WO2008016095A1

WO2008016095A1 - PREVENTIVE OR REMEDY FOR KERATOCONJUNCTIVAL DISORDERS CONTAINING Nrf2 ACTIVATOR AS THE ACTIVE INGREDIENT

Info

Publication number: WO2008016095A1
Application number: PCT/JP2007/065126
Authority: WO
Inventors: Masatsugu Nakamura; Shin-Ichiro Hirai; Atsushi Yoshida
Original assignee: Santen Pharmaceutical Co., Ltd.
Priority date: 2006-08-02
Filing date: 2007-08-02
Publication date: 2008-02-07

Abstract

It is intended to provide a novel preventive or remedy for keratoconjunctival disorders. An Nrf2 activator regulates the induction of the expression of genes of a phase II xenobiotic-metabolizing enzyme, an antioxidant enzyme and so on in corneal epithelial cells and thus exerts an excellent amelioration effect on a corneal disorder model. Therefore, it is useful as a preventive or remedy for keratoconjunctival disorders such as dry eye, punctate keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, superior limbic keratoconjunctivitis, filamentary keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, non-infectious conjunctivitis and so on.

Description

Specification

An agent for the prophylaxis or treatment of keratoconjunctival disorders comprising Nrf 2 activator as an active ingredient

[0001] The present invention relates to dry eye comprising puncta activating substance as an active ingredient, punctate surface keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis dry, upper ring keratoconjunctivitis, filamentous The present invention relates to an agent for the prophylaxis or treatment of keratoconjunctival disorders such as keratoconjunctivitis, infectious keratitis, noninfectious keratitis, infectious conjunctivitis, noninfectious conjunctivitis and the like.

Background art

[0002] A living body regulates the balance between oxidation and antioxidation, and functions by maintaining the intracellular redox state constant. The state in which the balance between oxidation and antioxidation is inclined in the direction of oxidation is called oxidation stress. Oxidative stress modifies and damages biopolymers such as lipids, DNA and proteins, causing cell dysfunction and cell death, causing carcinogenesis, hypertension, arteriosclerosis, cranial nerve disease, inflammation, asthma, skin disease, It is known to be deeply involved in the onset and progression of age-related chronic diseases such as age-related macular degeneration and cataract. Therefore, the defense mechanism that maintains a constant level of oxidation-antioxidation is a vital defense system for the living body, and enhancing the ability to protect against oxidative stress is useful for the prevention and treatment of these diseases. It is thought! /, Ru (nonpatent literature 1).

In recent years, it has been clarified that the K _e apl-Nrf2 system plays a central role in the oxidative stress defense mechanism of mammals. Nrf2 (NF-E2 related factor 2) is a glutathion S transferase that directly detoxifies electrophilic substances, quinone oxidoreductase and レ, 、代謝代謝酵素 Phase 2 enzyme and hemoxigenase, and peroxidase キシン! It is a transcription factor that induces / regulates the expression of genes such as oxidases. In steady state (unstimulated state), Nrf2 forms a complex with Keapl (Kelch-like ECH associated protein 1) in the cytoplasm and is in the inactive state to oxidative stress such as electrophilic substance or reactive oxygen species. When exposed, the interaction with Keapl is diminished (the dissociation is promoted) and it shifts to the nucleus. Nrf 2 transferred to the nucleus forms a heterodimer with a small Maf (Musculo pauro otic Fibrosarcoma) group factor, and the target sequence is the antioxidant response. It binds to a promoter called antioxidant responsive element (ARE) and induces gene expression of the above-mentioned enzymes. Thus, it is known that Nrf2 uniformly controls protection against oxidative stress in the living body and detoxification of xenobiotics (Non-patent Document 2).

[0004] Moreover, in mice in which the Keapl-Nrf 2 system is disrupted (Nrf2 gene-deficient mice), the balance of oxidation-antioxidation in the cells is lost, and the oncogenic potential (progesteral carcinogenesis due to benzpyrene, 2) Torosamine causes bladder cancer etc.), foreign body / sensitivity to oxidative stress (hepatotoxicity caused by acetoaminophen, lung injury caused by exposure to high oxygen etc.), inflammation 'immune system abnormality (glomerulonephritis, systemic self It has been reported to show immune diseases, cigarette-induced emphysema etc.). Thus, it is suggested that functional loss of Nrf 2 causes oxidative stress and induces cancer, hypertension, brain neurodegenerative diseases, inflammation and inflammation, and / or other diseases (Non-patent Document 3).

From these findings, activation of Nrf 2 is considered to be very effective in the prevention and amelioration of diseases associated with oxidative stress. For example, Orchibrazo or sulforaphan known as a substance that activates Nrf 2 suppresses foresite carcinogenesis caused by benzpyrene, and Orchibraz, a substance that activates Nrf 2, suppresses bladder carcinogenesis caused by nitrosamine. Is reported! /, (Non-patent document 3).

[0006] Also, Non-Patent Documents 4 to 5 and Patent Documents 3 to 4 show the usefulness of Nrf 2 activators for age-related macular degeneration and cataract, which are ophthalmic diseases associated with oxidative stress. . Age-related macular degeneration is a degeneration accompanied by age-related changes in the macula, and it is known that retinaldehyde (all-trans- retinaldehyde) accumulated by exposure to ultraviolet light and oxidative stress damage retinal pigment cells. Non-Patent Document 4 and Patent Document 1 report that Nrf 2 activators such as sulforaphan and bis 2-hydroxybenzylideneacetone suppress this cell damage. Non-Patent Document 5 reports that curcumin, an Nrf2 activator, suppresses galactose-induced cataract. Patent Documents 2 and 3 describe a method for treating drusen formation of age-related macular degeneration, Nb 2 activator, retinopathy associated with diabetic retinopathy and glaucoma, and optic neuropathy.

