Radical manufacturing method, spore sterilization method and therapeutic agent for cancer

Abstract

To provide a new method capable of easily sterilizing spores with high safety.SOLUTION: A radical manufacturing method includes a treatment step of irradiating a radical generating source with light, generating a radical, and treating spores with the radical, wherein a peak wavelength of the irradiation light in the light irradiation exceeds a UV wavelength and is 600 nm or less. A spore sterilization method includes a treatment step of irradiating a radical generating source with light, generating a radical, and treating spores with the radical, wherein a peak wavelength of the irradiation light in the light irradiation exceeds a UV wavelength and is 600 nm or less. The peak wavelength is, for example, 405 to 470 nm.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing radicals, a method for sterilizing spores, and a therapeutic agent for cancer. [Background technology]

[0002] Various disinfectants and germicides are used to prevent infections and other diseases. However, some bacteria (also called spore-forming bacteria or spore-forming bacteria) form spores, which are extremely durable cell structures. Well-known examples of spore-forming bacteria include Bacillus anthracis, Clostridium tetani, and Clostridium boturinum. These spore-forming bacteria exist in a vegetative form during proliferation. However, when placed in a harsh environment such as dryness, high heat, the presence of growth inhibitors, or radiation, they cease proliferation and transform into spores that barely exchange nutrients with the external environment, thereby surviving. When the environment returns to normal, they transform into a vegetative form and resume proliferation. For this reason, it is difficult to truly sterilize these spore-forming bacteria, as they are inhibited from starting to grow in normal environments by, for example, common alcohol-based disinfectants or heating (Non-Patent Document 1).

[0003] Currently, methods for sterilizing spore-forming bacteria include, for example, glutaraldehyde and peracetic acid, but these substances are extremely harmful to cells and tissues, and therefore cannot be used on living organisms, and cannot be used on metallic medical instruments, etc., because they corrode them. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] MJ Leggett, G. McDonnell, SP Denyer, P. Setlow, and J.-Y. Maillard, Bacterial spore structures and their protective role in biocide resistance.Journal Applied Microbiology, 113: 485-498 (2012) Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a new method that can easily sterilize spores and the like with high safety. [Means for solving the problem]

[0006] In order to achieve the above object, the method for producing a radical of the present invention comprises the steps of: A treatment step of irradiating a radical generating source with light to generate radicals and treating spores with the radicals, The peak wavelength of the light irradiated in the light irradiation is in the range of exceeding UV wavelength and not exceeding 600 nm.

[0007] The method for sterilizing spores of the present invention includes a treatment step of irradiating a radical generating source with light to generate radicals and treating spores with the radicals, and is characterized in that the peak wavelength of the irradiated light in the light irradiation is in the range of 600 nm or less, exceeding the UV wavelength.

[0008] The instrument sterilization device of the present invention includes a reagent storage section, an instrument storage section, a connector, and a light irradiation section, the instrument storage section and the reagent storage section are connected to the connector so that the reagent stored in the reagent storage section is introduced into the instrument storage section; the reagent storage unit can store a reagent containing a radical generating source, The instrument storage section can store instruments to be sterilized, the light irradiating unit has a light source that irradiates light onto at least one of the reagent containing unit and the instrument containing unit, The light source is characterized in that the peak wavelength of the irradiated light is in the range of exceeding UV wavelengths and not exceeding 600 nm.

[0009] The cancer treatment drug of the present invention is characterized by comprising a radical generating source and treating cancer cells with radicals generated from the radical generating source. [Effects of the Invention]

[0010] As a result of extensive research, the present inventors have found that radicals can be generated from a radical generating source simply by irradiating light with a peak wavelength of 600 nm or less, rather than UV, which has an adverse effect on the human body and the natural world. According to the present invention, radicals can be generated from a radical generating source simply by irradiating light with a peak wavelength of 600 nm or less, which exceeds UV wavelengths, and the generated radicals can sterilize spores. Because the light irradiation conditions are not severe for the environment or living organisms, for example, the sterilization of spores can be performed with high safety for the handling environment, tools, handlers, etc. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the colony count of Bacillus subtilis spores in Example 1. [Figure 2] FIG. 2 is a graph showing the molar concentration of ClO2 − in Example 2. [Figure 3] FIG. 3 is a graph showing the changes over time in the chlorine dioxide radical concentration and the oxygen concentration in Example 3. [Figure 4] FIG. 4 is a graph showing the relationship between the maximum value of the molar concentration of chlorine dioxide radicals and the maximum value of the molar concentration of oxygen in Example 3. [Figure 5] 1 is a schematic diagram showing an outline of a sterilization device of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following description.

[0013] <Radical manufacturing method> As described above, the method for producing radicals of the present invention includes a generation step of irradiating a radical generation source with light to generate radicals, and is characterized in that the peak wavelength of the irradiated light in the light irradiation is in the range of longer than the UV wavelength and not more than 600 nm.

[0014] (1) Radical source The radical generating source is not particularly limited, and may be, for example, a source that generates radicals under non-solid conditions, specifically, a source that generates radicals by light irradiation, etc. For the radical generating source and the radical generating catalyst described later, for example, WO2017 / 104797 can be cited.

[0015] The radical generating source may include, for example, at least one selected from the group consisting of halogen ions, hypohalite ions, halite ions, halogen ions, and perhalite ions, and preferably includes a halogen ion. The halogen ion is preferably, for example, a chlorite ion. The radical generating source may be, for example, the ion or a salt of the ion. Examples of the salt of the ion include sodium salts and potassium salts.

[0016] The radical generating source may include, for example, an oxoacid or its salt, and specific examples include, for example, a halogen oxoacid or its salt. Examples of the oxoacid include boric acid, carbonic acid, orthocarbonic acid, carboxylic acid, silicic acid, nitrous acid, nitric acid, phosphorous acid, phosphoric acid, arsenic acid, sulfurous acid, sulfonic acid, sulfinic acid, chromic acid, dichromate, and permanganic acid. Examples of the halogen oxoacid include chlorine oxoacids such as hypochlorous acid, chlorous acid, chloric acid, and perchloric acid; bromine oxoacids such as hypobromous acid, bromous acid, bromic acid, and perbromic acid; and iodine oxoacids such as hypoiodous acid, iodous acid, iodic acid, and periodic acid.

[0017] The radical generating source may include, for example, an electron donor-acceptor linked molecule. The electron donor-acceptor linked molecule is not particularly limited, and for example, the electron donor moiety may be one or more electron donating groups, and the electron acceptor moiety may be one or more aromatic cations. The aromatic cation may be a single ring or a fused ring, and the aromatic ring may or may not contain a heteroatom and may or may not have a substituent other than the electron donating group. The aromatic ring forming the aromatic cation is not particularly limited, and for example, the number of ring-constituting atoms is a 5- to 26-membered ring.

[0018] Examples of the aromatic ring that forms the aromatic cation include a pyrrolinium ring, a pyridinium ring, a quinolinium ring, an isoquinolinium ring, an acridinium ring, a 3,4-benzoquinolinium ring, a 5,6-benzoquinolinium ring, a 6,7-benzoquinolinium ring, a 7,8-benzoquinolinium ring, a 3,4-benzoisoquinolinium ring, a 5,6-benzoisoquinolinium ring, a 6,7-benzoisoquinolinium ring, a 7,8-benzoisoquinolinium ring, and rings in which at least one of the carbon atoms that constitute these rings is substituted with a heteroatom.

[0019] (2) Radical-generating catalyst The light irradiation in the generating step of the present invention may be carried out in the presence of a radical generating catalyst in the radical generating source.

[0020] The radical generating catalyst is not particularly limited, and examples thereof include a radical generating catalyst containing at least one of ammonium and its salt, and a radical generating catalyst containing an organic compound having at least one of a Lewis acid and a Bronsted acid.

[0021] The radical generating catalyst may be, for example, a cationic surfactant such as a quaternary ammonium type cationic surfactant. Examples of the quaternary ammonium type cationic surfactant include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, hexadecyltrimethylammonium bromide, dequalinium chloride, edrophonium, didecyldimethylammonium chloride, tetramethylammonium chloride, tetrabutylammonium chloride, benzyltriethylammonium chloride, oxitropium, carbachol, glycopyrronium, safranine, sinapine, tetraethylammonium bromide, and hexadecyltrimethylammonium bromide. Examples of the anti-inflammatory agent include trimethylammonium, suxamethonium, sphingomyelin, denatonium, trigonelline, neostigmine, paraquat, pyridostigmine, phellodendrine, pralidoxime methyl iodide, betaine, betanin, bethanechol, betalain, lecithin, and cholines (choline chlorides such as benzoylcholine chloride and lauroylcholine chloride hydrate, phosphocholine, acetylcholine, choline, dipalmitoylphosphatidylcholine, and choline bitartrate).

[0022] (3) Light irradiation In the production method of the present invention, the generating step is, as described above, a step of irradiating a radical generating source with light to generate radicals, and the peak wavelength of the irradiated light in the light irradiation is in the range of longer than UV wavelength and not more than 600 nm. The light irradiation of the radical generating source may be carried out, for example, in the coexistence of the radical generating catalyst with the radical generating source.

