JP5969872B2 - Inhibitor of corneal epithelial cell death containing flavin adenine dinucleotide or a salt thereof as an active ingredient - Google Patents

Description

  The present invention is a corneal epithelial cell death inhibitor containing as an active ingredient a flavin adenine dinucleotide (FAD) or a salt thereof (hereinafter collectively referred to as “FAD etc.”), The present invention relates to an inhibitor, wherein the corneal epithelial cell death is induced by ultraviolet irradiation.

  The cornea is a tissue in direct contact with the outside world and is constantly exposed to various stresses. There are three types of external stresses, ultraviolet A wave (wavelength: 320 nm to less than 400 nm), ultraviolet B wave (wavelength: 290 nm to less than 320 nm), and ultraviolet C wave (wavelength: 200 nm to less than 290 nm). Are known. Japanese Ophthalmology Bulletin, 57, 734-738 (2006) (Non-Patent Document 1) includes an ultraviolet B wave (hereinafter also referred to as “UV-B”) and an ultraviolet C wave (hereinafter also referred to as “UV-C”). Is absorbed in the corneal epithelium and UV-C is not a problem because it is absorbed in the ozone layer, while it is described that UV-B is clinically problematic.

  In addition, Exp. Eye Res. , 90 (2), 216-22 (2010) (non-patent document 2) discloses that UV-B induces apoptosis (cell death) of corneal epithelial cells. Document 1 discloses that UV-B leads to corneal epithelial damage, corneal edema, turbidity, and the like.

On the other hand, FAD is a coenzyme-type vitamin B ₂ that is directly involved in cellular redox, and is known to promote corneal enzyme respiratory metabolism. Furthermore, the Furabitan ^® ophthalmic solution 0.05% attachment (Non-Patent Document 3), the disodium salt of FAD is useful for keratitis which is implicated vitamin B ₂ deficiency or metabolic disorders It is described that there is.

However, the effect of FAD on corneal epithelial cell death induced by ultraviolet rays has not been clarified. Rather, WO99 / 43347 (Patent Document 1) contains a disodium salt of FAD. Flabitan ^{(registered trademark)} ophthalmic solution is exemplified as an ophthalmic agent that induces corneal epithelial disorder.

International Publication No. 99/43347

Bulletin of Japanese Ophthalmology, 57, 734-738 (2006) Exp. Eye Res. , 90 (2), 216-22 (2010) Flabitan (registered trademark) ophthalmic solution 0.05%

  As described above, it is an interesting subject to search for drugs that suppress corneal epithelial cell death induced by ultraviolet irradiation.

  The present inventors conducted extensive research to search for drugs that suppress corneal epithelial cell death induced by ultraviolet irradiation. As a result, flavin adenine dinucleotide disodium salt (hereinafter also referred to as “the present compound”) is prominent. The present invention has been completed by finding that it has an excellent cell death inhibitory effect.

  That is, the present invention relates to an inhibitor of corneal epithelial cell death containing flavin adenine dinucleotide or a salt thereof (FAD or the like) as an active ingredient, wherein the corneal epithelial cell death is induced by ultraviolet irradiation ( Hereinafter also referred to as “this agent”.

  Another aspect of the present invention is an inhibitor of corneal epithelial cell death containing FAD or the like as an active ingredient, wherein the corneal epithelial cell death is induced by ultraviolet B wave irradiation.

  Moreover, the other aspect of this invention is this agent whose density | concentrations, such as FAD, are 0.01-1% (w / v).

  Moreover, the other aspect of this invention is this agent whose density | concentrations, such as FAD, are 0.05% (w / v).

Moreover, the other aspect of this invention is this agent whose dosage form is ophthalmic administration.
Moreover, the other aspect of this invention is this agent whose dosage form is an eye drop.

  As will be described later, since FAD containing this compound remarkably suppresses the decrease in the number of viable corneal epithelial cells induced by ultraviolet irradiation, it becomes an inhibitor of corneal epithelial cell death induced by ultraviolet irradiation. sell.