However, there has been no report on examining the pharmacological action of Nrf2 activating substances on corneal and conjunctival disorders such as dry eye, corneal ulcer, keratitis and conjunctivitis. In particular, Nrf2 activity There are no reports to examine the preventive and ameliorating effects of chemical substances on dry eye.

Patent Document 1: International Publication 03/51313 brochure

Patent Document 2: International Publication 2005/063249 Pamphlet

Patent Document 3: International Publication No. 2005/063295 brochure

Non-patent literature l: Biomed. Pharmacother. 57, 251-60 (2003)

Non-patent literature 2: J. Biol. Chem., 278, 14, 12029-38 (2003)

Nonpatent literature 3: A separate volume "medical history of medicine" medicine of redox stress defense, 46-9 (2005) Nonpatent literature 4: Proc. Natl. Acad. Sci., 101, 10446-51 (2004)

Non-patent literature 5: Mol. Vis., 9, 223-30 (2003)

Disclosure of the invention

Problem that invention tries to solve

[0008] Therefore, with regard to Nrf2 activators, searching for new medical applications is an interesting task.

Means to solve the problem

The inventors of the present invention have conducted intensive studies to search for new pharmaceutical uses of Nrf2 activators, and it has been reported that compounds having the ability to activate Nrf2 S, human corneal epithelium We also found that in cells, we control the gene expression of foreign phase 2 enzymes and antioxidative enzymes. Furthermore, the present inventors have found that the Nrf2 activating substance exerts an excellent preventive and ameliorating effect on corneal disorders in a corneal disorder healing efficacy test using a corneal disorder model, and has led to the present invention.

That is, according to the present invention, dry eye, punctate superficial keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, upper ring keratoconjunctivitis containing Nrf2 activator as an active ingredient. It is an agent for the prophylaxis or treatment of keratoconjunctival disorders such as filamentous keratoconjunctivitis, infectious keratitis, noninfectious keratitis, infectious conjunctivitis, non-infectious conjunctivitis and the like.

[0011] The Nrf2 activating substance of the present invention is a compound having an action of activating the transcription factor Nrf2, and the activation of Nrf2 forms a complex with Nrf2 to form a cytoplasm. Dissociating from the trapped Keapl. In addition, as a result of this dissociation, Nrf2 is transferred to the nucleus to induce gene expression of foreign-phase metabolizing system phase 2 enzymes, antioxidative enzymes, etc. 2 Activators also have the effect of promoting the transfer of Nrf2 to the nucleus.

The Nrf2 activating substance of the present invention is, for example, (1) Michael reaction acceptor, (2) diphenols, quinones, butylated hydroxysoles, butylated hydroxytoluenes, (3) (4) hydroperoxides, (5) mercaptans, in particular vicinal dithiols in which two thiol groups are adjacent, (6) 1,2 dithiolthiones, (7) thiocarbamates (8) Arsenic compounds, (9) Heavy metals, (10) Diaryl sulfide, Indole-3-indole rubinol, Epigallocatechin gallate, Ellagic acid, Ethoxyquin, Carotenoids, Conjugated polyenes, 4, 4 Dimethyl- 3 , 4-dihydro-2H-1, 2-benzoselenadines, isohumulonic acid, isomerized hop extract (WO2006 / 043671), salts thereof, etc. (Biochemistry, 76 (4), P. 339-48 (2004) , PNAS 98, 6, 34 04-9 (2001), protein nucleic acid enzyme, 44 (15), P. 2390-5 (1999), WO 2005/06 3249, WO 2005/06 3295).

Examples of the Michael reaction acceptor include compounds having a carbon-carbon double bond linked to an electron withdrawing group such as a carbonyl group, a tolyl group or a nitro group (<<, 13 unsaturated carbonyl compound etc.) Specifically, maleic acid derivatives (Jechiru maleic acid, maleic phosphate dimethyl like), fumaric acid derivatives, 1 twelve Torrox 1 cyclohexene Shikuro, 2- Mechiren 4-butyrolactone, 15 Dokishi eighteen ¹² ^'14 — Prostaglandin J, 7 (E) — [6—

2

(4-Methylphenyl) hexyl] -5-oxo-1-cyclopentene-1-ylidene] Methyl putanoate (PNAS 103, 3, 768-73 (2006)), coumarins (3 hydroxycoumarin etc.), curcumin And bis (2-hydroxybenzylidene) acetone, a compound represented by the following general formula (I) (Cancer Res. 65, 11, 4789-98 (2005)), and the like.

[Formula 1]

[In the formula,

R R R R R R and R are the same or different and are a hydrogen atom or an alkyl group

One, two, three, four, five, six seven

Show

X represents a hydrogen atom, a halogen atom, a nitro group, a cyano group, an aldehyde group or a carboxyl group,

Y represents a cyano group or CO-Z,

Z represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group, an amino group or an imidazole ring

And an aromatic heterocyclic ring selected from the group consisting of triazole ring and benzoimidazole ring.

