JP7699426B2 - Ophthalmic composition for protecting cells from photodamage - Google Patents

Description

The present invention relates to an ophthalmic composition for protecting cells from damage caused by light.

In recent years, the spread of personal computers, smartphones, and LEDs has led to an increase in exposure to blue light. Because blue light has strong energy, it is known that exposure to blue light can cause symptoms such as eye fatigue, eye pain, and eye inflammation (for example, Non-Patent Document 1).

Asia-Pacific Journal of Ophthalmology, 4(2), p80-85, 2015

As it is predicted that exposure to blue light will further increase in the future, the present inventors have discovered that if cells in the eye can be protected from damage caused by blue light, it may be possible to alleviate the above-mentioned symptoms. The present invention aims to provide an ophthalmic composition that can protect cells from damage caused by light rays such as blue light.

The present inventors have found that at least one component selected from the group consisting of sulfites, vitamin E, and zinc sulfate can protect corneal epithelial stem cells from damage caused by blue light.

The present invention provides, for example, the following inventions.
[1]
An ophthalmic composition for protecting cells from damage caused by light, comprising at least one member selected from the group consisting of sulfites and vitamin E's.
[1]
The ophthalmic composition according to [1], wherein the light is blue light.
[3]
The ophthalmic composition according to [1] or [2], wherein the cells are stem cells.
[4]
The ophthalmic composition according to [3], wherein the stem cells are corneal epithelial stem cells.
[5]
A corneal tissue regeneration agent for preventing damage caused by light, comprising at least one member selected from the group consisting of sulfites and vitamin E.
[6]
A method for evaluating the protective effect of stem cells against damage caused by blue light, comprising the steps of irradiating stem cells with blue light and measuring the cell viability of the stem cells.

The present invention also provides, for example, the following inventions.
[2-1]
An ophthalmic composition for protecting cells from damage caused by light, comprising at least one member selected from the group consisting of sulfites, vitamin E, aminoethylsulfonic acid and its salts, flavin adenine dinucleotide and its salts, pyridoxine and its salts, neostigmine and its salts, naphazoline and its salts, aspartic acid and its salts, zinc sulfate, and vitamin A.
[2-2]
The ophthalmic composition according to [2-1], wherein the light is blue light.
[2-3]
The ophthalmic composition according to [2-1] or [2-2], wherein the cells are stem cells.
[2-4]
The ophthalmic composition according to [2-3], wherein the stem cells are corneal epithelial stem cells.
[2-5]
An agent for regenerating corneal tissue from damage caused by light, comprising at least one member selected from the group consisting of sulfites, vitamin E, aminoethylsulfonic acid and its salts, flavin adenine dinucleotide and its salts, pyridoxine and its salts, neostigmine and its salts, naphazoline and its salts, aspartic acid and its salts, zinc sulfate, and vitamin A.

The present invention also provides, for example, the following inventions.
[3-1]
An ophthalmic composition for protecting cells from damage caused by light, comprising at least one member selected from the group consisting of sulfites, vitamin E, and zinc sulfate.
[3-2]
The ophthalmic composition according to [3-1], wherein the light is blue light.
[3-3]
The ophthalmic composition according to [3-1] or [3-2], wherein the cells are stem cells.
[3-4]
The ophthalmic composition described in [3-3], wherein the stem cells are corneal epithelial stem cells.
[3-5]
An agent for regenerating corneal tissue from damage caused by light, comprising at least one member selected from the group consisting of sulfites, vitamin E, and zinc sulfate.

The present invention provides an ophthalmic composition that can protect cells from damage caused by light.

The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the following embodiments.

In this specification, unless otherwise specified, the unit of content "%" means "w/v%" and is synonymous with "g/100 mL."

1. Ophthalmic composition for protecting cells from damage caused by light rays
The ophthalmic composition according to this embodiment contains at least one member selected from the group consisting of sulfites, vitamin E, and zinc sulfate (also referred to as "component (A)").

In this specification, "light" refers to light of any wavelength, including ultraviolet light, visible light, infrared light, etc. The ophthalmic composition according to this embodiment can be suitably used for protecting cells from damage caused by blue light, which has a wavelength of 380 to 500 nm.

The cells that can be protected from damage caused by light by the ophthalmic composition according to this embodiment are not particularly limited as long as they are cells that may be damaged by exposure to light, and examples thereof include corneal epithelial cells and retinal epithelial cells. In addition, the ophthalmic composition according to this embodiment is preferably used to protect stem cells that have both the ability to self-replicate and the ability to differentiate into different types of cells, and an example of such stem cells is corneal epithelial stem cells.

[Component (A)]
[Sulfites]
There are no particular limitations on the sulfites, so long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable.

Examples of sulfites include sulfurous acid salts and pyrosulfite salts. Examples of sulfurous acid salts and pyrosulfite salts include salts with inorganic bases [e.g., ammonium salts, salts with alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), iron, and other metals]. Specific examples of sulfites include sodium hydrogen sulfite, sodium pyrosulfite, sodium sulfite, potassium hydrogen sulfite, potassium pyrosulfite, potassium sulfite, calcium sulfite, ammonium sulfite, and iron sulfite.

