apatite; for 10g / m ² )
1.5 (negative material)
Urethane binder only processed non-woven fabric
1.6 (unprocessed group)
Virus solution (serum-free culture); 20-fold diluted solution (serum-free medium)
The hydrosilver-titanium processed powder (1.3 and 1.4) is used as a reference material.

2. Medium / Reagent used The following medium / reagent was used.
2.1 Serum medium (FBS-added D-MEM medium) (TMPRSS2-expressing cell passage only + G418)
(Additional addition; MEM essential amino acid, sodium pyruvate, antibiotic (penicillin / streptomycin))
It was used as a cell passage medium, a growth medium, and an infectious titer test medium.
2.2 Serum-free medium (VP-SFM medium)
(Additional addition; L-glutamine, antibiotics (penicillin / streptomycin))
It was used as a diluting solution for virus by dipping method other than the medium for virus culture.
2.3 Trypsin (for cell detachment)
2.4 Agarose (for infectious titer test)
2.5 Formalin solution (for infectious titer test)
2.6 Crystal violet stain (for infectious titer test)

3. Cells used
3.1 VeroE6 / TMPRSS2 (TMPRSS2-expressing African green monkey kidney-derived cells) and VeroE6 (African green monkey kidney-derived cells). The cells used in the laboratory were used.
VeroE6 / TMPRSS2 was used for serum-free culture of the virus, and VeroE6 cells with a clear plaque image were used for infectious titer measurement.
There is no limit on the number of cell passages in virus cultures and infectious titer tests.

4. Used virus strain
4.1 SARS-CoV-2_Laboratory isolate (wild strain)

5. Test method and results
5.1 Preparation of cells for virus culture Subculture of VeroE6 / TMPRSS2 cells in serum medium (2.1) in T-75 and T-175 flasks, 6 × per 10 storage tubes (1.5 mL cryotubes) 10 ⁶ cells / tube (1 mL / tube) was frozen (-70 ° C or lower) and stored (stored during the experimental period). One of them was restored and it was confirmed that the storage condition was good. The cells were separately subcultured in a T-75 or T-175 flask and used to prepare the SARS-CoV-2 virus solution.
1A and 1B are micrographs of cells (VeroE6 / TMPRSS2 cells and VeroE6 cells) used in the experiment.

5.2 Confirmation of the effect of test material eluate on cell proliferation
5.2.1 Recovery of eluate from test material Of each test material, two sheet materials (1.1, 1.2, 1.5; 3.2 x 3.2 cm sections each) (double the amount of the evaluation experiment) were placed in a petri dish with a diameter of 5 cm. And 2 mL of serum medium (2.1) was put on it. In addition, weigh 200 mg of powder material (1.3, 1.4) (twice the amount of the evaluation experiment) into a 15 mL centrifuge tube, add 2 mL of serum medium (2.1) to each and shake, and heat each dish and centrifuge tube to 37 ° C (2). It was allowed to stand for 6 hours in a CO ₂ incubator). After the treatment, the petri dish was decanted, and the centrifuge tube was centrifuged at 1,000 rpm (9,100 g) for 5 minutes, and 0.7 mL of each supernatant was collected.
Each test material 1.1 to 1.4 was not sterilized, and 1.5 was pretreated with a UV lamp for sterilization for 30 minutes.

5.2.2 Confirmation of effects of material eluate on cells VeroE6 / TMPRSS2 cell cells were seeded on two 6-well plates at 5 × 10 ⁵ cells per well, and the eluate was added the next day. The eluate solution was added to the cultured cells at 0.3 mL / well (the eluate concentration was about 3 times the amount of the infectious titer test). The sample was duplicated. After the addition, the culture was continued for 2 days, the growth state was observed, and the number of cells was also measured on the 2nd day, which was compared with the negative control. In addition, since the powder material was centrifuged at 1,000 rpm for 5 minutes and a large amount of fine powder was mixed in, the supernatant was re-examined by centrifuging at 10,000 rpm (9,100 g) for 5 minutes.
Negative control and no change were observed in the cell status on the 1st and 2nd days in the eluate of each material, and the average number of cells in the 2 wells on the 2nd day was the same as that of the negative control (negative control 3.0 × 10 ⁶ cells). It was 3.2 to 3.6 × 10 ⁶ cells / well for each test material against / well. No change was seen in the powder re-examination (6 times the total infectious titer test was added). From these results, it was judged that the eluate from each test material did not give morphology or growth inhibition of VeroE6 / TMPRSS2 cells for virus infectious titer measurement used in this experiment.
In addition, in each culture material, no growth of microorganisms was observed in the serum medium (2.1) culture for 2 days during the culture period.
2A to 2F are views showing the results of shape comparison (2nd day) with the eluate-added cells.