The aromatic heterocycle defined above may be substituted by a halogen atom, an alkyl group or a phenyl group. ]

Examples of diphenols, quinones, butylated hydroxysoles and butylated hydroxylones are exemplified by catechol, taercetin, carnosol, rosmanol, epirosmanol, carnozic acid, etc. Diphenols, resveratrol, hydroquinone, tert-butyl hydroquinone, 2-tert-butylhydroxyisonone, 3-tert-butynole-hydroxyanisole, 3, 5-di, which has an internal structure and can be oxidized to quinone. tert butyl hydroxytoluene etc. are mentioned. Isothiocyanates are compounds which contain isothiocyanate (S (C = N) groups and are widely present in plants and their seeds. As an example of isothiocyanates, 1-isothiocyanato-4 (methylsulfier) butane (hereinafter, also referred to as “sulforaphane”), 6 And the like, and also include duccosinolate which is a precursor of isothiocyanates, dulcolaphanin (precursor of sulforaphan), and the like. Examples of hydroperoxides include tammen hydroperoxide, tert butyl hydroperoxide and the like. Examples of mercaptans include 2,3 dimercaptopropanol, 1,2 ethanedithiol and the like! /, And two thiol groups adjacent to each other! /, Vicinal dithiols and the like. As an example of 1, 2 dithiol thiones, 4-methyl-5 pyrazine 3H-1, 2-dithiol 3-thione (hereinafter, also referred to as "oltipraz"), 1, 2-dithiol thione, acetonetol trithion etc. It can be mentioned. Examples of thiocarbamates include pyrrolidine thiocarbamate and the like. As an example of the arsenic compound, trivalent arsenous acid (NaAsO) etc.

Examples include compounds having the property of binding to two adjacent thiol groups with an arsenic compound of two. Examples of heavy metals such as HgCl (Hg ²⁺ ), CdCl (Cd ²⁺ ), ZnCl (Zn ²⁺ ), etc.

2 2 2

And heavy metals that react with

[0015] Preferably, tert-butyl hydroquinone, ortipraz, 4,4 dimethyl-3,4 dihydric acid 1 H 2, 1-benzoselenadine, sulfo pha en, carnozic acid, carnosol, curcumin, a table of the following general formula [I] Compounds are mentioned.

As preferable examples of the compound represented by the general formula [I], 2 Shenau 3, 12 dioxooreana 1, 9 (11) Gen 28 Oic Acid (hereinafter referred to as “CDD 0” represented by the following Formula [II] (Also referred to as “CD”), a 2-cyano 3, 12 dioxooreana one represented by the following formula [III] 1, 9 (11) Gen 28 acid acid methyl ester (hereinafter also referred to as “CDDO-Me”), or 1 [2 SHANO 3, 12 dioxooreana 1, 9 (11)-GEN-28 oil] imidazole (hereinafter also referred to as "CDDO-Im") represented by [IV] can be mentioned.

[Formula 2]

As mentioned above, the Nrf 2 activators of the present invention include those of various chemical structures, and no common point is found in the chemical structures. However, on the other hand, it is mentioned that the SH (thiol) group is reactive as a property which is almost common to various substances having different structures. The fact is that the activation power S of Nrf2, S, and the three-dimensional three-dimensional structure of chemical substances In recognition of its reactivity (electrophilicity), it suggests that it is!

[0018] In fact, the following hypotheses have been established as the molecular mechanism of Nrf2 activation.

In steady state, Nrf2 is trapped in the cytoplasm by interacting with Keapl's Kelch domain and is in an inactive state. Keapl has a region called IVR, and the action of the electrophilic substance in this region promotes the conformational change of the Kelch domain and attenuates the interaction with Nrf 2, thereby dissociating Nrf 2. It is believed to be More specifically, it is believed that IVR has a highly reactive cysteine residue and promotes the change in the conformation of Kelch domain by the covalent bond between this cysteine residue and an electrophilic substance. (Biology, 76 (4), P. 339- 48 (2004)).

That is, the Nrf2 activating substance of the present invention broadly refers to a electrophilic compound having reactivity with SH group.

[0020] Whether or not the substance is an Nrf 2 activating substance can also be examined by using the amount of Nrf 2 in the nucleus as an indicator, as in the test method described later (Example 1).

The compounds represented by the general formula [I] can be prepared according to the usual methods in the field of synthetic organic chemistry, and the methods described in JP 2002-530272 and λ ¥ 2004/064723 have been described. Based on! /, You can even manufacture S power.

[0022] Here, each group and word specified in the present specification are shown below. The term "alkyl" refers to linear or branched alkyl having 1 to 8 carbon atoms, preferably linear to branched alkyl having from 6 to 6 carbon atoms. Specific examples thereof include methyl, ethyl, η-propyl, η-butyl, η-pentyl, η-hexyl, isopropyl, isobutyl, isopentyl, isohexyl, sec-butyl, t-butyl and the like. "Alkoxy" represents a linear or branched alkoxy having 1 to 8 carbon atoms, and preferably represents a linear or branched alkoxy having from 6 to 6 carbon atoms. Specific examples thereof include methoxy, ethoxy, n-propoxy, η-butoxide, η-pentoxy, n-hexenoryloxy, isopropoxy, isobutoxy, isopentoxy, isohexyloxy, sec-butoxy, t-butoxy and the like. The halogen atom is fluorine, chlorine, bromine or iodine.

The salts of the present compound are not particularly limited as long as they are pharmaceutically acceptable salts, and sodium salts, potassium salts, lithium salts, calcium salts, magnesium salts, zinc salts, iron salts, manga And salts with inorganic acids such as hydrochloric acid, nitric acid and sulfuric acid, salts with organic acids such as acetic acid, fumaric acid, maleic acid, oxalic acid and tartaric acid, etc., and quaternary ammonium salts Included in the salts in the invention. The compounds may be in the form of hydrates and solvates. In addition, optical isomers, geometric isomers, tautomers, polymorphs and the like of the present compound are included in the scope of the present invention.