[0023] According to the present invention, by setting the peak wavelength within the above-mentioned range, for example, it is possible to generate products such as radicals from the radical generating source by the light irradiation while sufficiently suppressing the effects on the environment and the human body. The generated radicals can be applied to, for example, various applications that utilize radicals. Note that the light irradiation conditions and the like can be referenced from the description of the spore sterilization method described below.

[0024] An example of such an application is the sterilization of spores, which will be described later. The application of the present invention is not limited thereto, and the present invention can also be applied to the sterilization of fungi, bacteria, viruses, etc. The application field of the present invention is not particularly limited, and the present invention can be used in any field where sterilization treatment is performed. Examples of such fields include various fields such as the medical field, the food field, the agriculture field, the dairy field, and the processing field.

[0025] Examples of fungi include Basidiomycota (mushrooms), Ascomycota (yeasts and molds), Diplocomycota, and Chytridiomycota. In the case of application to the agricultural field, examples of targets for sterilization treatment include the Basidiomycota, such as Rigidoporus microporus, which causes root rot of rubber trees, cacao, and cassava, and Sphaerotheca smut of wheat and barley; and the Ascomycota, such as Mycosphaerella fijiensis, which causes Sigatoga disease of bananas, Fusarium (e.g., F. oxysporum, which causes Panama disease of bananas) which causes wilt diseases of bananas, tomatoes, sweet potatoes, beans, and the like; Fusarium (e.g., F. oxysporum, which causes dry rot of onions; F. solani, which causes dry rot of potatoes; F. Fujikuroi, which causes bakanae disease of rice); and Ascomycota (e.g., Erysiphaceae), which cause powdery mildew and the like. When applied to the agricultural field, for example, the method of the present invention may be applied to the plant itself, or to the soil.

[0026] <Sterilization method> As described above, the method for sterilizing spores of the present invention includes a treatment step of irradiating a radical generating source with light to generate radicals and treating spores with the radicals, and is characterized in that the peak wavelength of the irradiated light in the light irradiation is in the range of 600 nm or less, exceeding the UV wavelength.

[0027] In the present invention, "sterilization of spores" means reducing the number of spores capable of proliferation.

[0028] (1) Spores The spores to which the present invention can be applied are not particularly limited, and examples thereof include spores of spore-forming bacteria. Examples of the spore-forming bacteria include gram-positive bacilli such as the genus Bacillus and Clostridium. Examples of the genus Bacillus include Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, and examples of the genus Clostridium include Clostridium tetani, Clostridium boturinum, Clostridium perfringens, and Clostridium difficile.

[0029] (2) Treatment process In the sterilization method of the present invention, the treatment step is a step of irradiating a radical generating source with light to generate radicals and treating spores with the radicals. The irradiation of the radical generating source with light may be carried out, for example, in the coexistence of the radical generating catalyst with the radical generating source.

[0030] As described above, the irradiated light has a peak wavelength in the range of 600 nm or less, exceeding the UV wavelength. In the present invention, UV (ultraviolet light) refers to light with a wavelength of 100 nm to 380 nm. According to the present invention, by setting the peak wavelength within the aforementioned range, for example, it is possible to sterilize highly resistant spores while sufficiently suppressing the effects on the environment and the human body, and by generating radicals and other products from the radical generating source through the light irradiation. For example, at the laboratory level, UV irradiation with a peak wavelength in the UV wavelength is commonly used as a sterilization treatment. However, due to the effects on living organisms and the environment, a clean bench or the like is used, and a shield or the like is installed during light irradiation to block the outside world. However, since the wavelength of the light used in the present invention is as described above, the installation of a shield or the like is not necessary. Furthermore, while spores cannot be sufficiently killed by UV irradiation, the present invention can kill spores without the risks associated with UV irradiation, as shown in the examples described below.

[0031] The lower limit of the peak wavelength of the irradiated light exceeds the aforementioned UV wavelength, specifically, for example, exceeds 380 nm, preferably 400 nm or more, and more preferably 405 nm or more, 415 nm or more, 440 nm or more, or 455 nm or more, since these wavelengths can sufficiently suppress the effects on the environment and living organisms. The upper limit of the peak wavelength of the irradiated light is 600 nm or less, for example, 520 nm or less, 500 nm or less, or 480 nm or less, and preferably 470 nm or less, 465 nm or less, or 460 nm or less, since these wavelengths can sufficiently generate products such as radicals that are effective for spore sterilization from the radical generating source. The lower limit of the wavelength range included in the irradiation light is, for example, more than 380 nm, 400 nm or more, or more than 400 nm, and for the same reasons as for the peak wavelength, more preferably, for example, 405 nm or more, 415 nm or more, or 440 nm or more, and the upper limit of the wavelength range included in the irradiation light is, for example, 600 nm or less, 520 nm or less, 500 nm or less, or 480 nm or less, and for the same reasons as for the peak wavelength, for example, 470 nm or less, 465 nm or less, or 460 nm or less. The spectral half width (full width at half maximum) of the irradiation light is, for example, in the range of 2 to 100 nm, 5 to 50 nm, or 12 to 35 nm.

[0032] The type of light source for the light irradiation is not particularly limited, and for example, an LED can be used. When an LED is used, its peak wavelength and spectral half width are, for example, the same as those described above.

[0033] The illuminance of the irradiation light is not particularly limited, and may be, for example, 1 to 500 mW / cm 2 , 2-300mW / cm 2 , 100-200mW / cm 2 The irradiation energy of the irradiation light is not particularly limited, and is, for example, 0.05 to 4000 J / cm 2 , 1~2000J / cm 2 , 100~400J / cm 2 is.

[0034] The treatment step can be exemplified by a first method and a second method. First, the first method will be exemplified. The treatment step of the first method includes a step of irradiating a radical generating source with light in the absence of spores to generate radicals, and a step of treating spores with the radicals. In other words, the first method is a method in which radicals are generated in advance and then the spores are treated with the radicals.

[0035] The conditions for light irradiation in the absence of spores, i.e., temperature, time, etc., are not particularly limited and can be appropriately set depending on, for example, the type of spores to be treated later, the type of treatment target in which spores may be present, the type of the radical source, etc. The temperature is not particularly limited and may be, for example, 0 to 100°C or 4 to 50°C, or may be so-called room temperature. The time is not particularly limited and may be, for example, 1 minute to 48 hours or 1 minute to 24 hours.

[0036] The form of the radical generating source reagent is not particularly limited, and examples thereof include a fluid form. When the reagent is a fluid reagent containing the radical generating source, it may be, for example, a liquid or a gel, and a liquid is preferred from the viewpoint of ease of handling. The liquid may be used in the form of a mist obtained by spraying, or may be used in a dispersed state by forming nanoparticles. The fluid reagent can be prepared, for example, by mixing the radical generating agent with a solvent (fluid solvent). Examples of the solvent include water (e.g., purified water, ion-exchanged water, pure water, sterilized water, etc.), a buffer solution, an alcohol such as ethanol, an acetic acid solvent, etc. The buffer solution is not particularly limited, and examples thereof include potassium phosphate buffer. The solvent may be used alone or in combination of two or more types. The pH of the fluid reagent is not particularly limited, and examples thereof are pH 5 to pH 10, pH 6 to pH 9, and pH 7 to pH 8. The acetic acid solvent, for example, functions as a solvent and also as a Lewis acid or Bronsted acid for the second radical generating catalyst.

[0037] In the reagent, the radical generating source may be dissolved in the solvent, or may be in a non-dissolved state, or may be in a dispersed, suspended, precipitated, or diffused state. When the reagent is a gel, for example, the radical generating source may be in a colloidal state.

[0038] In the reagent, the concentration of the radical source is not particularly limited and is, for example, 0.5 to 80 mmol / L, 1 to 40 mmol / L, or 4 to 15 mmol / L.

[0039] When the radical generating catalyst is used in combination, the reagent containing the radical generating source may further contain the radical generating catalyst, or the reagent containing the radical generating source and the reagent containing the radical generating catalyst may be used in combination. When the radical generating source and the radical generating catalyst are used in combination, the molar ratio of the radical generating source to the radical generating catalyst is, for example, 1:0.01 to 100, 1:0.1 to 50, or 1:1 to 5.

[0040] The reagent may further contain other substances, for example, and examples of the other substances include a pH adjuster and a buffering agent.

[0041] In the first method, the amount of the reagent to be added to the target is not particularly limited and can be appropriately set depending on the type of the target, the type of spores to be sterilized, etc. The amount of the reagent to be added can be indirectly indicated, for example, by the amount of the radical generating source contained in the reagent before the light irradiation, and can be expressed, for example, by the area cm of the target. 2 In addition, when the target is a liquid, the amount of the reagent to be added can be indirectly indicated, for example, by the amount of the radical generating source contained in the reagent before the light irradiation, and is, for example, 0.1 to 100 μmol, 1 to 20 μmol, or 5 to 10 μmol per cm of the volume of the target. 3 per, 0.5 to 500 μmol, 5 to 100 μmol, and 25 to 50 μmol.