It is a graph which shows the survival rate at the time of treating a corneal epithelial cell with this compound before UV-B irradiation.

  Flavin adenine dinucleotide is a compound represented by the following formula (1).

  The salt of flavin adenine dinucleotide is not particularly limited as long as it is a pharmaceutically acceptable salt. Salt with inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid , Fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, adipic acid, gluconic acid, glucoheptic acid, glucuronic acid, terephthalic acid, methanesulfonic acid, lactic acid, hippuric acid, 1,2-ethanedisulfonic acid, isethionic acid, Organics such as lactobionic acid, oleic acid, pamoic acid, polygalacturonic acid, stearic acid, tannic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, lauryl sulfate, methyl sulfate, naphthalene sulfonic acid, sulfosalicylic acid Salt with acid; quaternary ammonium salt with methyl bromide, methyl iodide, etc .; bromine ion Salt with halogen ions such as lithium ion, chlorine ion and iodine ion; salt with alkali metal such as lithium, sodium and potassium; salt with alkaline earth metal such as calcium and magnesium; metal salt with iron and zinc; ammonia And salts thereof; triethylenediamine, 2-aminoethanol, 2,2-iminobis (ethanol), 1-deoxy-1- (methylamino) -2-D-sorbitol, 2-amino-2- (hydroxymethyl) -1 , 3-propanediol, procaine, salts with organic amines such as N, N-bis (phenylmethyl) -1,2-ethanediamine, and the like.

  As a salt of flavin adenine dinucleotide, a sodium salt is preferable, and a flavin adenine dinucleotide disodium salt represented by the following formula (2) is particularly preferable.

  Moreover, flavin adenine dinucleotide or a salt thereof (FAD etc.) may take the form of a hydrate or a solvate.

  When a geometric isomer or an optical isomer exists in flavin adenine dinucleotide, the isomer or a salt thereof is also included in the scope of the present invention. Moreover, when a proton tautomer exists in flavin adenine dinucleotide, the tautomer or a salt thereof is also included in the scope of the present invention.

  If flavin adenine dinucleotide or a salt thereof (including a hydrate or solvate form) has a crystalline polymorph and a group of crystalline polymorphs (a crystalline polymorphic system), the crystalline polymorph Forms and crystal polymorph groups (crystal polymorph systems) are also within the scope of the present invention. Here, the crystal polymorphism group (crystal polymorphism system) means that the crystal form changes depending on the conditions and state of production, crystallization and storage of these crystals (including the formulated state in this state). Means the individual crystal forms at each stage and the whole process.

  Flavin adenine dinucleotide or a salt thereof can be produced according to a usual method in the field of synthetic organic chemistry. Moreover, what is marketed by Sigma etc. can also be used.

  In the present invention, “suppression of corneal epithelial cell death induced by ultraviolet irradiation” not only means suppressing the decrease in the number of viable corneal epithelial cells induced by ultraviolet irradiation, but also It also means preventing or treating corneal epithelial damage, corneal edema, turbidity, etc. accompanied by a decrease in the number of cells (ie, corneal epithelial cell death).

  In the present invention, “ultraviolet light” means three kinds of ultraviolet A wave (wavelength: 320 nm to less than 400 nm), ultraviolet B wave (wavelength: 290 nm to less than 320 nm), and ultraviolet C wave (wavelength: 200 nm to less than 290 nm). To do. As described in the background section, it is known that “ultraviolet B wave (UV-B)” is clinically problematic as one that induces corneal epithelial cell death.

This drug can be formulated as a mixed ophthalmic preparation by mixing other active ingredients and pharmaceutically acceptable additives in FAD, etc., using a widely used technique. In addition, a pharmaceutically acceptable additive can be added and formulated as a single ophthalmic preparation using a widely used technique. In Japan, an ophthalmic solution such as “Flavitan ^{(registered trademark)} ophthalmic solution 0.05%” containing the present compound at a concentration of 0.05% (w / v) as an active ingredient is commercially available.

  In the present invention, the agent is administered to the eye topically. Examples of the administration form of this agent include ophthalmic administration (including ophthalmic ointment), subconjunctival administration, intraconjunctival sac administration, subtenon sac administration and the like, and ophthalmic administration is particularly preferable.