Sulfites are preferably sodium hydrogen sulfite, sodium pyrosulfite, or potassium pyrosulfite, with sodium pyrosulfite being more preferred.

Sulfites that are commercially available can also be used. Sulfites can be used alone or in combination of two or more.

The content of sulfites in the ophthalmic composition according to this embodiment is preferably 0.0001 to 0.5 w/v%, more preferably 0.0005 to 0.1 w/v%, and even more preferably 0.001 to 0.05 w/v%, based on the total amount of the ophthalmic composition.

[Vitamin E]
The vitamin E is not particularly limited as long as it is medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable. Specific examples of the vitamin E include tocopherol, tocotrienol, and derivatives thereof, and salts thereof. Tocopherol and tocotrienol may be any of α-, β-, γ-, and δ-, and may be either d- or dl-isomer.

Examples of derivatives of vitamin E include esters with organic acids such as tocopherol acetate, tocopherol succinate, tocopherol nicotinate, and tocopherol linoleate.

Examples of salts of vitamin E include organic acid salts (lactate, acetate, butyrate, trifluoroacetate, fumarate, maleate, tartrate, citrate, succinate, malonate, methanesulfonate, toluenesulfonate, tosylate, palmitate, stearate, etc.), inorganic acid salts (for example, hydrochloride, sulfate, nitrate, hydrobromide, phosphate, etc.), salts with organic bases (for example, salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, amino acids, tripyridine, picoline, etc.), salts with inorganic bases (for example, ammonium salts, salts with alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and metals such as aluminum, etc.).

Preferred vitamin E compounds are d-α-tocopherol, dl-α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, vitamin E acetate (e.g., tocopherol acetate), vitamin E nicotinate, vitamin E succinate, and vitamin E linoleate, with tocopherol acetate (e.g., d-α-tocopherol acetate, dl-α-tocopherol acetate, etc.) being more preferred.

Vitamin E may be either natural or synthetic. Commercially available vitamin E may also be used. Vitamin E may be used alone or in combination of two or more types.

The content of vitamin E in the ophthalmic composition according to this embodiment is preferably 0.0001 to 0.5 w/v%, more preferably 0.0025 to 0.15 w/v%, and even more preferably 0.01 to 0.05 w/v%, based on the total amount of the ophthalmic composition.

[Zinc sulfate]
There are no particular limitations on the zinc sulfate, so long as it is medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable.

The content of zinc sulfate in the ophthalmic composition according to this embodiment is not particularly limited, and is set appropriately depending on the type and content of other blended ingredients, the formulation form, etc. The content of zinc sulfate is, for example, based on the total amount of the ophthalmic composition, preferably 0.001 to 1 w/v%, more preferably 0.005 to 0.5 w/v%, and particularly preferably 0.01 to 0.25 w/v%.

In another embodiment, the ophthalmic composition according to this embodiment contains at least one selected from the group consisting of sulfites, vitamin E, aminoethylsulfonic acid and its salts, flavin adenine dinucleotide and its salts, pyridoxine and its salts, neostigmine and its salts, naphazoline and its salts, aspartic acid and its salts, zinc sulfate, and vitamin A (also referred to as "component (A')"). Note that the sulfites, vitamin E, and zinc sulfate in this embodiment are as described in [component (A)].

[Component (A')]
[Aminoethylsulfonic acid and its salts]
There are no particular limitations on the aminoethylsulfonic acid (taurine) and its salts, so long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable.

Examples of salts of aminoethylsulfonic acid include salts with organic bases (e.g., salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, and picoline), and salts with inorganic bases (e.g., ammonium salts, and salts with metals such as alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), and aluminum).

As aminoethylsulfonic acid and its salts, aminoethylsulfonic acid is preferred.

Commercially available aminoethylsulfonic acid and its salts can also be used. One type of aminoethylsulfonic acid and its salts can be used alone, or two or more types can be used in combination.

The content of aminoethylsulfonic acid and its salts in the ophthalmic composition according to this embodiment is preferably 0.001 to 10 w/v%, more preferably 0.01 to 5 w/v%, and even more preferably 0.1 to 1.0 w/v%, based on the total amount of the ophthalmic composition.

[Flavin adenine dinucleotide and its salts]
Flavin adenine dinucleotide (vitamin B2) and salts thereof are not particularly limited, so long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable.

Examples of salts of flavin adenine dinucleotide include salts with inorganic bases and salts with organic bases, preferably alkali metal salts, and particularly preferably sodium salts (sodium flavin adenine dinucleotide).

The content of flavin adenine dinucleotide and its salts in the ophthalmic composition according to this embodiment is preferably 0.001 to 1.0 w/v%, more preferably 0.01 to 0.5 w/v%, even more preferably 0.03 to 0.1 w/v%, and even more preferably 0.03 to 0.05 w/v%, based on the total amount of the ophthalmic composition.