[0042] In the first method, the treatment step involves, for example, first irradiating the reagent containing no spores with light to generate radicals in the reagent. The radical-containing reagent from which the radicals have been generated is then added to a target containing spores (including targets in which spores may be present), and the spores in the target are treated with the radicals. According to this method, even if it is difficult to irradiate the target with light, the spores in the target can be treated with the radicals by adding a radical-containing reagent from which radicals have been generated in advance to the target. Specifically, this method is useful, for example, when a light source for irradiating light is not available at the location where the target is located.

[0043] The method for adding the radical-containing reagent to the target is not particularly limited, and examples thereof include dripping, applying, spraying, or scattering the target, immersing the target in the reagent, or filling the space in which the target is placed with the reagent. Other methods include impregnating a carrier such as gauze or absorbent cotton with the reagent and contacting the target with the radical. The target in which the spores are present is not particularly limited, and examples thereof include solids, liquids, and semisolids. Specific examples of the method include objects and places that humans touch (e.g., doorknobs, tables, bedside tables, clothing, toilets, cars, and bathtubs), tools used by humans (e.g., cutting boards and knives), machinery (e.g., agricultural equipment), humidifiers, food and beverages, soil, farmland, water, wastewater, and swimming pools.

[0044] Next, a second method will be exemplified. In the second method, the treatment step includes irradiating a radical generating source with light in the presence of spores to generate radicals and treating the spores with the radicals. In other words, the second method is a method in which radical generation and treatment of spores with the radicals are carried out in parallel.

[0045] The conditions for light irradiation in the presence of spores, i.e., temperature, time, etc., are not particularly limited and can be appropriately set depending on, for example, the type of spores to be treated, the type of treatment target in which spores may be present, the type of the radical source, etc. The temperature is, for example, 0 to 100°C, or 4 to 50°C, and treatment may be performed at so-called room temperature. The time is, for example, 1 minute to 24 hours.

[0046] The form of the radical generating source reagent is not particularly limited, and may be, for example, a fluid form, and the examples given in the first method can be used.

[0047] In the reagent, the concentration of the radical source is not particularly limited and is, for example, 0.5 to 80 mmol / L, 1 to 20 mmol / L, or 2 to 7 mmol / L.

[0048] In the second method, for example, first, the reagent is added to an object in which spores are present (including an object in which spores may be present), and the reagent is irradiated with light. The method for adding the reagent to the subject is not particularly limited, and the examples given in the first method can be used.

[0049] According to the second method, for example, the reagent is dropped onto the target and irradiated with light, thereby generating radicals and treating spores in the target with the generated radicals. This allows for, for example, simpler treatment. Furthermore, according to the second method, for example, effective products generated by irradiating the reagent with light act immediately on spores. Therefore, compared to the first method, for example, the amount of products (radicals, etc.) that disappear before acting on spores can be reduced, thereby reducing the amount of the radical source in the reagent. Examples of active ingredients generated by irradiating the reagent with light include, in addition to the radicals, singlet oxygen, etc. According to the second method, for example, singlet oxygen, which has a short half-life, can also act immediately on spores, in addition to the radicals. Therefore, even if the concentration of the radical source is set lower than in the second method, spores can be sterilized as efficiently. This also allows for cost reduction, for example. There are no particular limitations on the method for measuring singlet oxygen. For example, a total of 650 μL of sample prepared by adding powdered NaClO, 13 μL of 1 mol / L potassium phosphate buffer, and 637 μL of DO to a final concentration of 14.5 mmol / L is placed in a vial, and the sample is irradiated with 405 nm light using a measuring device (QE-5000, manufactured by Otsuka Electronics) to confirm emission of light at 1270 nm.

[0050] In the second method, the amount of the radical generating source to be added to the target is not particularly limited and can be appropriately set depending on the type of the target, the type of spores to be sterilized, etc. The amount of the radical generating source to be added is, for example, 2 In addition, when the target is a liquid, the amount of the radical generating source added is, for example, 0.1 to 100 μmol, 1 to 10 μmol, or 3 to 5 μmol per cm of the volume of the target. 3 per, 0.5 to 500 μmol, 5 to 50 μmol, and 15 to 30 μmol.

[0051] The application fields of the present invention are not limited in any way and can be used in any field where spore sterilization is necessary or useful. Examples of such fields include the medical field, food industry, agriculture, dairy farming, and processing. In the medical field, for example, sterilization of surgical instruments such as surgical fields, gastroscopes, catheters, nebulizers, scalpels, and laparoscopes, dialysis equipment, and sterilization of excrement such as collected blood and urine are possible. The sterilization method of the present invention can also be performed on living organisms, such as humans and non-human animals. The site where the reagent is added to the living organism is not particularly limited, and can be, for example, a location where spores may be present, such as skin, cells, tissues, and organs. The sterilization method of the present invention can be applied, for example, in vivo or in vitro.

[0052] By irradiating the radical generating source with light, radicals, singlet oxygen, superoxide ions, hydrogen peroxide, and the like are generated.

[0053] <How to treat spore infection> The method for treating spore infection of the present invention includes a treatment step of irradiating a radical-generating source with light to generate radicals and treating the affected area with the radicals, wherein the peak wavelength of the irradiated light is in the range of longer than UV wavelength and not more than 600 nm. The same description of the sterilization method of the present invention can be used for the infection treatment method of the present invention.

[0054] The infection treatment method of the present invention may be a first method in which the treatment step includes, like the sterilization method of the present invention, a step of irradiating a radical generating source with light in the absence of an affected area to generate radicals and a step of administering the radicals to the affected area to treat the affected area, or a second method in which the treatment step involves simultaneous generation of radicals and treatment of the affected area. That is, the first method can be said to be a method in which radicals are generated in advance in a reagent containing a radical generating source, and the reagent containing radicals is administered to the affected area to sterilize spores in the affected area with the radicals, while the second method can be said to be a method in which a reagent containing a radical generating source is administered to the affected area, radicals are generated by light irradiation, and the generated radicals are simultaneously used to sterilize spores in the affected area.

[0055] In the present invention, "treatment" includes the meaning of preventing infection in an affected area by killing spores, suppressing the spread of infection by killing spores in an infected affected area, curing the infection, etc., and may be any of these depending on the purpose. In the present invention, "affected area" includes not only an area that is already infected, but also an area that may become infected, and may be any of these depending on the purpose.

[0056] In the present invention, the subject of administration is, for example, a human or a non-human animal excluding a human. Examples of the non-human animal include a mouse, rat, rabbit, monkey, dog, cow, etc. The affected area is not particularly limited and may be, for example, skin, cells, tissue, organ, etc.

[0057] <Sterilizer> The instrument sterilization device of the present invention includes a reagent storage section, an instrument storage section, a connector, and a light irradiation section, and the instrument storage section and the reagent storage section are connected to the connector so that the reagent stored in the reagent storage section is introduced into the instrument storage section, the reagent storage section can store a reagent including a radical generation source, and the instrument storage section can store an instrument to be sterilized, and the light irradiation section has a light source that irradiates light onto at least one of the reagent storage section and the instrument storage section, and the light source has a peak wavelength of the irradiated light in the range of 600 nm or less, exceeding UV wavelengths.

[0058] The sterilization device of the present invention is a device that performs the predetermined light irradiation described above, and therefore can be used, for example, to sterilize spores on instruments, as described above. Furthermore, the targets of sterilization are not limited to spores, and the device can also be used to sterilize various bacteria, fungi, viruses, etc. in addition to spores.

[0059] The reagent contained in the reagent storage unit may contain the radical generating source. The form of the reagent is not particularly limited, and the description of the spore sterilization method of the present invention can be used. Preferably, the radical generating source is a fluid reagent contained in the fluid solvent. When the reagent is a fluid reagent, for example, the reagent storage unit may initially contain the radical generating source in the form of a fluid reagent, or the fluid reagent may be generated within the reagent storage unit. In the latter case, for example, the reagent storage unit is a storage unit for a fluid solvent, and when the radical generating source is added to the stored fluid solvent, a fluid reagent containing the radical generating source can be generated. Specifically, the fluid solvent can be stored in the storage unit, and the radical generating source can be added to the reagent storage unit to produce a fluid reagent containing the radical generating source within the reagent storage unit. This form enables, for example, a simple long-term transport of a fluid reagent when the radical generating source is solid. Furthermore, the reagent storage section may further include, for example, a stirring section, and by operating the stirring section while the fluid solvent and the radical generating source are stored, a fluid reagent containing the radical generating source can be effectively prepared.

[0060] The types of instruments that can be sterilized by the sterilization device of the present invention are not particularly limited, and the instrument storage section may be of any size and shape that can accommodate the intended instruments. Examples of such instruments include endoscopes such as laparoscopes and thoracoscopes, gastroscopes, and dialysis treatment components (e.g., blood purifiers, tubing, etc.). The sterilization device of the present invention may also be mounted on the instruments themselves used in surgery, etc.