In the present invention, the keratoconjunctival disorder refers to a condition in which the cornea or the conjunctiva is damaged due to various factors such as tear abnormality, metabolic abnormality, external injury, etc. For example, dry eye, point Superficial keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, upper limb keratoconjunctivitis, filamentous keratoconjunctivitis, infective keratitis, noninfectious keratitis, infective conjunctivitis, noninfection Sexual conjunctivitis etc. are mentioned. Furthermore, in the present invention, dry eye refers to tear reduction, xerostomia, oryalgia, Shiedaren syndrome, keratoconjunctivitis sicca, Stevens Johnson syndrome, lacrimal gland dysfunction, meibomian gland dysfunction, blepharitis, VDT (Visual Display Terminal) Check the keratoconjunctival disorder associated with work, surgery, medicine, trauma, contact lens wearing, etc. or a symptom associated with the keratoconjunctival disorder.

[0025] The agent for preventing or treating corneal and conjunctival disorders of the present invention can be administered orally or parenterally (such as eye drops and percutaneous administration). Dosage forms include eye drops, eye ointments, gels, skin ointments, injections, tablets, capsules, granules, fine granules, powders and the like. These can be formulated using widely used techniques. For example, in the case of eye drops, tonicity agents such as sodium chloride and concentrated glycerin, buffering agents such as sodium phosphate and sodium acetate, polyoxyethylene sorbitan monohydrate, polyoxyl stearate, polyoxyethylene hydrogenated castor Surfactants such as oils, stabilizers such as sodium citrate and sodium edetate, preservatives such as benzalkonium chloride and benzene, etc. can be formulated as needed. The pH may be within the range acceptable for the ophthalmic preparation, but a range of 4 to 8 is preferred.

An eye ointment can be prepared using a commonly used base such as white petrolatum, liquid paraffin, etc. In the case of oral preparations such as tablets, capsules, granules, fine granules and powders, extenders such as lactose, crystalline cellulose, starch and vegetable oil, lubricants such as magnesium stearate and talc, hydroxypropyl cellulose Binder such as Polyvinyl pyrrolidone If necessary, a disintegrating agent such as calcium hydroxide, a coating agent such as hydroxypropyl methyl cellulose, macrogol, silicone resin or the like, a gelatin film or the like may be added.

[0027] The present invention also relates to a method for preventing or treating keratoconjunctival disorder, comprising administering to a patient an effective amount of Nrf2 activator.

The dosage of the Nrf2 activating substance of the present invention may generally be changed in the case of an oral preparation, depending on the dosage form, the severity of the condition of the patient to be administered, the age, the body weight, and the judgment of the physician. It is possible to administer 0. 0;! To 5000 mg, preferably <0.1 to 2500 mg, more preferably <0.5 to 5 per adult; per single or several divided doses of OO mg per adult. . Also, in the case of eye drops or penetrants, it is possible that: 0. 000001; 10% (w / v), preferably 0. 00001-1% (w / v), more preferably 0 · 0001-0. 1 % (w / v) of the active ingredient concentration can be administered once or several times a day.

Effect of the invention

The following test was carried out: tert-Butyl hydroquinone which has been reported to have Nrf2 activating ability, Oltipras', sulforafuan, carnozic acid, carnosone acid, carnosolole, canorectin, CDDO, CDDO- Me and CDDO- Im However, in human corneal epithelial cells, expression is regulated by Nrf2! /, Target gene glutamate cysteine ligase regulation sub-unit (Glutamate-cysteine ligase, modifier subunit: hereinafter also referred to as "GCLM"), thioredoxin reductase (Thioredoxin Reductase: hereinafter also referred to as “TRxR”), Heme oxygenase 1 (Heme oxidogenase 1: hereinafter also referred to as “H01”) and NAD (P) H quinone oxidoreductase (NAD (P) H quinone oxidoreductase 1: Hereafter, it was shown to increase the expression amount of “NQ 01”. That is, the Nrf2 activating substance induces and controls gene expression of foreign substance metabolism system phase 2 enzymes (for example, NQOl) and antioxidant enzymes (for example, HO 1) also in human corneal epithelial cells. Similarly, the expression levels of target genes GCLM, TRxR, HOI and NQOl, whose expression is regulated by Nrf2, in human corneal epithelial cells in 4,4 Dimethyl-3,4 Dihydro-2H-1,2-Benzo selenazine Indicated. That is, 4,4 Dimethyl-3, 4-Dihydro-2H-1, 2-Benzo selenazine has the ability to activate Nrf2, and it is a foreign phase metabolising phase 2 enzyme in human corneal epithelial cells and Inducible control of gene expression of antioxidant enzymes.

Further, when the following pharmacological test was carried out, Nrf2-activating substances, tert-butylhydroquinone, ortiplusa ', sulforaphane, carnosonere, curcumin, 4, 4-dimethynole-3, 4-dihydro-2H-1, 2-benzoserenadine , CDDO, CDDO-Me and CDDO-Im showed superior improvement in the corneal injury model. That is, the Nrf2 activator is dry eye, punctate surface keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, conjunctival epithelial defect, dry keratitis, upper ring keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, It is useful as a preventive or therapeutic agent for keratoconjunctival disorders such as noninfectious keratitis, infectious conjunctivitis and noninfectious conjunctivitis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] The following shows the results of Examples and Formulation Examples, but these examples are for the purpose of better understanding the present invention, and do not limit the scope of the present invention.

[Example 1]

Inspection method of Nrf2 activating substance

The amount of nuclear Nrf2 can be used as an indicator to determine whether it has Nrf2 activation ability.