[Pyridoxine and its salts]
There are no particular limitations on pyridoxine (vitamin B6) and its salts, so long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable.

Examples of salts of pyridoxine include organic acid salts (e.g., lactate, acetate, butyrate, trifluoroacetate, fumarate, maleate, tartrate, citrate, succinate, malonate, methanesulfonate, toluenesulfonate, tosylate, palmitate, stearate, etc.) and inorganic acid salts (e.g., hydrochloride, sulfate, nitrate, hydrobromide, phosphate, etc.).

As pyridoxine and its salts, pyridoxine and its inorganic acid salts are preferred, and pyridoxine hydrochloride is more preferred.

Commercially available pyridoxine and its salts can also be used. Pyridoxine and its salts can be used alone or in combination of two or more types.

The content of pyridoxine and its salts in the ophthalmic composition according to this embodiment is preferably 0.001 to 1.0 w/v%, more preferably 0.01 to 0.5 w/v%, even more preferably 0.02 to 0.2 w/v%, and even more preferably 0.05 to 0.1 w/v%, based on the total amount of the ophthalmic composition.

[Neostigmine and its salts]
Neostigmine and its salts are not particularly limited as long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable. An example of a neostigmine salt is neostigmine methylsulfate. As neostigmine and its salts, neostigmine methylsulfate is preferred.

Neostigmine and its salts may be commercially available. Neostigmine and its salts may be used alone or in combination of two or more.

The content of neostigmine and its salts in the ophthalmic composition according to this embodiment is preferably 0.00005 to 0.05 w/v%, more preferably 0.0001 to 0.01 w/v%, even more preferably 0.0005 to 0.005 w/v%, and even more preferably 0.001 to 0.005 w/v%, based on the total amount of the ophthalmic composition.

[Naphazoline and its salts]
Naphazoline and its salts are not particularly limited as long as they are medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable. Examples of naphazoline salts include naphazoline hydrochloride and naphazoline nitrate. Naphazoline and its salts are preferably naphazoline hydrochloride. Naphazoline and its salts may be used alone or in combination of two or more.

The content of naphazoline and its salts in the ophthalmic composition according to this embodiment is preferably about 0.00001 to 0.1 w/v%, more preferably about 0.0001 to 0.01 w/v%, and even more preferably about 0.0003 to 0.003 w/v%, based on the total amount of the ophthalmic composition.

[Aspartic acid and its salts]
Aspartic acid is a known compound as an acidic amino acid also called 2-aminobutanedioic acid. Aspartic acid and its salts are not particularly limited as long as they are medicamentarily, pharmacologically (pharmaceutical) or physiologically acceptable. Aspartic acid may be in any of the L-, D-, and DL-forms, but is preferably in the L-form.

Examples of salts of aspartic acid include salts with inorganic bases (e.g., ammonium salts; salts with metals such as alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), and aluminum), salts with organic bases (e.g., salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, and picoline), etc. As aspartic acid and its salts, salts of aspartic acid with inorganic bases are preferred, alkali metal salts and alkaline earth metal salts of aspartic acid are more preferred, and potassium aspartate, magnesium aspartate, and magnesium potassium aspartate are even more preferred. Aspartic acid and its salts may be used alone or in combination of two or more.

The content of aspartic acid and its salts in the ophthalmic composition according to this embodiment is not particularly limited, and is set appropriately depending on the type and content of other blended ingredients, the formulation form, etc. The content of aspartic acid or its salts is, for example, preferably 0.01 to 20 w/v%, more preferably 0.01 to 5.0 w/v%, and even more preferably 0.2 to 2.0 w/v%, based on the total amount of the ophthalmic composition.

[Vitamin A]
There are no particular limitations on the vitamin A, so long as it is medicamentously, pharmacologically (pharmaceutical), or physiologically acceptable. Specific examples of vitamin A include retinol (vitamin A1), 3-dehydroretinol (vitamin A2), and derivatives thereof, as well as salts thereof.

Examples of derivatives of vitamin A include esters with monocarboxylic acids such as retinol palmitate, retinol acetate, retinol butyrate, retinol propionate, retinol octylate, retinol laurate, retinol oleate, and retinol linoleate.

Examples of salts of vitamin A include organic acid salts [e.g., monocarboxylates (acetate, trifluoroacetate, butyrate, palmitate, stearate, etc.), polycarboxylates (fumarate, maleate, succinate, malonate, etc.), oxycarboxylates (lactate, tartrate, citrate, etc.), organic sulfonates (methanesulfonate, toluenesulfonate, tosylate, etc.)], inorganic acid salts (e.g., hydrochloride, sulfate, nitrate, hydrobromide, phosphate), salts with organic bases (e.g., salts with organic amines such as methylamine, triethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, picoline, etc.), salts with inorganic bases [e.g., ammonium salts; salts with metals such as alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), and aluminum, etc.].