[0061] In the sterilizer of the present invention, the location of the light source is not particularly limited, as long as it can irradiate light into the interior of at least one of the reagent storage unit and the instrument storage unit. When the light source is located in a location where it can irradiate light onto the reagent storage unit, for example, first, a flowable reagent containing the radical generating source is stored in the reagent storage unit, the light source irradiates the flowable reagent in the reagent storage unit with light, and the flowable reagent containing radicals generated by the light irradiation is introduced via the connector into the instrument storage unit containing the instrument. When the light source is located in a location where it can irradiate light onto the instrument storage unit, for example, first, a flowable reagent containing the radical generating source is stored in the reagent storage unit, the flowable reagent is introduced via the connector into the instrument storage unit containing the instrument, and the light source irradiates the flowable reagent introduced into the instrument storage unit with light. In this way, the instrument stored in the instrument storage unit can be sterilized using the radicals.

[0062] In the sterilization apparatus of the present invention, the reagent storage unit or the instrument storage unit that is irradiated with light to generate radicals may or may not be light-shielded. In the case of UV irradiation, for example, because of known effects on the human body and the environment, it is common to place a light-shielding object to prevent UV exposure to the worker. On the other hand, in the present invention, since the specified light can be irradiated with a high level of safety, the light-shielding is not essential. Therefore, the sterilization apparatus of the present invention can also reduce manufacturing costs, for example.

[0063] An example of the sterilizer of the present invention is shown in Figure 5. Figure 5 is merely an example, and the present invention is not limited thereto. Figure 5(A) is a perspective view showing the outline of the sterilizer 1, and Figure 5(B) is a perspective view of the instrument storage section 12 of the sterilizer 1 with the lid 121 open.

[0064] As shown in FIG. 5(A), the sterilization apparatus 1 includes a reagent storage section 11, an instrument storage section 12 with an openable / closable lid 121, and a connector 13 connecting the two. An LED light source 14 is disposed inside the instrument storage section 12. As shown in FIG. 5(B), the LED light source 14 is disposed on the side of the interior of the instrument storage section 12, but is not limited to this. For example, the LED light source 14 may be disposed on the lid 121 of the instrument storage section 12 so that it faces the interior. The sterilization instrument 1 can be used, for example, as follows: First, the fluid reagent is stored in the reagent storage section 11 of the sterilization apparatus 1. Then, the lid 112 of the instrument storage section 12 is opened, and an instrument 13 (a gastroscope is exemplified in FIG. 5(B)) is stored therein. The lid 121 is then closed, and the fluid reagent is introduced from the reagent storage section 11 into the instrument storage section 12 via the connector 13. Next, the LED light source 14 irradiates the fluid reagent introduced into the instrument storage section 12 with light. This causes radicals to be generated from the radical generator, which sterilizes the instrument 13 placed inside the instrument housing section 12. While Fig. 5 shows a gastroscope as an example, the present invention is not limited to this and can be applied to various instruments that require sterilization.

[0065] <Cancer treatment drugs and cancer treatment methods> As described above, the cancer therapeutic agent of the present invention is characterized by including a radical generating source and treating cancer cells with radicals generated from the radical generating source.

[0066] The cancer treatment method of the present invention is characterized by comprising a cancer cell treatment step of generating radicals from a radical generating source and treating cancer cells with the radicals.

[0067] As shown in the experiments in the Reference Examples below, light irradiation in the presence of a radical source can produce a bactericidal effect on yeast and Candida cells. In this method, the target sites for yeast and Candida cells may be the respiratory chain and GTP synthesis pathway present in mitochondria.

[0068] Animal (e.g., human) tissues are composed of cells belonging to the same eukaryotes as yeast and Candida. Therefore, the intracellular structure of animals is very similar to that of eukaryotes such as yeast and Candida, and in particular, the mitochondrial respiratory chain and ATP synthase are very similar. Therefore, it is presumed that the present invention can kill not only cell spores but also cancer cells.

[0069] Cancer cells rely on glycolysis for energy production and have a lower utilization rate of the respiratory chain compared to normal cells (Warburg effect). However, mitochondria are present in cancer cells, and the respiratory chain is still functioning, albeit at a low level. Furthermore, cancer cells are highly sensitive to reactive oxygen species (ROS), which may explain their reliance on glycolysis for energy production. Therefore, radicals or ROS generated by the present invention may cause greater damage to cancer cells than normal cells.

[0070] Furthermore, cancer cells have approximately twice the amount of GTP synthesis as normal cells due to active peptide synthesis in ribosomes, and it is known that this GTP synthesis pathway is targeted by anticancer drugs in some cases. If the target sites of the radicals or ROS produced by the present invention are the respiratory chain and GTP synthesis pathway present in mitochondria as described above, the radicals or ROS may cause greater damage to cancer cells than to normal cells.

[0071] When there is no difference in sensitivity between normal cells and cancer cells to the radicals or ROS produced by the present invention, it is preferable to narrow the range of light irradiation using, for example, an LED, to only cancer cells or cancer tissues, and to selectively irradiate only cancer cells or cancer tissues as much as possible. In this way, it is possible to selectively damage cancer cells or cancer tissues while suppressing damage to normal cells.

[0072] In addition, the following document states that when yeast (not spores) is irradiated with a 455 nm LED, strong energy (526 J / cm 2 ), the survival rate is said to be one-tenth of that of normal cells. The following document states that LED light hits flavins in cells, generating singlet oxygen. According to the present invention, by using a radical generator, the energy of light irradiation can be kept extremely low compared to the following document. This makes it possible, for example, to selectively damage cancer cells or cancer tissues while suppressing damage to normal cells. 405nm and 450nm Photoinactivation of Saccharomyces cerevisiae K. Hoenes, M. Hess, P. Vatter, B. Spellerberg and M. Hessling European Journal of Microbiology and Immunology, 8(4):142-148 (2018).

[0073] The cancer therapeutic agent of the present invention may be capable of generating radicals, for example, when the radical generating source is irradiated with light.

[0074] In the cancer therapeutic drug of the present invention, for example, the peak wavelength of the irradiated light in the light irradiation may be in the range exceeding UV wavelengths and not exceeding 600 nm.

[0075] In the cancer therapeutic drug of the present invention, for example, the peak wavelength of the irradiated light in the light irradiation may be in the range of 405 to 470 nm.

[0076] In the cancer therapeutic drug of the present invention, for example, the lower limit of the wavelength range included in the irradiation light may be 380 nm or more.

[0077] In the cancer therapeutic agent of the present invention, for example, the light irradiation may be light irradiation with an LED.

[0078] In the cancer treatment drug of the present invention, for example, the LED may have a peak wavelength in the range of longer than UV wavelength and not more than 600 nm, and a spectral half width (full width at half maximum) of 12 to 35 nm.

[0079] The cancer therapeutic agent of the present invention may, for example, generate radicals from the radical generating source in the absence of cancer cells and treat cancer cells with the radicals.

[0080] The cancer therapeutic agent of the present invention may, for example, generate radicals from the radical generating source in the presence of cancer cells and treat the cancer cells with the radicals.

[0081] The cancer therapeutic agent of the present invention may be, for example, a fluid drug containing the radical source.

[0082] In the cancer therapeutic agent of the present invention, for example, the fluid agent may be a liquid or a gel.

[0083] In the cancer therapeutic agent of the present invention, for example, the solvent of the fluid agent may be water or a buffer solution.

[0084] In the cancer therapeutic agent of the present invention, the pH of the fluid agent may be 5 to 10, for example.

[0085] In the cancer therapeutic agent of the present invention, for example, the radical source may contain at least one selected from the group consisting of halogen ions, hypohalite ions, halite ions, halogen ions, and perhalite ions.

[0086] In the cancer therapeutic agent of the present invention, for example, the haloid ion may be a chlorite ion.

[0087] In the cancer therapeutic agent of the present invention, for example, the cancer cells may be epithelial cancer cells, and the cancer may be epithelial cancer.

[0088] In the cancer therapeutic agent of the present invention, for example, the epithelial cancer may be cancer of at least one tissue selected from the group consisting of the urinary system, reproductive system, digestive system, circulatory system, and respiratory system.

[0089] In the cancer therapeutic agent of the present invention, for example, the epithelial cancer may be cancer of at least one tissue selected from the group consisting of bladder cancer, uterine cancer, cervical cancer, ovarian cancer, fallopian tube cancer, testicular cancer, prostate cancer, colon cancer, small intestine cancer, duodenal cancer, gastric cancer, esophageal cancer, laryngeal cancer, oral cancer, tongue cancer, pharyngeal cancer, liver cancer, pancreatic cancer, gallbladder cancer, splenic cancer, peritoneal cancer, lung cancer, and eye cancer.

[0090] The form of the cancer therapeutic agent of the present invention is not particularly limited, and may be, for example, a solid, powder, semi-solid, liquid, etc. In the case of a solid, it may be, for example, a tablet, a capsule, etc.

[0091] The method for producing the cancer therapeutic drug of the present invention is not particularly limited, and can be produced, for example, by a method similar to or similar to a general pharmaceutical production method. Specifically, for example, all components of the cancer therapeutic drug of the present invention may be simply mixed. Furthermore, for example, after mixing all the components, the mixture may be compressed into a tablet or encapsulated.