Hepatic cancer cells (eg, HepG2) can be used to evaluate whether a compound has the ability to activate Nrf2. For example, monolayer HepG2 cells are cultured in alpha MEM medium containing 10% fetal bovine serum, and when reaching subconfluent, medium is replaced with the medium containing the test compound. After 24 hours of culture, the nuclear extract is collected as described in the method of Abhinav. Jain et al. (J. Biol. Chem., 280, 32, 29158-29168 (2005)), and immunoblotted. Determine the amount of Nrf2. If the amount of Nrf2 in the nuclear extract of cells treated with the test compound is increased as compared to control cells without the test compound, it can be judged that the compound has Nrf2 activating ability.

Second Embodiment

Xenobiotic metabolism system in human corneal epithelial cells of compounds reported to have Nrf2 activating ability and 4,4 dimethyl-3,4-dihydro-2H 1, 2-benzoserenadine. Gene expression induction of phase 2 enzyme and antioxidant enzyme. I examined the ability.

Expression analysis of a gene whose expression is regulated by Nrf 2 Using human corneal epithelial cells, the effect on the expression of a target gene known to be upregulated upon activation of Nrf 2 was examined.

[0035] (Experimental method)

The cells used were SV40 immortalized human corneal epithelial cells (HCE-T) obtained from RIKEN BioResource Center. 6 Ueno plates were seeded with 1 × 10 ⁵ HCE-T and cultured for 3 days at 37 ° C., 5% CO. Culture 40 _§ 15% fetal bovine serum (ICN)

2

DMEM / Ham's F12 (Nacalai Tester) containing / mL gentamycin (Gibco) was used. The culture broth was removed, tert-butyl hydroquinone (100 M), ortibraz (100%), 4, 4-dimethyl-3, 4-dihydro-2-, 1-, 2-benzoselenadine (15%), sulforaphane Change to a culture medium containing (15%), carnozic acid (15%), carnosol (15%), curcumin (15 ^), CDDO (IOONM), CDDO-Me (ΙΟΟ) or CDDO-Im (ΙΟ) And cultured at 37 ° C., 5% CO 2 for 12 hours. Each

2

The compound was dissolved in DMSO and adjusted to the above concentration by diluting 1000 times with culture solution. In addition, a culture solution containing only DMSO was used as a base culture solution.

[0036] After 12 hours of culture, total RNA is extracted from the cells using RNeasy Mini Kit (manufactured by Qiagen), and Oligo (dT) primer (manufactured by Invitrogen) and M-MLV Reverse Transcriptase dnvitrogen) are used. cDNA was synthesized. Using the primers of the following sequence and the QuantiTectTM SYBR Green PCR Kit (manufactured by Qiagen) with the synthesized cDNA as a template, various types can be used with the ABI PrismTM 7000 Sequence Detection System (manufactured by Applied Systems Inc.) The gene expression level was measured. The gene expression level was corrected using Glyceraldehyde 3-phosphate dehydrogenase (hereinafter referred to as “GAPDH”), which is a housekeeping key gene, as an internal standard.

[0037] The DNA primers shown in Table 1 were used for gene expression analysis of tert-butyl hydroquinone (hereinafter, also referred to as "tBHQ") and Olchibraz.

[Table 1] table 1

[0039] 4, 4 Dimethyl-3, 4 Dihydro-2H-1, 2 Benzoselenadine (hereinafter, also referred to as ": BXT-51072"), Snorephoraffan, Carnozic acid, Carnosol, Kunorekmin, CDDO, CDDO- Me and CDDO — In the gene expression analysis of Im, the DNA primers shown in Table 2 were used.

[Table 2] [Table 2]

[0041] (Result)

Assuming that the expression amount of the base culture solution is 1, the gene expression amount (average of the number of examples 3) of each 7 compounds is shown in Table 3.

[Table 3] Table 3

(Discussion)

As apparent from Table 3, in all the compounds used in this test, an increase in the expression level of the target gene GCLM, TRxR, NQOl and HOI induced and controlled by Nrf2 was observed. Compounds such as tBHQ, oltipraz, sulforafuan, carnozic acid, carnosol, clorectin, CDDO, CDDO-Me and CDDO-Im have been reported to activate Nrf 2 in various cells. These Nrf 2) It has been shown that the activator also regulates and regulates the gene expression of foreign phase 2 enzymes and anti-oxidant enzymes in human corneal epithelial cells. In addition, it was shown that BXT-51072 induces and regulates gene expression of foreign-type metabolic system phase 2 enzymes and antioxidant enzymes in human corneal epithelial cells, as in the case of the above-mentioned Nrf2-activating substance. Therefore, it was suggested that BXT-51072 has Nrf 2 activation ability. From the above results, Michael reaction acepter represented by curcumin, CDDO, CDDO-Me and CDDO-Im, diphenols represented by carnozic acid and carnosol, quinones represented by tBHQ, and isothi represented by sulforaphane Nrf2 activators such as ionates, 1,2-dithiolethione represented by Orchibraz, and BXT-51072 Gene expression of foreign phase 2 phase enzyme and antioxidant enzyme also in human corneal epithelial cells Induction control It was shown to do.

[Example 3]

Using the corneal injury model, the effect of Nrf2 activators on corneal injury was examined.

[0045] Therapeutic efficacy test for corneal disorder

Using male SD rats, a corneal injury model was prepared according to the method of Fujihara et al. (Invest. Ophthalmol. Vis. Sci 42 (1) 96-100 (2001)). Miyata et al.'S method (Ophthalmology Clinic, 48 (2), 183-188 (1994)) after modification of the corneal injury model (Modified Ophthalmology, 21 (1), 87-90 (2004)) The rate of improvement of corneal disorder was determined.