As vitamin A, a retinol derivative is preferred, an ester of retinol with a monocarboxylic acid is more preferred, retinol palmitate and retinol acetate are even more preferred, and retinol palmitate is even more preferred.

As vitamin A, synthetic substances may be used, or extracts obtained from natural substances (such as vitamin A oil) may be used. Vitamin A oil is fatty oil obtained from animal tissues containing retinol, or a concentrate thereof, or a product obtained by appropriately adding vegetable oil to either of these. As vitamin A, commercially available products may also be used. One type of vitamin A may be used alone, or two or more types may be used in combination.

The content of vitamin A in the ophthalmic composition according to this embodiment is, based on the total amount of the ophthalmic composition, preferably 5,000 to 200,000 IU/100 mL, more preferably 10,000 to 100,000 IU/100 mL, even more preferably 15,000 to 60,000 IU/100 mL, even more preferably 20,000 to 55,000 IU/100 mL, even more preferably 40,000 to 55,000 IU/100 mL, and particularly preferably 45,000 to 55,000 IU/100 mL.

"IU" refers to the international unit calculated by the method described in the Vitamin A Quantitative Method in the Japanese Pharmacopoeia, 17th Edition. For example, the monographs of the Japanese Pharmacopoeia, 17th Edition, state that retinol acetate contains at least 2.5 million units of vitamin A per gram, and retinol palmitate contains at least 1.5 million units of vitamin A per gram.

[Combination of Component (A) or Component (A')]
The ophthalmic composition according to the present embodiment may contain two or more of the above-mentioned (A) or (A') components in combination. This allows the cell protection effect against damage caused by light to be more pronounced. Specific examples of the combination of the (A) or (A') components include, but are not limited to, a combination of sulfites (particularly sodium pyrosulfite) and vitamin A (particularly retinol palmitate), a combination of sulfites (particularly sodium pyrosulfite) and vitamin E (particularly tocopherol acetate), a combination of zinc sulfate and aminoethylsulfonic acid (taurine), and a combination of zinc sulfate and vitamin E (particularly tocopherol acetate).

The pH of the ophthalmic composition according to this embodiment is not particularly limited as long as it is within a medicamentously, pharmacologically (pharmaceutical) or physiologically acceptable range. The pH of the ophthalmic composition according to this embodiment may be, for example, 4.0 to 9.5, preferably 4.0 to 9.0, more preferably 4.5 to 9.0, even more preferably 4.5 to 8.5, even more preferably 5.0 to 8.5, and particularly preferably 5.0 to 8.0.

The ophthalmic composition according to this embodiment can be adjusted to an osmotic pressure ratio within a range acceptable to the living body, if necessary. The appropriate osmotic pressure ratio can be appropriately set depending on the purpose, formulation form, method of use, etc. of the ophthalmic composition, and can be, for example, 0.4 to 5.0, preferably 0.6 to 3.0, more preferably 0.8 to 2.2, and even more preferably 0.8 to 2.0. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to 286 mOsm (the osmotic pressure of a 0.9 w/v% sodium chloride aqueous solution) based on the 17th Revised Japanese Pharmacopoeia, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for measuring osmolality ratios (0.9 w/v% sodium chloride aqueous solution) can be prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650°C for 40-50 minutes, allowing it to cool in a desiccator (silica gel), and then accurately weighing out 0.900 g of it and dissolving it in purified water to make exactly 100 mL, or a commercially available standard solution for measuring osmolality ratios (0.9 w/v% sodium chloride aqueous solution) can be used.

The viscosity of the ophthalmic composition according to this embodiment is not particularly limited as long as it is within a range that is medicamentally, pharmacologically (pharmaceutical), or physiologically acceptable. The viscosity of the ophthalmic composition according to this embodiment is preferably 1 to 10,000 mPa·s, more preferably 1 to 8,000 mPa·s, even more preferably 1 to 1,000 mPa·s, even more preferably 1 to 1000 mPa·s, and particularly preferably 1 to 20 mPa·s, as measured at 20°C using a rotational viscometer (RE550 type viscometer, manufactured by Toki Sangyo Co., Ltd., rotor: 1°34'×R24).