[0092] The cancer therapeutic agent of the present invention may or may not contain optional components other than the radical generating source. As described above, the optional components are not particularly limited and may be, for example, the same as or equivalent to common drugs. When the cancer therapeutic agent of the present invention is in liquid form, examples of the optional components include water, pH buffer solution, and physiological saline.

[0093] The cancer therapeutic agent of the present invention may further contain, for example, one or more pharmaceutically acceptable additives as the optional ingredient. That is, for example, the sulfated polysaccharide or sulfated oligosaccharide may be formulated with one or more pharmaceutically acceptable additives prior to administration. The additives are not particularly limited, but include, for example, inactive substances such as carriers, diluents, flavorings, sweeteners, lubricants, solubilizers, suspending agents, binders, tablet disintegrants, and encapsulating materials. In addition to these, any additives commonly used in the pharmaceutical field may also be used as appropriate.

[0094] When the cancer therapeutic agent of the present invention is orally administered, the additives that can be used include, for example, the substances listed below. Carriers include, for example, lactose, starch, sucrose, glucose, sodium carbonate, mannitol, sorbitol, calcium carbonate, calcium phosphate, calcium sulfate, methylcellulose, etc. Disintegrants include, for example, corn flour, starch, methylcellulose, agar, bentonite, xanthan gum, alginic acid, etc. Binders include, for example, gelatin, natural sugar, beta-lactose, corn sweetener, natural and synthetic gums, gum arabic, tragacanth gum, sodium alginate, carboxymethylcellulose, polyethylene glycol, wax, etc. Lubricants include, for example, magnesium stearate, sodium stearate, stearic acid, sodium oleate, sodium benzoate, sodium acetate, table salt, talc, etc.

[0095] The cancer therapeutic agent of the present invention may be prepared by mixing the radical generating source and, if necessary, other optional ingredients with a diluent or encapsulating the radical generating source in a carrier. The carrier may be in the form of a capsule, a small bag, paper, or other container. The carrier may also serve as a diluent and may be a solid, semi-solid, or liquid that acts as a vehicle. The form of the pharmaceutical of the present invention is not particularly limited, and may be in various forms, such as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and packaged sterile powders.

[0096] The administration form of the cancer therapeutic agent of the present invention is not particularly limited, and can be administered orally or parenterally depending on the purpose. The form when administered orally is not particularly limited, and forms commonly used by those skilled in the art can be selected, such as regular and enteric-coated tablets, capsules, pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups, solid or liquid aerosols, and emulsions. The form when administered parenterally is also not particularly limited, and forms commonly used by those skilled in the art can be selected, such as intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, injection, infusion, etc.

[0097] Furthermore, the single dose and administration interval of the cancer therapeutic agent of the present invention are not particularly limited and can be appropriately selected depending on the purpose. They can be selected by those skilled in the art taking into consideration various factors, including the patient's age, weight, sex, medical condition, pathology, administration route, the patient's level of metabolic and excretory function, the dosage form used, and the specific oxoacid, oxoacid ion, or oxoacid salt to be administered.

[0098] Next, the cancer treatment method of the present invention is characterized by including a cancer cell treatment step of generating radicals from a radical generating source and treating cancer cells with the radicals.

[0099] In the cancer treatment method of the present invention, for example, in the cancer cell treatment step, the radical generating source may be irradiated with light to generate radicals.

[0100] In the cancer treatment method of the present invention, for example, the peak wavelength of the irradiated light in the light irradiation may be in the range exceeding UV wavelengths and not exceeding 600 nm.

[0101] In the cancer treatment method of the present invention, for example, the peak wavelength of the irradiated light in the light irradiation may be in the range of 405 to 470 nm.

[0102] In the cancer treatment method of the present invention, for example, the lower limit of the wavelength range included in the irradiation light may be 380 nm or more.

[0103] In the cancer treatment method of the present invention, for example, the light irradiation may be light irradiation with an LED.

[0104] In the cancer treatment method of the present invention, for example, the LED may have a peak wavelength in the range of longer than UV wavelength and not more than 600 nm, and a spectral half width (full width at half maximum) of 12 to 35 nm.

[0105] In the cancer treatment method of the present invention, the treatment step may include, for example, a step of generating radicals from the radical generating source in the absence of cancer cells, and a step of treating cancer cells with the radicals.

[0106] In the cancer treatment method of the present invention, the treatment step may include, for example, generating radicals from the radical generating source in the presence of cancer cells and treating the cancer cells with the radicals.

[0107] In the cancer treatment method of the present invention, for example, the radical generation from the radical source may be radical generation from a fluidity reagent containing the radical source.

[0108] In the cancer treatment method of the present invention, for example, the flowable reagent may be a liquid or a gel.

[0109] In the cancer treatment method of the present invention, for example, the solvent of the flowable reagent may be water or a buffer solution.

[0110] In the method for treating cancer of the present invention, the flowability reagent may have a pH of 5 to 10, for example.

[0111] In the cancer treatment method of the present invention, for example, the radical source may include at least one selected from the group consisting of halogen ions, hypohalite ions, halite ions, and perhalate ions.

[0112] In the cancer treatment method of the present invention, for example, the haloid ion may be a chlorite ion.

[0113] In the cancer treatment method of the present invention, for example, the cancer cells may be epithelial cancer cells, and the cancer may be epithelial cancer.

[0114] In the cancer treatment method of the present invention, for example, the epithelial cancer may be cancer of at least one tissue selected from the group consisting of the urinary system, reproductive system, digestive system, circulatory system, and respiratory system.

[0115] In the cancer treatment method of the present invention, for example, the epithelial cancer may be cancer of at least one tissue selected from the group consisting of bladder cancer, uterine cancer, cervical cancer, ovarian cancer, fallopian tube cancer, testicular cancer, prostate cancer, colon cancer, small intestine cancer, duodenal cancer, gastric cancer, esophageal cancer, laryngeal cancer, oral cancer, tongue cancer, pharyngeal cancer, liver cancer, pancreatic cancer, gallbladder cancer, splenic cancer, peritoneal cancer, lung cancer, and eye cancer.

[0116] In the cancer treatment method of the present invention, for example, the radical source may be a radical source contained in the cancer treatment drug of the present invention, and the cancer treatment method may further include an administration step of administering the cancer treatment drug to a patient.

[0117] The present invention further provides a method for using the therapeutic agent of the present invention for the treatment of cancer.

[0118] The method for using the cancer therapeutic agent of the present invention may include, for example, an administration step of administering the cancer therapeutic agent of the present invention to a patient.

[0119] The present invention further provides a method for producing the cancer therapeutic drug of the present invention, using the radical generating source. As described above, the method for producing the cancer therapeutic drug of the present invention is not particularly limited.

[0120] In the present invention, patients to be administered with a cancer therapeutic drug or treated by a cancer treatment method include, for example, humans or non-human animals, such as mice, rats, rabbits, monkeys, dogs, and cows. [Example]

[0121] Examples of the present invention will be described below, but the present invention is not limited to the following examples.

[0122] [Example 1] Bacillus subtilis spores were sterilized by the method of the present invention, and a reduction in the number of spores was confirmed.

[0123] (1) Checking radical concentration Sodium chlorite solution (Fujifilm Wako Pure Chemical Industries, chlorite ion standard solution) was used as the radical source. The molar concentration of sodium chlorite was set to 14.5 mmol / L, and the pH was adjusted to 7.0, 7.5, or 8.0 by adding 20 mmol / L potassium phosphate buffer to prepare reagents. 1 mL of each reagent was placed in a DispoCell UV (trade name: Double-sided Transparent Semi-Micro #1941, Nikko Hansen Co., Ltd.), and light was irradiated from the side of the cell using an LED. The LED used was a commercially available product (trade name: M455L4, Thorlabs Japan Co., Ltd.) with a peak wavelength of 455 nm and a spectral half-width of 15 nm. The distance between the side of the cell where the light was irradiated and the LED light output surface was 0.3 cm, and the irradiation conditions were an irradiance of 157 mW / cm. 2 The irradiation time was 40 minutes. The irradiation energy under these conditions was 0.157 (W / cm2 )×40(minutes)×60(seconds)=377(J / cm 2 ) was calculated.

[0124] Then, a two-beam spectrophotometer (product name UV-2400PC, Shimadzu Corporation) was used to measure the absorbance of the reagent in the cell from 450 nm to 240 nm, and the molar concentrations of chlorite ions and chlorine dioxide radicals were quantified. These results are shown in Table 1 below. Table 1 below shows the concentration results from six measurements (n=6) under each pH condition. As shown in Table 1 below, the generation of chlorine dioxide radicals was confirmed by irradiating the reagent with light using an LED. Furthermore, the more acidic the pH of the reagent, the greater the amount of chlorine dioxide radicals generated.

[0125] [Table 1]

[0126] (2) Sterilization of spores It was confirmed that radicals were generated by the light irradiation under the above-mentioned predetermined conditions, and the resulting sterilizing effect on spores was confirmed.