(Method of Experiment)

Male SD rats were given general anesthesia with Nembutal administration, then extraorbital lacrimal gland was removed and corneal damage was induced over 2 months.

[0047] Next, the Nrf2 activators tBHQ, altipraz, BXT-51072, sulforaphane, carnosol and curcumin were administered as follows.

[0048] tBHQ instillation group:

Saline solution containing tBHQ (62511 M) was instilled in both eyes 6 times a day for 14 days (8 animals per group).

Orchibraz Eyedrops:

Physiological saline containing alchibraz (500 M) was instilled in both eyes 6 times a day for 14 days (group of 4 animals: 8 eyes).

[0050] BXT-51072 instillation group:

A saline solution containing BXT-51072 (25 M) was instilled in both eyes six times a day for 14 days (group of 4 animals: 8 eyes).

[0051] Snolev Olafan Eye Drops:

A saline solution containing sulfoorophane (25 M) was instilled in both eyes 6 times a day for 14 days (group of 4 animals: 8 eyes).

Carnosol instillation group:

Saline solution containing carnosol (25 M) was instilled in both eyes 6 times a day for 14 days (group of 4 animals: 8 eyes). Curcumin instillation group:

Saline solution containing curcumin (25 M) was instilled in both eyes 6 times a day for 14 days (8 animals per group).

[0054] CDDO instillation group:

Saline solution containing CDDO (l 11 M) was instilled in both eyes 6 times a day for 7 days (group of 4 animals: 8 eyes).

[0055] CDDO— Me Eye Drop Group:

Saline solution containing CDDO-Me (0.1 μM) was instilled in both eyes 6 times a day for 7 days (8 animals in 4 groups).

[0056] CDDO— Im Instillation Group:

[0057] As a control group, physiological saline is applied to both eyes six times a day for seven days (CDDO, CDD O-Me and CDDO-Im controls) or 14 days (tBHQ, Oltipraz ,, BXT 51072, sulfolafuan) (Carnosol and curcumin control) was instilled (4 animals per group, 8 eyes).

Seven or 14 days after the start of instillation, the injured part of the cornea was stained with fluorescein.

For each of the upper, middle and lower portions of the cornea, the degree of staining with fluorescein was scored according to the following criteria, and the improvement rate of the corneal disorder was calculated from the average value of the total score in each portion. Also for normal eyes, the average value of the total score of each part was calculated.

(Criteria)

0: not stained

1: Staining is sparse, and each spot-like stained part is separated.

2: Staining is moderate, and a part of spot-like stained parts are adjacent.

3: Staining is dense, and each spot-like stained portion is adjacent to /!

[0060] (Result)

Based on the average score of the control group (saline) as a standard (improvement rate: 0%) The improvement rates of tBHQ, Oltipraz, BXT-51072, sulfolafuan, carnosol, curcumin, CDDO, CDDO-Me, and CDDO-Im are shown in Table 4. The average score is the average of 8 cases each.

[0061] Improvement rate (%) = {(control)-(test compound)} / degree of failure X 100

Degree of disability = {(control)-(normal eye)}

[Table 4] Table 4

(Discussion)

As evident from the above-mentioned results of the healing efficacy test of the corneal injury using rats, tBHQ, Oltipraz, BXT-51072, sulfolofaan, carnosol, curcumin, CDDO, CDDO-Me and CDDO-Im significantly improve corneal injury. did. That is, Mykenore reaction oxidators represented by Talcmin, CDDO, CDDO-Me and CDDO-Im, Diphenols represented by Carnosol, Quinones represented by tBHQ, Isothiocyanates represented by Sulforaphane It has been shown that it has an improving effect on Nrf2 activator power S such as 1,2-dithiolthiones represented by ortho-blaz and BXT-51072 and corneal disorder.

[Formulation Example] Although the preparation of the present invention will be described in more detail by way of formulation examples, the present invention is not limited to these preparation examples.

Prescription example 1 Eye drop

In 100 ml

Carnosol lOmg

Sodium chloride 900 mg

Sterile purified water

[0065] By changing the addition amount of carnosol, the concentrations were 0.10% (w / v), 0. 03% (w / v), 0.1% (w / v), 0. 3% (w) / v), 1% (w / v) eye drops can be prepared.

Prescription example 2

In 100 ml

Honoreviz l OOmg

International medicine 800 mg

Hydrogen phosphate disodium lOOmg

Sodium dihydrogen phosphate

Sterile purified water

[0067] The concentration is 0 · 05% (w / v), 0.3% (w / v) by changing the amount of addition of orchibraz

An eye drop of 0.5% (w / v) and 1% (w / v) can be prepared.

Prescription example 3 Eye salve

100g of liquid paraffin 10. Og

White vaseline

[0069] By changing the addition amount of sulfoorophane, the concentration becomes 0.05% (w / v), 0.1% (w

/ v), 0.5% (w / v), 1% (w / w) eye ointment can be prepared.

Formulation Example 4 Tablet

in lOOmg

Knolekmin lmg Lactose 66. 4 mg

Magnesium stearate 0.6 mg

Curcumin and lactose are mixed in a mixer, and carboxymethyl cellulose and hydroxypropyl cellulose are added to the mixture to granulate, and the resulting granules are dried and sized, and the sized granules are stearic acid. Add magnesium, mix and tablet with a tablet press. In addition, by changing the amount of curcumin added, tablets having a content of 0.1 mg, 10 mg and 50 mg in 100 mg can be prepared.