The ophthalmic composition according to the present embodiment may contain a suitable amount of a combination of various pharmacologically active ingredients and physiologically active ingredients in addition to the above-mentioned ingredients, as long as the effects of the present invention are not impaired. The ingredients are not particularly limited, and examples thereof include active ingredients in ophthalmic drugs described in the 2017 Guide to Manufacturing and Sales Approval Standards and Application Practice for Prescription and Non-prescription Drugs (supervised by the Japan Society of Regulatory Science). Specific examples of ingredients used in ophthalmic drugs include the following ingredients.
Antiallergic agents: for example, sodium cromoglycate, tranilast, pemirolast potassium, etc.
Antihistamines: for example, iproheptine, diphenhydramine hydrochloride, chlorpheniramine maleate, ketotifen fumarate, olopatadine hydrochloride, levocabastine hydrochloride, etc.
Amino acids: for example, monoaminomonocarboxylic acids such as glycine, alanine, aminobutyric acid, aminovaleric acid, aminocaproic acid, etc.; monoaminodicarboxylic acids such as aspartic acid, glutamic acid, etc. or salts thereof; diaminomonocarboxylic acids such as arginine, lysine, etc. or salts thereof.
Steroids: for example, fluticasone propionate, fluticasone furoate, mometasone furoate, beclomethasone propionate, flunisolide, etc.
Decongestants: for example, tetrahydrozoline hydrochloride, tetrahydrozoline nitrate, epinephrine, epinephrine hydrochloride, ephedrine hydrochloride, phenylephrine hydrochloride, dl-methylephedrine hydrochloride, and the like.
Ocular muscle regulating drugs: for example, cholinesterase inhibitors having an active center similar to that of acetylcholine, specifically, tropicamide, helenien, atropine sulfate, etc.
Anti-inflammatory agents: for example, glycyrrhetinic acid, dipotassium glycyrrhizinate, pranoprofen, methyl salicylate, glycol salicylate, allantoin, tranexamic acid, ε-aminocaproic acid, berberine chloride, berberine sulfate, sodium azulene sulfonate, lysozyme, licorice, etc.
Astringents: for example, zinc oxide, zinc lactate, etc.
Fat-soluble vitamins (excluding vitamin E): For example, retinol palmitate, retinol acetate, etc.
Water-soluble vitamins: for example, cyanocobalamin, panthenol, calcium pantothenate, sodium pantothenate, etc.
Local anesthetics: for example, lidocaine, procaine, etc.
Others: For example, sulfamethoxazole, sulfamethoxazole sodium, etc.

In the ophthalmic composition according to the present embodiment, various additives may be appropriately selected according to the purpose and the formulation form, and one or more of them may be combined and contained in an appropriate amount in accordance with a conventional method, so long as the effect of the present invention is not impaired. Examples of such additives include various additives described in the Pharmaceutical Additives Encyclopedia 2007 (edited by the Japan Pharmaceutical Additives Association). Representative components include the following additives.
Carrier: For example, an aqueous solvent such as water or aqueous ethanol.
Chelating agents: for example, ethylenediaminediacetic acid (EDDA), ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), and the like.
Bases: For example, octyldodecanol, titanium oxide, potassium bromide, plastibase, etc.
pH adjusters: for example, hydrochloric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, triethanolamine, diisopropanolamine, etc.
Surfactants: for example, nonionic surfactants such as tyloxapol, polyoxyethylene sorbitan fatty acid esters, polyoxyl stearates, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, and poloxamers; anionic surfactants such as polyoxyethylene alkyl ether phosphates, polyoxyethylene alkyl ether sulfates, alkylbenzene sulfonates, alkyl sulfates, and N-acyltaurine salts; and zwitterionic surfactants such as lauryl dimethylaminoacetate betaine.
Cooling agents: for example, menthol, menthone, camphor, borneol, geraniol, cineole, citronellol, carvone, anethole, eugenol, limonene, linalool, linalyl acetate, thymol, cymene, terpineol, pinene, camphene, isoborneol, fenchene, nerol, myrcene, myrcenol, linalool acetate, lavandulol, eucalyptus oil, bergamot oil, peppermint oil, cool mint oil, spearmint oil, peppermint oil, fennel oil, cinnamon oil, rose oil, camphor oil, etc.
Thickeners: for example, cellulose-based polymer compounds such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose; polyvinyl-based polymer compounds such as polyvinylpyrrolidone and polyvinyl alcohol; carboxyvinyl polymers; guar gum; hydroxypropyl guar gum; gum arabic; karaya gum; xanthan gum; agar; alginic acid and its salts (sodium salt, etc.); mucopolysaccharides such as heparinoids, heparin, heparin sulfate, heparan sulfate, heparinoid, hyaluronic acid and its salts (sodium salt, etc.), and chondroitin sulfate and its salts (sodium salt, etc.); starch; chitin and its derivatives; chitosan and its derivatives; carrageenan; monosaccharides such as glucose, etc.
Buffers: for example, borate buffers, phosphate buffers, carbonate buffers, acetate buffers, lactate buffers, succinate buffers, citrate buffers, Tris buffers, AMPD buffers, and the like.
Stabilizers: for example, edetic acid, edetate salts (disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate), sodium formaldehyde sulfoxylate (Rongalit), aluminum monostearate, glycerin monostearate, cyclodextrin, monoethanolamine, dibutylhydroxytoluene, etc.
Preservatives: for example, alkyl polyaminoethyl glycine quaternary ammonium salts (e.g., benzalkonium chloride, benzethonium chloride, etc.), chlorhexidine gluconate, polydonium chloride, zinc chloride, sodium benzoate, ethanol, chlorobutanol, sorbic acid, potassium sorbate, sodium dehydroacetate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, biguanide compounds (specifically, polyhexanide hydrochloride (polyhexamethylene biguanide), alexidine, etc.), Gloquil (trade name, manufactured by Rhodia), etc.
Isotonicity agents: for example, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, potassium acetate, sodium acetate, sodium bicarbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, glycerin, propylene glycol, etc.
Sugar alcohols: for example, xylitol, sorbitol, mannitol, etc. These may be in the d-, l- or dl-form.
Oils: for example, vegetable oils such as sesame oil, castor oil, soybean oil, olive oil, etc.; animal oils such as squalane, etc.; mineral oils such as liquid paraffin, Vaseline, etc.