  The dosage form of this agent is not particularly limited as long as it can be used for topical ophthalmic administration, and examples thereof include eye drops, eye ointments, injections, patches, gels, insertion agents, and the like. Is preferred. In addition, these can be prepared using the normal technique currently used widely in the said field | area. Furthermore, in addition to these preparations, the present therapeutic agent can also be made into preparations for DDS (drug delivery system) such as preparations for intraocular implants and microspheres.

  Eye drops include isotonic agents such as sodium chloride, potassium chloride, concentrated glycerin; buffering agents such as sodium phosphate, sodium acetate, epsilon-aminocaproic acid; polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, polyoxy Surfactants such as ethylene hydrogenated castor oil; stabilizers such as sodium citrate and sodium edetate; preservatives such as benzalkonium chloride and paraben can be selected and used as necessary. The pH may be in the range acceptable for ophthalmic preparations, but is usually preferably in the range of 4-8.

  The eye ointment can be prepared using a widely used base such as white petrolatum or liquid paraffin.

  The injection is selected as necessary from an isotonic agent such as sodium chloride; a buffering agent such as sodium phosphate; a surfactant such as polyoxyethylene sorbitan monooleate; and a thickener such as methylcellulose. Can be prepared.

  The intercalating agent is prepared by pulverizing and mixing a biodegradable polymer such as hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxyvinyl polymer, polyacrylic acid and the like together with the compound, and compressing the powder. If necessary, excipients, binders, stabilizers, pH adjusters and the like can be used.

  The intraocular implant preparation can be prepared using a biodegradable polymer, for example, a biodegradable polymer such as polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, and hydroxypropylcellulose.

  The dose of this drug can be appropriately changed according to the dosage form, the severity of the symptoms of the patient to be administered, age, weight, doctor's judgment, etc. For example, when an eye drop is selected, 0.005 -5% (w / v), preferably 0.01-1% (w / v), more preferably 0.02-0.5% (w / v), still more preferably 0.05% (w / v) This drug containing FAD at a concentration of v) is instilled 1-2 drops at a time.

  The concentration of FAD or the like described above may be the concentration of flavin adenine dinucleotide (free form) or the concentration of a flavin adenine dinucleotide salt.

  The usage of this drug can be appropriately changed according to the dosage form, the severity of the symptoms of the patient to be administered, age, weight, doctor's judgment, etc.For example, when an eye drop is selected as the dosage form, It can be administered to the eye topically in 1-10 times a day, preferably 2-8 times a day, more preferably 3-6 times a day.

  The results of pharmacological tests and formulation examples are shown below, but these examples are for better understanding of the present invention and do not limit the scope of the present invention.

[Pharmacological test]
When corneal epithelial cells were treated with FAD before ultraviolet (UV-B) irradiation, it was evaluated whether or not the decrease in the number of living cells due to ultraviolet irradiation was suppressed.

(Test method)
SV40 immortalized human corneal epithelial cells (HCE-T: RIKEN, BioResource Center, Cell No .: RCB2280) were seeded in a 96-well plate (1 × 10 ⁴ cells / well), and 10% FBS-containing DMEM / F- Cultured for 12 days in 12 media. On the next day, the DMEM / F-12 medium containing 10% FBS was added to 0.0125% (w / v) or 0.05% (w / v) flavin adenine dinucleotide disodium salt PBS, 0.5% (w / v). v) Chondroitin sulfate sodium-containing PBS or PBS, and then cultured at 37 ° C. for 60 minutes (hereinafter referred to as “0.0125% FAD group”, “0.05% FAD group”, “0.5”, respectively) % CS group ”or“ base group ”). Thereafter, corneal epithelial cells were irradiated with UV-B (80 mJ / cm ² ) for about 1 minute. The medium of each group was again replaced with 10% FBS-containing DMEM / F-12 medium, and cultured at 37 ° C. for 24 hours. Then, CellTiter96 ^{(registered trademark)} AQueous One Solution Cell Proliferation Assay (manufactured by Promega, catalog number: The number of viable cells was measured using G3580), and the survival rate was calculated according to the following formula 1. The flavin adenine dinucleotide disodium salt and chondroitin sulfate sodium salt used in this test were purchased from Kyowa Hakko Bio Co., Ltd. and Maruha Nichiro Foods, Inc., respectively.