Industrial applicability

[0072] The present invention provides dry eye containing puncta Nrf2 activator as an active ingredient, punctate surface keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis dry, upper ring keratoconjunctivitis, filamentous It is possible to provide an agent for preventing or treating keratoconjunctival disorders such as keratoconjunctivitis, infectious keratitis, noninfectious keratitis, infectious conjunctivitis, noninfectious conjunctivitis and the like.

Claims

The scope of the claims

[1] An agent for the prophylaxis or treatment of corneal and conjunctival disorders, which comprises an Nrf2 activating substance as an active ingredient.

[2] Nrf2 activating substance is Michael reaction acceptor, diphenols, quinones, butylated hydroxybisols, butylated hydroxytoluenes, isothiocyanates, hydroxy peroxide, mercaptans, 1, 2- Dithiolthiones, Thiocarbamates, Arsenic Compounds, Heavy Metals, Diaryl Sulfide, Indole-3- Forces Rubinol, Epigalocatechin Gallate, Ellagic Acid, Ethoxyquin, Carotenoids, Conjugated Polyenes, 4, 4 Dimethyl- 3 The preventive or therapeutic agent according to claim 1, which is selected from the group consisting of, 4 dihydro-2H-1, 2 benzoserenadine, isohumulonic acid and an isomerized hop extract.

[3] Michael reaction force force jetyl maleate, dimethyl maleate, 1 to 2

1-Cyclohexene, 2 Methylene-4-Butylolone Acetone, 15 Dioxy Δ ¹² 'Prostaglandin J

2, 7 (E)-[6- (4 methyl phenyl) hexyl] 5-oxo methyl 3- cyclopentene 1 ylidene] heptanoate, 3-hydroxycoumarin, talcamine, bis (2- hydroxybenzylidene) acetone and the following general Selected from the group consisting of compounds represented by formula (I), and / or

The diphenolenole is selected from the group consisting of diphenolenotribe power S, force techonere, taeno recetin, force nore sonolee, rosmano single nore, epilos manol, carnozic acid and resveratrol, and / or

The quinones are selected from the group consisting of hydroquinone and tert butyl hydroquinone and / or

Isothiocyanates power S, 1 isothiocyanate 4 (methyl sulfier) butaneto, selected from the group consisting of gonorechocinolate and gonore coraphanin, and / or 1,2 dithiolthiones The preventive or therapeutic agent according to claim 2, wherein the agent is selected from the group consisting of 4-methyl-5 pyrazole 3H-1,2 dithiol 3 thione and 1,2 dithiol thione, and acetone tritrion.

[Formula 1]

[In the formula,

One, two, three, four, five, six seven

Show

Y represents a cyano group or CO-Z,

[4] Nrf2 activating substance is tert-butylhydroquinone, 4-methyl-5 pyrazoline 3H—

1,2 dithiol-3 thione, 4,4 dimethyl-3,4 dihydro-2H-1,2 benzoserenadine, 1 isothiocyanato-4 (methylsulfinyl) butane, carnodinic acid, carnosol, curcumin and the general formula [I] The prophylactic or therapeutic agent according to claim 3, which is selected from the group consisting of the represented compounds and salts thereof.

[5] The compound represented by the general formula [I] is 2-cyano-3, 12 dioxooreana--1, 9 (11)

Jen 28 oic acid, 2 sianho 3, 12 dioxooreana 1, 9 (11) Diene 28 oic acid methyl ester or 1 [2 siano 3, 12-dioxoolean-1, 9 (11) gen 28 oil] in imidazole The prevention according to claim 4 Or a therapeutic agent.

[6] The prophylactic or therapeutic agent according to claim 4, wherein the keratoconjunctival disorder is dry eye.

[7] Keratoconjunctival disorders include punctate superficial keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, upper limb keratoconjunctivitis, filamentous keratoconjunctivitis, infective keratitis, not infected The prophylactic or therapeutic agent according to claim 4, which is sexual keratitis, infectious conjunctivitis or noninfectious conjunctivitis.

[8] The prophylactic or therapeutic agent according to claim 4, wherein the administration form is eye drop administration or oral administration.

[9] The preventive or therapeutic agent according to claim 4, wherein the dosage form is an eye drop, an eye ointment, a tablet, a fine granule or a capsule.

[10] A method for the prophylaxis or treatment of keratoconjunctival disorder, comprising administering an effective amount of Nrf2 activator to a patient.

[11] Nrf2 activating substances are Michael reaction acceptors, diphenols, quinones, butylated hydroxybisols, butylated hydroxytoluenes, isothiocyanates, hydroxy peroxide, mercaptans, 1, 2- Dithiolthiones, Thiocarbamates, Arsenic Compounds, Heavy Metals, Diaryl Sulfide, Indole-3- Forces Rubinol, Epigalocatechin Gallate, Ellagic Acid, Ethoxyquin, Carotenoids, Conjugated Polyenes, 4, 4 Dimethyl- 3 11. The method according to claim 10, which is selected from the group consisting of: 4 dihydro-2H-1, 2 benzoserenadine, isohumulonic acid and an isomerized hop extract.