When the ophthalmic composition according to this embodiment contains water, the water content is, for example, preferably 80 w/v% or more and less than 100 w/v%, more preferably 85 w/v% or more and 99.5 w/v% or less, and even more preferably 90 w/v% or more and 99.2 w/v% or less, based on the total amount of the ophthalmic composition, from the viewpoint of more significantly achieving the effects of the present invention.

The water used in the ophthalmic composition according to this embodiment may be any water that is medicamentally, pharmacologically (pharmaceutical) or physiologically acceptable. Examples of such water include distilled water, tap water, purified water, sterile purified water, water for injection, and distilled water for injection. These definitions are based on the 17th edition of the Japanese Pharmacopoeia.

The ophthalmic composition according to this embodiment can be prepared by adding and mixing a desired amount of component (A) (or component (A')) and, if necessary, other components to a desired concentration. For example, it can be prepared by dissolving or dispersing those components in purified water, adjusting the pH and osmotic pressure to a predetermined level, and sterilizing the composition by filtration sterilization or the like.

The ophthalmic composition according to this embodiment can be in various formulation forms depending on the purpose. Examples of the formulation form include a liquid, a gel, a semi-solid (ointment, etc.), etc.

The ophthalmic composition according to this embodiment can be used, for example, as eye drops (also called eye drops or eye drops. Eye drops include eye drops that can be applied while wearing contact lenses), artificial tears, eyewash (also called eyewash or eyewash. Eyewash includes eyewash that can be applied while wearing contact lenses), and a composition for contact lenses [contact lens wearing solution, contact lens care composition (contact lens disinfectant, contact lens preservative, contact lens cleaner, contact lens cleaning preservative), etc.]. The term "contact lenses" includes hard contact lenses and soft contact lenses (including both ionic and non-ionic, and both silicone hydrogel contact lenses and non-silicone hydrogel contact lenses).

The ophthalmic composition according to this embodiment is preferably an eye drop (including eye drops that can be applied while wearing contact lenses) because it can more significantly exert the effects of the present invention. When the ophthalmic composition according to this embodiment is an eye drop, the method of use and dosage are not particularly limited as long as they are effective and have few side effects, but for adults (15 years or older) and children aged 7 years or older, an example of a method of use is to apply 1 to 3 drops, 1 to 2 drops, or 2 to 3 drops at a time, 5 to 6 times a day.

The ophthalmic composition according to the present embodiment is provided in any container. The container for containing the ophthalmic composition according to the present embodiment is not particularly limited, and may be made of glass or plastic, for example. Plastic is preferable. Examples of plastic include polyethylene terephthalate, polyarylate, polyethylene naphthalate, polycarbonate, polyethylene, polypropylene, polyimide, copolymers of monomers constituting these, and mixtures of two or more of these. Polyethylene terephthalate is preferable. The container for containing the ophthalmic composition according to the present embodiment may be a transparent container that allows the inside of the container to be seen, or may be an opaque container that makes it difficult to see the inside of the container. A transparent container is preferable. Here, the "transparent container" includes both a colorless transparent container and a colored transparent container.

A nozzle may be attached to the container that contains the ophthalmic composition according to this embodiment. The material of the nozzle is not particularly limited, and may be made of, for example, glass or plastic. It is preferably made of plastic. Examples of plastic include polybutylene terephthalate, polyethylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, copolymers of monomers that constitute these, and mixtures of two or more of these. From the viewpoint of further enhancing the effects of the present invention, the nozzle material is preferably polypropylene, polyethylene, polyethylene terephthalate, polybutylene terephthalate, or polyethylene naphthalate, and more preferably polyethylene.

The container that holds the ophthalmic composition according to this embodiment may be a multi-dose type that holds an amount for multiple uses, or a unit-dose type that holds an amount for a single use.

The ophthalmic composition according to this embodiment is preferably filled in a container having an internal volume of 4 to 30 mL, more preferably filled in a container having an internal volume of 5 to 20 mL, even more preferably filled in a container having an internal volume of 6 to 16 mL, and even more preferably filled in a container having an internal volume of 10 to 15 mL.