[0127] Bacillus subtilis was plated on a normal agar medium and cultured at 37°C for one week. Colonies were scraped from the agar medium, suspended in saline, washed, and centrifuged to recover the Bacillus subtilis, which was used as a spore. The spores were collected in saline at a concentration of approximately 6 x 10 6 The cell spore suspension was diluted with purified water as needed to adjust the cell spore concentration to the desired level.

[0128] Meanwhile, a reagent at pH 7.0 was prepared in the same manner as in (1) above, and irradiated with the LED to generate radicals. The reagent (chlorine dioxide radical concentration 600 μmol / L) from which radicals were generated was appropriately diluted with 10 mmol / L potassium phosphate buffer (pH 7.0) to prepare samples containing radicals at a predetermined concentration. 0.5 mL of each sample was mixed with 0.5 mL of the cell spore suspension, and the mixture was allowed to stand at room temperature for 30 minutes. The mixture after standing was then plated on brain heart agar medium and cultured at 37°C for 12 hours, after which the number of colonies was counted.

[0129] As control 1, a pH 7.0 reagent that had not been irradiated with light was added to the cell spore suspension, and the suspension was left to stand and cultured in the same manner, followed by counting colonies. As control 2, the reagent was not added, and the cell spore suspension was irradiated with LED light for 30 minutes under the irradiation conditions in (1) above, then cultured in the same manner, and the colonies were counted.

[0130] These results are shown in Figure 1. In Figure 1, the vertical axis represents the number of spores grown, and the horizontal axis represents the chlorine dioxide radical concentration in the spore suspension spread on the agar medium. In both Controls 1 and 2, no reduction in the number of spores was confirmed, whereas in the Example, as shown in Figure 1, it was confirmed that chlorine dioxide radicals were generated by light irradiation, and that the cell spores were killed in a manner dependent on the chlorine dioxide radical concentration. Furthermore, when the chlorine dioxide radical concentration was 300 μmol / L, the number of spores was 10 -6 Furthermore, in this example, the wavelength of the light irradiation does not include, for example, ultraviolet light that can have an effect on the human body, and the reagents used and the radicals generated can be used at concentrations that are safe for the human body, so it can be said that the sterilization of spores, which has been difficult to achieve in the past, can be achieved in an extremely safe manner.

[0131] [Example 2] A sodium chlorite solution (Fujifilm Wako Pure Chemical Industries, chlorite ion standard solution) was used as the radical source. Reagents with sodium chlorite molar concentrations of 14.5 mmol / L, 7.25 mmol / L, and 3.63 mmol / L were prepared using a 20 mmol / L potassium phosphate buffer solution (pH 7.0). As in Example 1(1), 1 mL of each reagent was placed in a DispoCell UV (trade name: Double-sided Transparent Semi-Micro #1941, Nikko Hansen Co., Ltd.), and light was irradiated from the side of the cell using an LED. The light irradiation was performed in the same manner as in Example 1, unless otherwise specified. The LED used was the same commercially available LED (trade name: M455L4, Thorlabs Japan Co., Ltd.) with a peak wavelength of 455 nm and a spectral half-width of 15 nm, as in Example 1. The distance between the side of the cell where light was irradiated and the light emission surface of the LED was 0.3 cm, and the irradiation conditions were an irradiance of 81.5 mW / cm. 2 or 157 mW / cm 2 Then, using a two-path spectrophotometer (product name UV-2400PC, Shimadzu Corporation), the absorption spectrum was measured over time from the start of irradiation, and the concentration of chlorite ions (ClO2 - ) was calculated, and the molar concentration of chlorine dioxide radicals (ClO2 · ) was calculated.

[0132] These results are shown in Figure 2. In the upper panel of Figure 2, the horizontal axis represents the irradiation time (the scale is the same as in the lower panel), and the vertical axis represents the ClO2 - In the lower graph of Figure 2, the horizontal axis is the irradiation time, and the vertical axis is the ClO2 · In both figures in Figure 2, the black plots indicate the irradiance of 81.5 mW / cm 2 The white plot shows the result of irradiance 157mW / cm 2 This is the result.

[0133] As shown in the bottom diagram of Figure 2, when the irradiance of LED irradiation is doubled, ClO2 ·The time to reach the maximum peak of the same molar concentration was shortened by half. - It was found that when the concentration of chlorite ions doubled, the height of the maximum peak approximately doubled. From these results, for example, by appropriately setting the concentration of chlorite ions in the reagent, the irradiance, etc., it is possible to adjust the concentration of chlorine dioxide radicals and the irradiation time required for maximum generation of chlorine dioxide radicals. Furthermore, it was confirmed that when light irradiation was performed in the same manner except that an LED with a peak wavelength of 405 nm was used, the time required for maximum peak generation of chlorine dioxide radicals could be shortened to approximately 1 / 10 to 1 / 15. From these results, it is possible to adjust the irradiation time required for maximum generation of chlorine dioxide radicals by changing the peak wavelength of the light source used for light irradiation.

[0134] It is presumed that the following reaction occurs when a reagent containing chlorite ions is irradiated with light under the above conditions, but this does not limit the present invention in any way. [ka]

[0135] Furthermore, as shown in FIG. 2, according to this example, simply ClO2 ― Not only did the concentration decrease, but after the increase in ClO2· concentration, ClO2 ― It was confirmed that the concentration of ClO2· decreased. Without light irradiation, the concentration of ClO2· decreased to about half in 2 hours. This is thought to be due to the diffusion of ClO2· from the water layer to the gas layer. Also, as shown in the above chemical reaction formula (Chemical Formula 1), ClO2· is converted to O2 - Thus, according to this example, it is thought that ClO2 - It was confirmed that only low concentrations of H2O2 remained, with almost no ClO2· or singlet oxygen (which has an extremely short lifespan). H2O2 is significantly less toxic to living organisms than ClO2·, and at the maximum concentration estimated to be produced, around 800 μM (0.0027 W / V%, 27 ppm), its cytotoxicity and biotoxicity are extremely low.

[0136] [Example 3] The generation of singlet oxygen was indirectly confirmed by irradiating a radical source with an LED.

[0137] A sodium chlorite solution (Fujifilm Wako Pure Chemical Industries, Ltd., chlorous acid standard solution) was used as the radical source. Using a 20 mmol / L potassium phosphate buffer solution (pH 7.0), reagents with sodium chlorite molar concentrations of 14.5 mmol / L, 7.25 mmol / L, and 3.63 mmol / L were prepared. The same spores as in Example 1 were added to each of the reagents at a concentration of 1 × 10 6 ~1×10 7 The reagents were added so that the spores were contained within the solution. Next, 300 μL of each concentration of reagent containing the spores was added to an oxygen measuring device equipped with an oxygen electrode. The oxygen measuring device was equipped with an oxygen electrode (trade name: Biological Dissolved Oxygen Monitor, Digital 1ch OXY018A) and a 2.5 mL Perspex electrode (OXY042A, Rank Brothers). An LED was attached to the top of the oxygen electrode and irradiated. A commercially available LED (trade name: M455L4, Thorlabs Japan Co., Ltd.) with a peak wavelength of 455 nm and a spectral half-width of 15 nm was used. The distance between the surface of the reagent solution to be irradiated and the light emission surface of the LED was 3 cm, and the irradiation conditions were 300 mW / cm. 2 Then, using a two-path spectrophotometer (product name UV-2400PC, Shimadzu Corporation), the absorption spectrum was measured over time from the start of irradiation, and the molar concentration of chlorine dioxide radicals (ClO2·) was calculated from the absorbance at the absorption maximum at a wavelength of 359 nm. The molar concentration of oxygen was measured using the oxygen meter.

[0138] Figure 3 shows the changes in chlorine dioxide radical concentration and oxygen concentration over time for a reagent with a sodium chlorite molar concentration of 14.5 mmol / L. In Figure 3, the vertical axis represents the molar concentrations of oxygen and chlorine dioxide radicals, and the horizontal axis represents irradiation time. Figure 4 shows the results for the maximum molar concentrations of oxygen and chlorine dioxide radicals for reagents with sodium chlorite molar concentrations of 14.5 mmol / L, 7.25 mmol / L, and 3.63 mmol / L. In Figure 4, the vertical axis represents the maximum molar concentration of chlorine dioxide radicals, and the horizontal axis represents the maximum molar concentration of oxygen. In Figure 4, the left plot represents the results for the 14.5 mmol / L reagent, the middle plot represents the results for the 7.25 mmol / L reagent, and the right plot represents the results for the 3.63 mmol / L reagent.

[0139] The results in Figures 3 and 4 revealed the following. As shown in Figure 3, within 20 minutes of irradiation with a 455 nm LED, the ClO2· concentration reached a maximum of approximately 0.7 mmol / L, and at almost the same time, the oxygen concentration also reached approximately 0.6 mmol / L. This shows that a 20-minute irradiation time produces a sufficient amount of ClO2· to sterilize spores. Furthermore, as shown in Figure 4, it became clear that the ClO2· concentration and oxygen concentration are proportional when they reach their maximum concentration. Furthermore, the generation of a sufficient amount of oxygen could be confirmed using an oxygen electrode, and this result can be said to indirectly prove the production of singlet oxygen. Furthermore, the generation of a sufficient amount of oxygen was confirmed using an oxygen electrode, and this result can be said to indirectly prove the production of singlet oxygen. Furthermore, the generation of a sufficient amount of oxygen from the spores (1 x 10 6 / 300 μL) in the presence of 3.63 mmol / L sodium chlorite (20 mmol / L phosphate buffer, pH 7.0) under LED 455 nm (irradiation conditions: 300 mW / cm 2 ), and after 30 minutes of irradiation, the spore count was reduced to 1 x 100 / 300 μL or less.