VeroE6 cell cells were seeded on two 6-well plates at 4 × 10 ⁵ cells per well, and the eluate was added 6 hours later. The eluate solution was added to the cultured cells at 0.3 mL / well (the eluate concentration was about 3 times the amount of the infectious titer test). The sample was duplicated. After the addition, the culture was continued for 4 days, the growth state was observed, and the number of cells was counted on the 4th day, which was compared with the negative control. As the powder material, the supernatant obtained by centrifuging at 10,000 rpm (9,100 g) for 5 minutes was used.
Negative control and no change were observed in the cell status on the 2nd and 4th days in the eluate of each material, and the average number of cells in the 2 wells on the 4th day was the same as that of the negative control (negative control 2.1 × 10 ⁶ cells). It was 2.2 to 2.5 × 10 ⁶ cells / mL for each test material per / mL (6 times the weight of the total infectious titer test). From these results, it was judged that the eluate from each test material did not give the morphology and growth inhibition of VeroE6 cells for measuring the viral infectivity used in this experiment.
In addition, in each culture material, no growth of microorganisms was observed in the serum medium (2.1) culture for 4 days during the culture period.
3A to 3F are views showing the results of shape comparison (4th day) with the eluate-added cells.

5.3 Preparation of virus solution for infectious titer test
5.3.1 Collection of virus solution In order to eliminate the reaction inhibition by the protein during the virus reaction with each test material, the virus was cultured in a serum-free medium (2.2) with as little protein contamination as possible, and an infectious titer test was performed. The virus solution for this was collected.
Vero E6 / TMPRSS2 cells were cultured in a T-175 flask in serum medium (2.1), the culture medium was removed from the cells that became confulent, the cells were washed with serum-free medium (2.2), and then the virus solution (4.1) provided in the laboratory. ) 500 μL (equivalent to 10 ⁶ pfu / mL and MOI = 0.01 assumed) was inoculated and allowed to stand for 1 hr. Then, 20 mL of serum-free medium (2.2) was added and virus culture was started. The pH dropped and CPE was observed on the second day after virus inoculation, but since live cells still remained, culture was carried out until the next day (3rd day), and the culture supernatant (20 mL) was collected. , Stored frozen (-70 ° C or lower).
FIG. 4 is a photograph before culturing (left of the screen) and after culturing (3rd day, right of the screen) when the virus was cultured in the VP-SFM medium.

5.3.2 Measurement of infectious titer of collected virus solution A plaque assay was performed on a 12-well plate using Vero E6 / TMPRSS2 cells using a virus (5.3.1 collected virus) cultured in serum-free medium (2.2). Assuming that the collected virus (5.3.1) is 10 ⁶ to ⁷ pfu / mL, the dilution is 10 ² -fold, 10 ³ -fold, 10 ⁴ -fold, 10 ⁵ -fold, 10 ⁶ -fold step dilution to 12-well plates. 100 μL of the solution was inoculated in 2 wells (Duplicate), cultured in D-MEM medium supplemented with agarose + FBS for 2 days, inactivated by formalin, stained with crystal violet, and the number of plaques was counted to calculate the virus infectivity.
FIG. 5 is a virus infectious titer test crystal violet stained image. The infectious titer of the virus solution collected on the third day after inoculation was 2.8 × 10 ⁷ pfu / mL.

5.4 Experiment to confirm antiviral activity using test materials
5.4.1 Treatment of virus solution with test material Figure 6 is a list of virus solution treatment conditions.

FIG. 7A is a conceptual diagram of the dipping method, and FIG. 7B is a conceptual diagram of the dropping method.
FIG. 8 is a layout drawing of the dipping method plate. As shown in FIG. 8, six plates containing the materials 1) to 5) were prepared in each well of the 6-well plate. 2 mL of the collected virus solution (5.3.1) diluted 20-fold with serum-free medium (2.2) was placed in each well, and the materials of 1) to 5) were immersed, and the following treatments A to C were performed by duplicate.
A: Refrigerated 24hr treatment B: Room temperature 6hr treatment C: Room temperature 6hr treatment + UV irradiation After washing out the soaked virus solution, the collected virus solution was frozen (-70 ° C or less) until the virus infectivity titer was measured.