The ophthalmic composition according to the present embodiment contains at least one selected from the group consisting of sulfites, vitamin E, and zinc sulfate, thereby protecting corneal epithelial stem cells from damage caused by light. In addition, the ophthalmic composition according to the present embodiment contains at least one selected from the group consisting of sulfites, vitamin E, aminoethylsulfonic acid and its salt, flavin adenine dinucleotide and its salt, pyridoxine and its salt, neostigmine and its salt, naphazoline and its salt, aspartic acid and its salt, zinc sulfate, and vitamin A, thereby protecting corneal epithelial stem cells from damage caused by light. Here, since the corneal epithelial stem cells are protected from damage caused by light, corneal epithelial cells and corneal tissue composed of the corneal epithelial cells are regenerated, the ophthalmic composition according to the present embodiment can also be suitably used as a corneal tissue regenerator against damage caused by light. Furthermore, since the corneal epithelial stem cells are protected from damage caused by light, the corneal epithelial cells and the corneal tissue composed of the corneal epithelial cells are restored, and therefore the ophthalmic composition according to this embodiment can be suitably used as an agent for restoring corneal tissue from damage caused by light. Furthermore, since the corneal epithelial stem cells are protected from damage caused by light, the corneal epithelial cells and the corneal tissue composed of the corneal epithelial cells are restored, and therefore the ophthalmic composition according to this embodiment can be suitably used as an agent for restoring corneal tissue from damage caused by light.

2. Method for evaluating the protective effect of stem cells against damage caused by blue light
The method for evaluating the protective effect of stem cells against damage caused by blue light according to this embodiment (also simply referred to as the "evaluation method according to this embodiment") includes a step of irradiating stem cells with blue light (also simply referred to as the "irradiation step"), and a step of measuring the cell viability of the stem cells (also simply referred to as the "measurement step").

In the irradiation process, the stem cells are exposed to blue light for a specified period of time.

Blue light with a wavelength in the range of 380 to 500 nm can be used for irradiating stem cells. The irradiation time of blue light on stem cells can be set appropriately within the range of, for example, 1 to 15 hours.

The stem cells are not particularly limited as long as they are stem cells that may be damaged by exposure to blue light, and examples of such stem cells include corneal epithelial stem cells.

In the measurement step, the cell viability of stem cells is measured in the absence and presence of the test substance.

In this specification, "cell viability" refers to the percentage of viable cell counts measured in test solutions containing or not containing a test substance after blue light irradiation, when the viable cell count of the control is taken as 100%. Here, the control refers to a test solution that is not irradiated with blue light and does not contain a test substance. When corneal epithelial stem cells are used as stem cells, the viable cell counts can be measured by irradiating each test solution with blue light, adding a cell count measurement reagent, culturing under specified conditions, and measuring the absorbance at a specified wavelength.

In the evaluation method according to the present embodiment, the measurement step may further include a step of calculating a cell protection rate based on the measured cell viability rate. Here, the "cell protection rate" refers to a numerical value calculated by the following formula:
(Formula) Cell protection rate (%)={(cell viability in the presence of test substance−cell viability in the absence of test substance)/cell viability in the absence of test substance}×100

The evaluation method according to this embodiment preferably includes a step of evaluating the protective effect of stem cells against damage caused by blue light based on the change in cell viability of the stem cells obtained in the measurement step (also referred to simply as the "evaluation step").

In the evaluation step, the protective effect of stem cells against damage caused by blue light is evaluated based on the change in cell viability of stem cells in the absence and presence of the test substance obtained in the measurement step. Specifically, when the cell viability in the presence of the test substance is increased compared to the cell viability in the absence of the test substance, the test substance can be evaluated as having a protective effect on stem cells against damage caused by blue light.

In the evaluation method according to this embodiment, when the measurement step includes a step of calculating the cell protection rate, the evaluation step evaluates the protective effect of stem cells against damage caused by blue light based on the calculated cell protection rate. Specifically, when the cell protection rate is a positive value, the test substance can be evaluated as having an effect of protecting stem cells against damage caused by blue light.

The present invention will be specifically explained below based on test examples, but the present invention is not limited to these.

[Test Example 1: Cell protection rate of mouse corneal epithelial stem cells by blue light irradiation (1)]
Each aqueous composition was prepared according to a conventional method using the composition shown in Table 1, and used as a test solution. The unit of each component in Table 1 is w/v %.

Mouse corneal epithelial stem cells (TKE2) were seeded on a culture plate (12 wells, Corning Japan) and cultured until confluent under conditions of 37°C, 5% _CO2 , and 90% humidity. KSFM (keratinocyte serum-free medium (Bovine Pituitary Extract, containing EGF), Thermo Fisher Scientific) medium was used as the culture medium.

The culture medium was removed from each well by suction, and 1 mL of each test solution was added to each well and incubated for 1 hour under conditions of 37° C. and 5% CO _2. Next, blue light (wavelength 470 nm) was irradiated for 14 hours under conditions of 37° C. and 5% CO ₂ , and then the number of viable cells in each well was measured.

The number of viable cells was measured by adding a cell counting reagent Cell Counting Kit-8 (manufactured by DOJINDO) to each well, incubating at 37°C and 5% _CO2 for 1 hour, and then measuring the absorbance at 450 nm. The percentage of the number of viable cells measured for each test solution when the number of viable cells in the control was taken as 100% was taken as the cell viability (%). The control was treated in the same manner as Comparative Example 1-1, except that blue light was not irradiated.