[0140] [Example 4] Yeast and Candida cells were sterilized by exposure to light in the presence of sodium chlorite, and the reduction in colony numbers was confirmed.

[0141] (1) Preparation of yeast cells The yeast was inoculated into a modified YPE liquid medium (2% polypeptone, 1% yeast extract, 2% ethanol, 0.2% glucose) and cultured at 30°C for 1 day. The bacterial solution was collected from the liquid medium, suspended in purified water, washed, and the yeast cells were collected by centrifugation. The cells were diluted to approximately 5 × 10 in 100 mmol / L potassium phosphate buffer (pH 7.0). 8 The cell suspension was diluted with 100 mmol / L potassium phosphate buffer solution as needed to adjust the cell concentration to the desired cell concentration.

[0142] (2) Preparation of a reagent (aqueous solution) containing sodium chlorite A sodium chlorite solution (Fujifilm Wako Pure Chemical Industries, chlorite ion standard solution) was used as the radical source. The molar concentration of sodium chlorite was set to 7.25 mmol / L, and a 20 mmol / L potassium phosphate buffer solution was added to prepare a reagent with a pH of 7.0.

[0143] (3) Irradiation of sodium chlorite solution with light The reagent (2) was irradiated with light from an LED with a peak wavelength of 455 nm and a spectral half-width of 15 nm in the same manner as in "(1) Confirmation of radical concentration" in Example 1, except that the molar concentration of sodium chlorite was set to 7.25 mmol / L. The irradiation conditions were an irradiance of 157 mW / cm 2 The irradiation time was 40 minutes. After the irradiation, the reagent in the cell was measured for absorption from 450 nm to 240 nm using a two-path spectrophotometer (trade name UV-2400PC, Shimadzu Corporation), and the molar concentrations of chlorite ions and chlorine dioxide radicals were quantified. The chlorite ions were 350 mmol / L, and the chlorine dioxide radicals were 375 μmol / L. In this way, a reagent containing chlorine dioxide radicals was prepared.

[0144] (4) Sterilization of yeast

[0145] The yeast was sterilized using two methods, "Experiment 1" and "Experiment 2," as follows.

[0146] (4-1) Experiment 1 The reagent containing chlorine dioxide radicals prepared in (3) above was diluted with 10 mmol / L potassium phosphate buffer (pH 7.0) to prepare a sample containing chlorine dioxide radicals at a concentration of 20 μmol / L. On the other hand, the cell suspension prepared in (1) was diluted with 10 mmol / L potassium phosphate buffer (pH 7.0) to prepare a sample containing chlorine dioxide radicals at a concentration of approximately 5 × 10 8 The concentration was adjusted to 0.5 x 10 cells / mL. Next, 0.5 mL of the sample containing chlorine dioxide radicals was mixed with 0.5 mL of the cell spore suspension, and the mixture was left standing at room temperature for 30 minutes. The mixture was then spread on a brain heart agar medium and incubated at 30°C for one day, after which the number of colonies was counted. As a result, the cell concentration was found to be approximately 2.5 x 10 7 The cell count was 1 / mL, confirming that the survival rate had decreased to approximately one-tenth.

[0147] (4-2) Experiment 2 A sodium chlorite solution (Fujifilm Wako Pure Chemical Industries, chlorite ion standard solution) was used as the radical source. A reagent with a molar concentration of sodium chlorite of 3.75 mmol / L was prepared using 20 mmol / L potassium phosphate buffer (pH 7.0). Then, as in Experiment 1, 0.5 mL of the reagent was added with a concentration of approximately 5 × 10 yeast cells. 8 0.5 mL of yeast solution containing 1000 cells / mL was added to make a 1 mL solution, which was placed in a DispoCell UV (trade name: Double-sided Transparent Semi-Micro #1941, Nikko Hansen Co., Ltd.), and light was irradiated from the side of the cell using an LED. Unless otherwise specified, the light irradiation was carried out in the same manner as in Experiment 1. That is, the LED used was the same commercially available LED (trade name: M455L4, Thorlabs Japan Co., Ltd.) with a peak wavelength of 455 nm and a spectral half-width of 15 nm as in Experiment 1, the distance between the side of the cell where light was irradiated and the light emission surface of the LED was 0.3 cm, and the irradiation conditions were an irradiance of 21 mW / cm. 2 The irradiation energy under these conditions was 37.8 J / cm. 2The treated solution was diluted appropriately and plated on brain heart agar medium. After incubation at 30°C for one day, the number of colonies was counted. As a result, the cell concentration was approximately 2.5 x 10 7 The cell count was 1 / mL, confirming that the survival rate had decreased to approximately one-tenth.

[0148] In addition, when cells were cultured in the same manner as in (1) to (4), except that Candida albicans was used instead of yeast, it was confirmed that the cell viability was similarly reduced to about one-tenth of the original level.

[0149] Furthermore, in yeast and Candida cells, a decrease in the electron transport activity of the respiratory chain was observed in proportion to the decrease in viability. Therefore, in this example, it is speculated that one of the targets of treatment with chlorine dioxide radicals in yeast and Candida cells is the respiratory chain in the mitochondria. When the respiratory chain is damaged, reactive oxygen species (ROS) are produced, leading to apoptosis and cell death.

[0150] (5) Control experiment The yeast or Candida cells were left to stand at 25°C for 1 hour in the same manner as in (1) to (4) above, except that the sodium chlorite aqueous solution in (3) above was not irradiated with light. When the cells were left to stand at 25°C for 1 hour in the same manner as in (1) to (4) above, the survival rate did not decrease. In addition, when the concentration of the cells prepared in the "(4) Spore sterilization treatment" above was about 2.5 x 10 8 The suspension containing chlorine dioxide radicals was irradiated with light at a peak wavelength of 455 nm and a spectral half-width of 15 nm at 37.8 J / cm in the same manner as in "(3) Irradiation of sodium chlorite solution with light" above, without mixing a sample containing chlorine dioxide radicals. 2 Irradiation did not reduce the viability of yeast or Candida cells.

[0151] According to these results, chlorine dioxide radicals (ClO2·) were not generated when sodium chlorite solution or light irradiation alone was used, but when both were used in combination, chlorine dioxide radicals were generated, which is thought to have had a bactericidal effect on cells. Furthermore, when sodium chlorite solution was used in combination with light irradiation, singlet oxygen was generated in addition to chlorine dioxide radicals, and it is possible that a synergistic effect of chlorine dioxide radicals and singlet oxygen resulted in a bactericidal effect on cells.

[0152] (6) Reference experiment The same treatments as in (1) to (4) were performed, except that a 2.90 mmol / L aqueous solution of cGMP (cyclic guanosine monophosphate) was used instead of the cell suspension. As a result, it was confirmed that a substance inhibiting the growth of yeast and Candida was produced. Based on the structure of the raw material cGMP, this product is likely to be a compound that inhibits the GTP synthesis pathway. Therefore, in this example, the target site of the treatment of yeast and Candida cells with chlorine dioxide radicals may be the GTP synthesis pathway in addition to the respiratory chain present in mitochondria.

[0153] [Example 5] Cancer cells were treated by the method of the present invention, and a reduction in the number of the cancer cells was confirmed.

[0154] HT-29 cells (human colon adenocarcinoma cells) were seeded into each well of a 96-well cell culture microplate (surface-treated) and grown in a CO2 incubator at 37°C with 5% CO2 until a cell density of approximately 2000 cells / well was reached. 90 μL of McCoy's 5a Medium (Fujifilm Wako Pure Chemical Corporation) was used as the cell culture medium. After each of the treatments (1) through (6) below, the medium was replaced with 90 μL of McCoy's 5a Medium and cultured in a CO2 incubator under the above conditions for 2 days. To determine viable cell counts, 10 μL of WST-8 solution (solution in the "WST-8 Cell Proliferation Kit"; Funakoshi Co., Ltd.) was added and the cells were incubated in a CO2 incubator at 37°C for 5 hours. The absorbance at 450 nm was measured using a microplate reader. The percentage of absorbance for each sample was calculated using treatment (1) as a control. The absorbance values ​​of untreated cells and treatment (1) were almost the same.

[0155] The substance name of "WST-8" in the WST-8 solution is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.