[Formula 1]
Survival rate (%) = (number of viable cells in each group / number of viable cells in UV-B non-irradiated group) × 100
(result)
The test results are shown in FIG. In FIG. 1, the value represents an average value ± standard error (N = 3).

(Discussion)
As is clear from FIG. 1, when corneal epithelial cells were pretreated with this compound before UV-B irradiation, a concentration-dependent inhibitory effect on corneal epithelial cell death was confirmed. On the other hand, in the group treated with sodium chondroitin sulfate known to protect the cornea surface exposed to ultraviolet rays (see Baicin ^{(registered trademark)} UV package insert), the same effect was not confirmed. That is, FAD was shown to suppress corneal epithelial cell death induced by ultraviolet irradiation.

[Formulation example]
The pharmaceutical agent of the present invention will be described more specifically with formulation examples, but the present invention is not limited to these formulation examples.

(Prescription Example 1)
Eye drops (FAD concentration: 0.05% (w / v)) Flavin adenine dinucleotide disodium salt in 100 ml 0.05 g
Sodium chloride 0.9g
Sodium hydrogenphosphate hydrate Appropriate amount Sterilized purified water Appropriate amount Add flavin adenine dinucleotide disodium salt and other components to sterilized purified water, and mix them well to prepare an eye drop. Eye drops whose FAD concentration is 0.005% (w / v), 0.01% (w / v) or 0.1% (w / v) by changing the amount of flavin adenine dinucleotide disodium salt added Agent can be prepared.

(Prescription example 2)
Eye ointment (FAD concentration: 0.05% (w / w)) Flavin adenine dinucleotide disodium salt in 100 g 0.05 g
Liquid paraffin 10mg
Appropriate amount of white petrolatum An ointment is prepared by adding flavin adenine dinucleotide disodium salt to uniformly melted white petrolatum and liquid paraffin, thoroughly mixing them, and then gradually cooling them. By changing the amount of flavin adenine dinucleotide disodium salt, the FAD concentration is 0.005% (w / w), 0.01% (w / w) or 0.1% (w / w) An ointment can be prepared.

  Flavin adenine dinucleotide or its salt significantly suppresses the decrease in the number of viable corneal epithelial cells induced by ultraviolet irradiation, and is expected to be an inhibitor of corneal epithelial cell death induced by ultraviolet irradiation. Is done.

Claims (8)

An inhibitor of corneal epithelial cell death containing flavin adenine dinucleotide or a salt thereof as an active ingredient (except when containing zinc) , the administration form is eye drop administration, and the dosage form is eye drop, An inhibitor, wherein the corneal epithelial cell death is induced by ultraviolet irradiation.   The inhibitor according to claim 1, wherein the ultraviolet rays are ultraviolet B waves.   The inhibitor according to claim 1 or 2, wherein the concentration of flavin adenine dinucleotide or a salt thereof is 0.01 to 1% (w / v).   The inhibitor according to claim 1 or 2, wherein the concentration of flavin adenine dinucleotide or a salt thereof is 0.05% (w / v). Use of flavin adenine dinucleotide or a salt thereof for producing an inhibitor of corneal epithelial cell death (excluding cases containing zinc), wherein the corneal epithelial cell death is induced by ultraviolet irradiation, and the dosage form is Use in which the administration is eye drops and the dosage form is eye drops . 6. Use according to claim 5, wherein the ultraviolet light is ultraviolet B wave . The use according to claim 5 or 6, wherein the concentration of flavin adenine dinucleotide or a salt thereof is 0.01 to 1% (w / v) . The use according to claim 5 or 6, wherein the concentration of flavin adenine dinucleotide or a salt thereof is 0.05% (w / v) .

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