FIG. 9 is a layout drawing of the dropping method plate. As shown in FIG. 9, six plates containing the materials of 6) to 9) were prepared in each well of the 6-well plate. In advance, 100 μL of the collected virus solution (5.3) was dropped directly onto the materials of 6), 7), 8) on 6 6-well plates. In the well of 9), 2 mL of the collected virus solution (5.3) was diluted 20-fold. The following processes A to C were performed in duplicate.
A: Refrigerated 24hr treatment B: Room temperature 6hr treatment C: Room temperature 6hr treatment + UV irradiation After A to C treatment, add 2 mL of washout solution (FBS-added D-MEM medium) to each well to wash out the virus, and the washout solution is the virus. It was cryopreserved (-70 ° C or lower) until the infectious titer was measured.

5.4.2 Measurement of virus infectivity of specimens The day before the virus infectivity test, cells (cells using VeroE6) were seeded on a 12-well plate in 2 × 10 ⁵ cells per well, and virus inoculation was carried out the next day.
For virus inoculation, the frozen virus sample (5.5.1) after each treatment was thawed, and then a virus solution diluted stairs at 100-fold, 10-fold, 100-fold, and 1,000-fold was inoculated at 100 μL / well. After standing for 1 hr, agarose + FBS-added D-MEM medium was added, and after culturing for 3 days, formalin inactivation, crystal violet staining, and the number of plaques were counted to calculate the virus infectivity titer.
FIG. 10 is a layout diagram of virus inoculation for measuring infectious titer.

Initially, it was planned to use VeroE6 / TMPRSS2 cells, but it was difficult to understand the number of plaques in the cells because CEP was too strong. Therefore, the plaque image was changed to Vero E6 with a clear image. In addition, it was confirmed in advance that the virus detection sensitivity was the same and that the reproducibility of the infectious titer by the sample was obtained. The virus sample (5.5.1) was thawed and serially diluted to 1,000-fold.

FIG. 11 is a virus infectious titer test plaque image (refrigerated 24 hr immersion treatment). The abbreviations in FIG. 11 have the following meanings.
Sh-NC: Control, Sh + 4: Test material (low concentration), Sh + 10: Test material (high concentration)

5.4.3 Evaluation of viral infectious titer and antiviral activity under each treatment condition of the test material The antiviral activity value is calculated by the following method.
Antiviral activity value (M) = regular log value (log (Va)) of control group pfu / mL value (Va) -common log value (log (Vb)) of sample group pfu / mL value (Vb).
i) For the infectious titer, the average value performed by duplicate was used.
ii) For the control group, if the difference between 9) the regular logarithmic value of the pfu / mL value of the additive-free group and 5) or 8) the regular logarithmic value of the pfu / mL value of the sheet NC group is> 1.0 under each treatment condition. The sheet NC is used as a control group.
iii) As there is no control material for hydrosilver titanium powder, as a reference value,
M = 9) Common logarithmic value of pfu / mL value in the additive-free group-The regular logarithmic value of pfu / mL value in each group.
iV) As for antiviral activity, it is judged that there is significant activity when M ≧ 2.0.

FIG. 12 is a list of virus infectious titers and antiviral activity values (immersion method).
FIG. 13 is a list of virus infectious titers and antiviral activity values (drop method).

6. Summary of results
6.1 Virus solution preparation Since protein is an inhibitor to confirm the antiviral activity of the sheet coating, SARS-CoV-2 (laboratory established wild strain) virus is inoculated into VeroE6 / TRPMSS2 cells and serum-free medium. By culturing in (2.2), a virus solution with less protein contamination was obtained. The virus infectious titer was 2.8 × 10 ⁷ pfu / mL, and a virus solution having an infectious titer equivalent to that of serum culture was obtained.