[0156] The conditions for each treatment (1) to (6) and the percentage of absorbance for each sample are as follows. The percentage of absorbance that decreases from 100% is estimated to be approximately equal to the cancer cell death rate (%). (1) 100 μL of PBS (pH 7.0) ** and left at room temperature for 30 minutes. ------Absorbance ratio 100% (2) Replace with 100 μL of PBS (pH 7.0) and incubate at room temperature with an LED (product name M455L3, Thorlabs Japan Co., Ltd.: wavelength 455 nm, irradiance 40 mW / cm2 ) for 30 minutes (irradiation energy: 72 J / cm 2 ). ------Absorbance ratio 96% (3) The mixture was replaced with 100 μL of 1.4 mM NaClO2-containing PBS (pH 7.0) and left at room temperature for 30 minutes. -------Absorbance ratio 97% (4) Replace with 100 μL of 1.4 mM NaClO2-containing PBS (pH 7.0), and then use an LED (product name M455L3, Thorlabs Japan Co., Ltd.: wavelength 455 nm, irradiance 40 mW / cm) at room temperature. 2 ) for 30 minutes (irradiation energy: 72 J / cm 2 ). ------Absorbance ratio 11% (5) The mixture was replaced with 100 μL of 2.8 mM NaClO2-containing PBS (pH 7.0) and left at room temperature for 30 minutes. -------Absorbance ratio 97% (6) Replace with 100 μL of 2.8 mM NaClO2-containing PBS (pH 7.0), and incubate at room temperature with an LED (product name M455L3, Thorlabs Japan Co., Ltd.: wavelength 455 nm, irradiance 40 mW / cm2). 2 ) for 30 minutes (irradiation energy: 72 J / cm 2 ). ------Absorbance ratio 6% ** The pH was adjusted to 7.0 and the osmolality was the same as that of standard pH 7.5 phosphate buffered saline (PBS) (pH 7.0) (NaCl: 140 mM, KCl: 0.43 mM, KH2PO4: 3.72 mM, Na2HPO4: 5.81 mM).

[0157] According to this example, in the presence of NaClO2 solution, 455 nm visible light was irradiated at 72 J / cm 2 It was confirmed that irradiation resulted in the death of approximately 89% (1.4 mM NaClO2) and 94% (2.8 mM NaClO2) of cancer cells (see treatments (4) and (6) above).

[0158] As described above, in this example, cancer cells were treated by the method of the present invention, and a reduction in the number of the cancer cells was confirmed.

[0159] As mentioned above, human tissues are composed of cells belonging to the same eukaryotes as yeast and Candida. Therefore, the structure of human cells is very similar to that of eukaryotes such as yeast and Candida, and the mitochondrial respiratory chain and ATP synthase in particular are very similar. According to this example, as in Example 4, it is believed that the combination of chlorite ions and purple LED (455 nm) quantitatively produces chlorine dioxide radicals and singlet oxygen, thereby killing human cancer cells.

[0160] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention. [Industrial Applicability]

[0161] According to the present invention, radicals can be generated from a radical generating source simply by irradiating light with a peak wavelength of 600 nm or less, exceeding the UV wavelength. Therefore, for example, spores can be sterilized by the generated radicals. Furthermore, the light irradiation conditions are not severe for, for example, the environment and living organisms, and therefore spore sterilization can be performed with high safety for the handling environment, tools, handlers, etc.

[0162] This application claims priority based on Japanese Patent Application No. 2019-181758, filed October 1, 2019, and Japanese Patent Application No. 2020-089254, filed May 21, 2020, the disclosures of which are incorporated herein in their entireties.

Claims

1. A treatment step of irradiating a radical generating source with light to generate radicals and treating spores with the radicals, A method for sterilizing spores, characterized in that the peak wavelength of the irradiated light in the light irradiation is in the range exceeding UV wavelengths and not exceeding 600 nm.

2. The sterilization method according to claim 1, wherein the peak wavelength is in the range of 405 to 470 nm.

3. The sterilization method according to claim 1 or 2, wherein the lower limit of the wavelength range included in the irradiation light is 380 nm or more.

4. The processing step Irradiating the radical generating source with light in the absence of spores to generate radicals; The sterilization method according to any one of claims 1 to 3, further comprising a step of treating spores with the radicals.

5. The processing step The sterilization method according to any one of claims 1 to 4, comprising the step of irradiating the radical generating source with light in the presence of spores to generate radicals and treating the spores with the radicals.

6. The sterilization method according to any one of claims 1 to 5, wherein the light irradiation is light irradiation by an LED.

7. The sterilization method according to claim 6, wherein the peak wavelength of the LED is in the range of more than UV wavelength and not more than 600 nm, and the spectral half width (full width at half maximum) is 12 to 35 nm.

8. The sterilization method according to any one of claims 1 to 7, wherein the light irradiation of the radical generating source is light irradiation of a flowable reagent containing the radical generating source.

9. The sterilization method according to claim 8, wherein the flowable reagent is a liquid or a gel.

10. The sterilization method according to claim 8 or 9, wherein the solvent of the flowable reagent is water or a buffer solution.

11. The sterilization method according to any one of claims 8 to 10, wherein the pH of the fluid reagent is 5 to 10.

12. 12. The sterilization method according to any one of claims 1 to 11, wherein the radical generating source comprises at least one selected from the group consisting of halogen ions, hypohalite ions, halite ions, and perhalate ions.

13. The sterilization method according to claim 12, wherein the haloid ion is a chlorite ion.

14. The sterilization method according to any one of claims 1 to 13, wherein the spores are spores of the genus Bacillus or Clostridium.

15. a reagent storage section, an instrument storage section, a connector, and a light irradiation section; the instrument storage section and the reagent storage section are connected to the connector so that the reagent stored in the reagent storage section is introduced into the instrument storage section; the reagent storage unit can store a reagent containing a radical generating source, The instrument storage section can store instruments to be sterilized, the light irradiating unit has a light source that irradiates light onto at least one of the reagent containing unit and the instrument containing unit, The instrument sterilization device is characterized in that the light source emits light with a peak wavelength in the range exceeding UV wavelengths and not exceeding 600 nm.

16. The sterilization apparatus according to claim 15, wherein the reagent is a flowable reagent and includes the radical generating source and a flowable solvent.

17. The reagent storage unit is a reservoir for a fluid solvent; The sterilization apparatus according to claim 15 or 16, wherein when the radical generating source is added to the stored fluid solvent, a fluid reagent containing the radical generating source is produced.

18. A cancer therapeutic agent comprising a radical generating source, wherein cancer cells are treated with radicals generated from the radical generating source.

19. The cancer therapeutic agent according to claim 18, which is capable of generating radicals by irradiating the radical generating source with light.

20. The cancer therapeutic drug according to claim 19, wherein the peak wavelength of the irradiated light is in the range exceeding UV wavelengths and not exceeding 600 nm.

21. The cancer therapeutic agent according to claim 20, wherein the peak wavelength is in the range of 405 to 470 nm.

22. The cancer therapeutic agent according to claim 20 or 21, wherein the lower limit of the wavelength range included in the irradiated light is 380 nm or more.

23. The cancer therapeutic agent according to any one of claims 19 to 22, wherein the light irradiation is light irradiation by an LED.

24. The cancer therapeutic agent according to claim 23, wherein the peak wavelength of the LED is in the range of exceeding UV wavelengths to 600 nm or less, and the spectral half width (full width at half maximum) is 12 to 35 nm.

25. generating radicals from the radical generating source in the absence of cancer cells; The cancer therapeutic agent according to any one of claims 18 to 24, wherein cancer cells are treated with the radical.

26. The cancer therapeutic agent according to any one of claims 18 to 24, wherein radicals are generated from the radical generating source in the presence of cancer cells, and the cancer cells are treated with the radicals.

27. The cancer therapeutic agent according to any one of claims 18 to 26, which is a fluid agent containing the radical generating source.

28. The cancer therapeutic agent of claim 27, wherein the fluid agent is a liquid or a gel.

29. The cancer therapeutic agent according to claim 27 or 28, wherein the solvent of the fluid agent is water or a buffer solution.

30. The cancer therapeutic agent according to any one of claims 27 to 29, wherein the pH of the fluid agent is 5 to 10.

31. The cancer therapeutic agent according to any one of claims 18 to 30, wherein the radical generating source comprises at least one selected from the group consisting of halogen ions, hypohalite ions, halite ions, and perhalate ions.

32. The cancer therapeutic agent of claim 31 , wherein the haloid ion is a chlorite ion.

33. The cancer therapeutic agent according to any one of claims 18 to 32, wherein the cancer cells are epithelial cancer cells and the cancer is an epithelial cancer.

34. The cancer therapeutic drug according to claim 33, wherein the epithelial cancer is cancer of at least one tissue selected from the group consisting of the urinary system, reproductive system, digestive system, circulatory system, and respiratory system.

35. The cancer therapeutic agent according to claim 33 or 34, wherein the epithelial cancer is cancer of at least one tissue selected from the group consisting of bladder cancer, uterine cancer, cervical cancer, ovarian cancer, fallopian tube cancer, testicular cancer, prostate cancer, colon cancer, small intestine cancer, duodenal cancer, gastric cancer, esophageal cancer, laryngeal cancer, oral cancer, tongue cancer, pharyngeal cancer, liver cancer, pancreatic cancer, gallbladder cancer, splenic cancer, peritoneal cancer, lung cancer, and eye cancer.

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