Yes

[12] Michael reaction force force jetyl maleate, dimethyl maleate, 1 to 2

1-Cyclohexene, 2 Methylene-4-Butyrone Acetone, 15 Dioxy Δ ¹² 'Prostaglandin J, 7 (E)-[6- (4 methylphenyl) hexyl] 5-oxo

2

Mono-cyclopentene 1-ylidene] heptanoate methyl, 3-hydroxycoumarin, talcine, bis (2-hydroxybenzylidene) acetone and a compound represented by the following general formula (I) are selected from the group consisting of and / or ,

And / or

Isothiocyanates S, 1 Isothiocyanate 4 _

6-(Methylsulfinyl) hexyl isothiocyanate

At least one selected from the group consisting of tonor, gonore cinolate and

12. dithiol-thiones, a force selected from the group consisting of 4-methyl-5-pyrajleu 3H-1, 2-dithiol 3-thione, 1,2-dithiolethione, and acetonetritrithione. Prevention or treatment methods.

[Formula 2]

[In the formula,

One, two, three, four, five, six seven

Show

Y represents a cyano group or CO-Z,

[13] Nrf2 activating substance is tert-butylhydroquinone, 4-methyl-5 pyrazoline 3H— 1, 2-Dithiole-3-thione, 4, 4-Dimethyl-3, 4-Dihydro-2H-1, 2-Benzoselenazine, 1 Isothiocyanato-4 (Methylsulfinyl) butane, Carnodinic Acid, Carnosol, Curcumin and General Formula The method according to claim 12, which is selected from the group consisting of a compound represented by [I] and a salt thereof.

[14] The compound represented by the general formula [I] is 2 Shenau 3, 12 Dioxooreana 1, 9 (11)

Gen 28 oic acid, 2 sianho 3, 12 dioxooreana 1, 9 (11) Diene 28 oic acid methyl ester or 1 [2 siano 3, 12-dioxooreana-1, 9 (11)-gent 28 oil] The method for preventing or treating according to claim 13, which is imidazole.

[15] The method for prevention or treatment according to claim 13, wherein the keratoconjunctival disorder is dry eye.

[16] Keratoconjunctival disorders include punctate superficial keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, upper limb keratoconjunctivitis, filamentous keratoconjunctivitis, infective keratitis, not infected The method for prophylaxis or treatment according to claim 4, which is keratitis, infectious conjunctivitis or noninfectious conjunctivitis.

[17] A method for the prophylaxis or treatment of keratoconjunctival disorder, which comprises instilling or orally administering an effective amount of an Nrf2 activator to a patient.

[18] The method for preventing or treating according to claim 10, wherein the dosage form is an eye drop, an eye ointment, a tablet, a fine granule or a capsule.

[19] Use of an Nrf2 activator for the manufacture of a preventive or therapeutic agent for keratoconjunctival disorder.

[20] The Nrf 2 activating substance is a Michael reaction acceptor, diphenols, quinones, butylated hydroxybisols, butylated hydroxytoluenes, isothiocyanates, hydroxy peroxide, mercaptans, 1, 2 —Dithiolthiones, Thiocarbamates, Arsenic Compounds, Heavy Metals, Diaryl Sulfide, Indole 3-Inhibil, Epigarocatechin Gallate, Ellagic Acid, Ethoxyquin, Carotenoids, Conjugated Polyenes, 4, 4 Dimethyl- 20. The use according to claim 19 selected from the group consisting of 3,4 dihydro-2H-1,2 benzoselenazine, isohumulonic acid and isomerized hop extract.

[21] Michael reaction force force jetyl maleate, dimethyl maleate, 1 to 2

1-Cyclohexene, 2 Methylene-4-Butylolone Acetone, 15 Dihydroxy Δ ¹² ' Prostaglandin J, 7 (E)-[6-(4 methyl phenyl) hexyl] 5-oxo

2

Isothiocyanates power S, 1 isothiocyanate 4 (methyl sulfier) butaneto, selected from the group consisting of gonorechocinolate and gonore coraphanin, and / or 1,2 dithiolthiones 21. The use according to claim 20, wherein the use is selected from the group consisting of 4-methyl-5- pyrazole 3H-1, 2 dithiol 3 thione and 1, 2 dithiol thione, and acetone tritrion.

[Chemical 3]

[In the formula,

One, two, three, four, five, six seven

Show

X represents a hydrogen atom, a halogen atom, a nitro group, a cyano group, an aldehyde group or a carboxyl group, Y represents a cyano group or CO z,

[22] Nrf2 activating substance is tert-butyl hydroquinone, 4-methyl-5 pyrazolyl 3H-1,2 dithiol-3 thione, 4,4 dimethyl-3,4 dihydro-2H-1,2 benzoserenadine, 1 isothiocyanate-4 22. The use according to claim 21 selected from the group consisting of (methylsulfinyl) butane, carnodinic acid, carnosol, curcumin and a compound represented by the general formula [I] and salts thereof.

[23] The compound represented by the general formula [I] is 2 Shenau 3, 12 Dioxooreana 1, 9 (11)

Gen 28 oic acid, 2 sianho 3, 12 dioxooreana 1, 9 (11) Diene 28 oic acid methyl ester or 1 [2 siano 3, 12-dioxooreana-1, 9 (11)-gent 28 oil] 23. Use according to claim 22 which is imidazole.

[24] The use according to claim 22, wherein the keratoconjunctival disorder is dry eye.

[25] Keratoconjunctival disorders include punctate superficial keratopathy, corneal epithelial defect, corneal erosion, corneal ulcer, corneal ulcer, conjunctival epithelial defect, keratoconjunctivitis sicca, upper limb keratoconjunctivitis, filamentous keratoconjunctivitis, infective keratitis, not infected 23. The use according to claim 22, which is sexual keratitis, infectious conjunctivitis or non-infectious conjunctivitis.

[26] The use according to claim 22, wherein the administration form is eye drop administration or oral administration.

[27] The use according to claim 22, wherein the dosage form is an eye drop, an eye ointment, a tablet, a fine granule or a capsule.