6.2 Confirmation of antiviral activity by test material Figure 14 is a list of antiviral activity of A1 sheet and A2 sheet. In FIG. 14, the antiviral activity value (M) was calculated using the following formula.
Antiviral activity value (M) = log (Va; control) -log (Vb; sample)
*: M ≧ 2.0 is considered to be significantly different.
It was determined that the eluate from each test material had no effect on the viral infectious titer test.
In this experiment, it was confirmed that the test material "A2 sheet (high concentration)" has significant antiviral activity against SARS-CoV-2 virus.
No significant antiviral activity was shown for "A1 sheet (low concentration)" under the experimental conditions, but there was a correlation between the application amount and antiviral activity compared to "A2 sheet (high concentration)". It was estimated that there was.
Since there is no control material for reference material 1 (low concentration) and reference material 2 (high concentration), the difference in the degree of non-specific adsorption is unknown, but plaque was not detected under any of the treatment conditions, and A1 and A2 sheets. It was presumed that it also has antiviral activity as a raw material for coating.
In this experiment, we investigated a system to confirm the antiviral activity of the test material against SARS-CoV-2 virus under each treatment condition, including the dipping method and the dropping method, but in terms of non-specific adsorption and reproducibility. It was judged that the immersion method could be adopted for future studies of antiviral activity. Under the treatment conditions of this experiment under UV irradiation, the untreated group (virus only) was below the detection level, and the system was not established.
In the confirmation test of the activity of the test material A2 sheet against the new coronavirus by the dipping method, it was confirmed that the test material had significant antiviral activity in both refrigerated 24 hours and room temperature 6 hours treatment. Since the antiviral activity value (M value) of the test material A2 sheet after refrigerated 24-hour treatment was 4.0 or more, assuming that the growth of the new coronavirus of the control (untreated test material) is 1, the new type of A2 sheet It was confirmed that the growth of coronavirus was suppressed to 0.0001 or less, and that the new coronavirus could be attenuated with a reduction rate of 99.99% or more. By the same calculation, it was confirmed that the new coronavirus can be attenuated by 99.9% or more by treating the test material A2 sheet at room temperature for 6 hours.
In the confirmation test of the activity of the test material A2 sheet against the new coronavirus by the dropping method, it was confirmed that the test material had a significant antiviral activity by treatment at room temperature for 6 hours. By the same calculation as above, it was confirmed that the new coronavirus can be attenuated by 99.9% or more by treating the test material A2 sheet at room temperature for 6 hours.

Normally, when the virus is reduced by the square of 10, the virus activity is considered to decrease with a dominant difference (effective for reducing the infectious titer). Therefore, in the confirmation test of the activity of the A2 sheet against the new coronavirus by the immersion method, the number of coronaviruses that existed in the 4th power of 10 at the start of the experiment decreased to the 4th power of 10 which is the decrease in the dominant difference. Calculated. As a result, the time from the start of the experiment to the time when the superiority difference was seen was 0.05 hours, that is, 3 minutes. This confirmed that the activity of the A2 sheet against coronavirus had an immediate effect. Similarly, for the A1 sheet by the immersion method, the time from the start of the experiment until the superiority difference was found was about 100 hours (4 to 5 days).

[Example 2]
A confirmation test of the inactivating ability of composite particles and processed non-woven particles (hereinafter also referred to as "X sheet") containing composite particles against the new coronavirus (SARS-CoV-2) was conducted at the Yokoyama Special Laboratory, RIKEN. It was outsourced to Shigeyuki Yokoyama under the supervision of Research Fellow.
[Purpose]
A sample of the membrane protein (S, M, E) present on the surface of the virus particle of the new coronavirus (SARS-CoV-2) is prepared in vitro, and chemically modified / denatured / decomposed by the X sheet to form a three-dimensional structure. Damage and denaturation were analyzed, and the virus inactivating ability of X-sheet was based on the viewpoint of protein conformation.

[Experimental system]
Experimental facility: RIKEN Yokoyama Special Laboratory Implemented by: RIKEN Yokoyama Special Laboratory

[experimental method]
1 Material Test product 1: Composite particle M1 (titanium oxide composition)
Test product 2: Composite particle M2 (titanium oxide composition)
Test product 3: Processed non-woven fabric provided with composite particles M1 (+4; 4 g / m ² coated, hereinafter also referred to as "X1 sheet"),
Test product 4: Processed non-woven fabric provided with composite particles M2 (+10; 10 g / m ² coated, also referred to as "X2 sheet")
Control product: Polyester non-woven fabric (titanium oxide composition unprocessed)
Template: Chemically synthesized DNA fragment encoding SARS-CoV-2 derived protein Protein preparation: Cell-free protein synthesis reagent