Next, the cell protection rate (%) of each Example was calculated according to the following (Equation 1).
(Formula 1) Cell protection rate (%)={(cell viability of Example−cell viability of Comparative Example 1-1)/cell viability of Comparative Example 1-1}×100
The results are shown in Table 1.

It was confirmed that the addition of sodium pyrosulfite protects corneal epithelial stem cells from damage caused by blue light.

[Test Example 2: Cell protection rate of mouse corneal epithelial stem cells by blue light irradiation (2)]
Each aqueous composition was prepared according to a conventional method using the composition shown in Table 2 to prepare a test solution. The unit of each component in Table 2 is w/v %.

Mouse corneal epithelial stem cells (TKE2) were seeded on a culture plate (12 wells, Corning Japan) and cultured until confluent under conditions of 37°C, 5% _CO2 , and 90% humidity. KSFM (keratinocyte serum-free medium (Bovine Pituitary Extract, containing EGF), Thermo Fisher Scientific) medium was used as the culture medium.

The culture medium was removed from each well by suction, and 1 mL of each test solution was added to each well and incubated for 1 hour under conditions of 37° C. and 5% CO _2. Next, blue light (wavelength 470 nm) was irradiated for 14 hours under conditions of 37° C. and 5% CO ₂ , and then the number of viable cells in each well was measured.

The number of viable cells was measured by adding a cell counting reagent Cell Counting Kit-8 (manufactured by DOJINDO) to each well, incubating at 37°C and 5% _CO2 for 1 hour, and then measuring the absorbance at 450 nm. The percentage of the number of viable cells measured for each test solution when the number of viable cells in the control was taken as 100% was taken as the cell viability (%). The control was treated in the same manner as Comparative Example 2-1, except that blue light was not irradiated.

Next, the cell protection rate (%) of each Example was calculated according to the following (Equation 2).
(Formula 2) Cell protection rate (%)={(cell viability of the example−cell viability of Comparative Example 2-1)/cell viability of Comparative Example 2-1}×100
The results are shown in Table 2.

It was confirmed that the addition of tocopherol acetate protects corneal epithelial stem cells from damage caused by blue light.

[Test Example 3: Cell protection rate of mouse corneal epithelial stem cells by blue light irradiation (3)]
Each aqueous composition was prepared according to a conventional method using the composition shown in Table 3, and used as a test solution. The unit of each component in Table 3 is w/v %.

Mouse corneal epithelial stem cells (TKE2) were seeded on a culture plate (12 wells, Corning Japan) and cultured until confluent under conditions of 37°C, 5% _CO2 , and 90% humidity. KSFM (keratinocyte serum-free medium (Bovine Pituitary Extract, containing EGF), Thermo Fisher Scientific) medium was used as the culture medium.

The culture medium was removed from each well by suction, and 1 mL of each test solution was added to each well and incubated for 6 hours under conditions of 37° C. and 5% CO _2. Next, after irradiating with blue light (wavelength 470 nm) for 14 hours under conditions of 37° C. and 5% CO ₂ , the number of viable cells in each well was measured.

The number of viable cells was measured by adding a cell counting reagent Cell Counting Kit-8 (manufactured by DOJINDO) to each well, incubating at 37°C and 5% _CO2 for 1 hour, and then measuring the absorbance at 450 nm. The percentage of the number of viable cells measured for each test solution when the number of viable cells in the control was taken as 100% was taken as the cell viability (%). The control was treated in the same manner as in Comparative Example 3-1, except that blue light was not irradiated.

Next, the cell protection rate (%) of each Example was calculated according to the following (Equation 3).
(Formula 3) Cell protection rate (%)={(cell viability of the example−cell viability of Comparative Example 3-1)/cell viability of Comparative Example 3-1}×100
The results are shown in Table 3.

It was confirmed that the addition of zinc sulfate protects corneal epithelial stem cells from damage caused by blue light.

[Formulation example]
Eye drops or eye drops for soft contact lenses (Formulation Examples 1 to 16) are prepared by a conventional method according to the formulations shown in Tables 4 and 5 below. The units of the amounts of each component in Tables 4 and 5 below are w/v % unless otherwise specified in the tables. In Tables 4 and 5, the amount of retinol palmitate shown is the amount per 100 mL.

Claims (3)

An ophthalmic composition for protecting corneal epithelial cells from damage caused by blue light , comprising at least one member selected from the group consisting of pyrosulfite and zinc sulfate (excluding those containing α-tocopheryl retinoate). The ophthalmic composition of claim 1 , wherein the corneal epithelial cells are corneal epithelial stem cells. A corneal tissue regeneration agent for preventing damage caused by blue light , which contains at least one member selected from the group consisting of pyrosulfite and zinc sulfate (excluding those containing α-tocopheryl retinoate).