2 Experimental design
2.1 Analysis of chemical modification / denaturation / degradation of viral protein and analysis of conformational disruption SDS-PAGE, Native-PAGE, mass spectrometry of treated samples of control group and test group were treated under each condition of X-sheet and viral protein. Analysis was performed by analysis (MALDI-TOF / MS), negative staining electron microscopic analysis, cryo-electron microscopic analysis, etc. By molecular dynamics calculation of the three-dimensional structure, the process of the three-dimensional structure collapse due to the damage of the viral protein was analyzed in detail.
2.1.1 Preparation Viral protein preparation: Three SARS-CoV-2 proteins (S, M, E) were synthesized by cell-free protein synthesis as fusion proteins with N-terminal FLAG-SUMO tags attached, and affinity was achieved. Purified by chromatography and gel filtration.
Preparation of X1 sheet and X2 sheet: The test product and the control product were sterilized (dry heat, EOG) and then inflated with PBS.
Dose: Equivalent to x1, x3, x10 doses of clinical dose (m ² / kg or m ² / surface area)

FIG. 15 shows the configuration.

1.1.2 Detection method SDS-PAGE, Native-PAGE and mass spectrometry (MALDI-TOF / MS) were performed on each treatment solution.
PAGE was detected by CBB staining or Western blot depending on the protein concentration. Electron microscopic analysis was performed mainly by negative staining, and if necessary, averaging of cryo-electron microscopy was also performed.
1.1.3 Evaluation method The evaluation was based on the difference in molecular weight and abundance between the test group and the control group.
For S protein and E protein whose three-dimensional structure is known, the cleavage / modification sites were mapped on the three-dimensional structure from the results of mass spectrometry. In addition, electron microscopic analysis was used to analyze how the three-dimensional structures of S and E proteins change due to cleavage and modification. Furthermore, the process of disruption of the three-dimensional structure of these cleaved / modified proteins was simulated by molecular dynamics calculation, and a movie of computer graphics was created and analyzed in detail.
In molecular dynamics calculations, the process of disruption of the three-dimensional structure of these cleavage / modification proteins for S proteins with known three-dimensional structures is described by Toru Terada, Associate Professor, Department of Applied Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo. With the support (BINDS), molecular dynamics simulation, that is, the movement of molecules was calculated and visualized by a computer. Since the S protein is very large in size, we used a coarse-grained model that treats about four atoms (excluding hydrogen atoms) as one particle.

16 and 17 are image diagrams of the evaluation method.

[Experimental result]
The results of SDS-PAGE and the results of molecular dynamics (MD) simulation are shown.

1.1 SDS-PAGE results (composite particle M1, composite particle M2)
FIG. 18 is a diagram showing the results of SDS-PAGE (+ βME), which is electrophoresis when the S domain sample solution is irradiated with artificial sunlight in the presence of composite particles M1 and composite particles M2. From FIG. 18, it was shown that the disconnection of the S domain started 1 minute later. The meanings of the abbreviations in FIG. 18 are as follows. C: Control (without composite particles M1 and M2), +4: Composite particle M1 (low concentration), +10: Composite particle M2 (high concentration)

1.2 SDS-PAGE results (X1 sheet, X2 sheet)
FIG. 19 is a diagram showing the results of SDS-PAGE (+ βME), which is an electrophoresis when an S domain sample solution is irradiated with artificial sunlight in the presence of an X1 sheet and an X2 sheet. From FIG. 19, it was shown that the disconnection of the S domain started 3 minutes later. The meanings of the abbreviations in the figure are as follows. C: Control (without X1, X2 sheet), +4: X1 sheet, +10: X2 sheet

1.3 SDS-PAGE results (percentage of disrupted protein over time)
FIG. 20A is a diagram showing the ratio of the amount of disintegrated protein over time to the control when the composite particles M1 and M2 are used. FIG. 20B is a diagram showing the ratio of the amount of disintegrated protein over time to the control when using the X1 and X2 sheets.

From FIG. 20A, it was shown that in the composite particles M1 and M2, cleavage started after 1 minute, and the S domain collapsed to about 10% after 6 minutes. From FIG. 20B, it was shown that in the X1 and X2 sheets, cleavage started after 3 minutes and the S domain was disrupted to 20% or less after 10 minutes.

2.1 Molecular Dynamics (MD) Simulation Figure 21 is a molecular dynamics (MD) simulation diagram of the new coronavirus spike protein. 21A to 21C show a view seen from the side, and FIGS. 21D to 21F show a view seen from above (outside). As shown in FIGS. 21A and 21D, the peplomer trimer in the native state was stable and the three-dimensional structure was maintained. As shown in FIGS. 21B and 21E, when the protein loop was cleaved, the three-dimensional structure instantly deteriorated and began to disintegrate. Then, as shown in FIGS. 21C and 21F, the three-dimensional structure rapidly deteriorated and collapsed. Molecular dynamics simulations of the cleaved S protein showed in a video the process by which the conformational collapse was triggered almost instantly (2 microseconds; 2 × ^10-6 seconds).

2.2 Molecular Dynamics (MD) Simulation Figures 22A to 22D are molecular dynamics (MD) simulation diagrams focusing on the peplomer protein S domain and ACE2 receptor. As shown in FIG. 22A, the peplomer protein S domain was bound to the ACE2 receptor. As shown in FIG. 22B, the native S domain was stable and the three-dimensional structure was maintained. When the protein loop was cleaved as shown in FIG. 22C, the three-dimensional structure instantly deteriorated and began to disintegrate. Then, as shown in FIG. 22D, the three-dimensional structure rapidly deteriorated and collapsed.

3. Summary From the above, cutting with composite particles M1, M2, and X1, X2 sheets promptly caused deterioration and collapse of the three-dimensional structure. Therefore, it was shown that the S domain cannot bind to ACE2.
In addition, it was demonstrated by biochemical analysis that the composite particles M1, M2, and X1, X2 sheets cleave the artificially synthesized new coronavirus spike protein at multiple sites in 3 to 10 minutes. .. Molecular dynamics simulations confirmed that this cleavage disrupts the peplomer conformation required for binding to the human cell receptor ACE2.
Envelope protein (E) and matrix protein (M) were also prepared by the cell-free protein synthesis method, and biochemical analysis was performed by the same method as S protein, and the same results were obtained.

The coronavirus killing agent according to the present invention can easily and economically kill a new type of coronavirus. Further, the coronavirus killing agent according to the present invention can kill the new coronavirus in a short time. The coronavirus killing agent according to the present invention can be applied to various members. Therefore, as one of the countermeasures against infectious diseases of the new coronavirus, it is possible to kill the new coronavirus in a short time by applying the coronavirus killing agent according to the present invention to a member which is expected to be infected by droplets or contact. Therefore, it is possible to effectively prevent the spread of the new coronavirus.

Claims

A coronavirus killing agent with composite particles
The coronavirus killing agent has a titanium oxide particle content of 70% by mass to 96% by mass and a silver particle content of more than 0.5% by mass and 3.6% by mass or less based on the total mass of the coronavirus killing agent. Including coronavirus killing agent.
The coronavirus killing agent according to claim 1, wherein the composite particles have a particle size of more than 0.1 μm and less than 0.3 μm as measured by a laser diffraction method, in which silver particles are bonded to the surface of titanium oxide particles. ..
Claim 1 or 2 in which the content of titanium oxide particles is 78 to 96% by mass and the content of silver particles is more than 0.5% by mass and 3.3% by mass or less based on the total mass of the coronavirus-killing agent. The listed coronavirus killing agent.
The coronavirus killing agent according to any one of claims 1 to 3, wherein the coronavirus is COVID-19.
The coronavirus killing agent is the coronavirus killing agent according to any one of claims 1 to 4, further comprising calcium phosphate particles.
The coronavirus killing agent according to any one of claims 1 to 5, wherein the particle size of the composite particle is 0.2 μm or more and 0.29 μm or less.
The coronavirus killing agent according to any one of claims 1 to 6, wherein the particle size of the composite particle is 0.24 μm or more and 0.27 μm or less.
A member containing the coronavirus-killing agent according to any one of claims 1 to 7.
The member according to claim 8, wherein the member is any one selected from the group consisting of non-woven fabric, woven fabric, knitted fabric, resin film, plastic, metal, ceramics, and air filter.
A medical device containing the coronavirus-killing agent according to any one of claims 1 to 7.
A mask containing the coronavirus killing agent according to any one of claims 1 to 7.
A medical sheet containing the coronavirus-killing agent according to any one of claims 1 to 7.
A coronavirus agent containing composite particles having a particle size of more than 0.1 μm and less than 0.3 μm as measured by a laser diffraction method, or a member containing the same, in which silver particles are bonded to the surface of titanium oxide particles. A coronavirus killing method that is placed where the virus is expected to be present.
The method for preventing the growth of coronavirus according to claim 13, wherein the coronavirus killing agent or a member containing the agent is the one according to claim 8 or 9.